Production strategies and environmental applications of fungal enzymes

INTERNATIONAL DOCTORAL SCHOOL OF THE USC Sandra González Rodríguez PhD Thesis Production strategies and environmental applications of fungal enzymes Santiago de Compostela, 2023 Doctoral Programme in Chemical and Environmental Engineering TESE DE DOUTORAMENTO PRODUCTION STRATEGIES AND ENVIRONMENTAL APPLICATIONS OF FUNGAL ENZYMES Sandra González Rodríguez ESCOLA DE DOUTORAMENTO INTERNACIONAL DA UNIVERSIDADE DE SANTIAGO DE COMPOSTELA PROGRAMA DE DOUTORAMENTO EN ENXEÑARÍA QUÍMICA E AMBIENTAL SANTIAGO DE COMPOSTELA 2023 DECLARACIÓN DA AUTORA DA TESE Production strategies and environmental applications of fungal enzymes Dna. Sandra González Rodríguez Presento a miña tese, seguindo o procedemento axeitado ó Regulamento, e declaro que: 1) A tese abarca os resultados da elaboración do meu traballo. 2) Na tese faise referencia ás colaboracións que tivo este traballo. 3) A tese é a versión definitiva presentada para a súa defensa e coincide coa versión enviada en formato electrónico. 4) Confirmo que a tese non incorre en ningún tipo de plaxio doutros autores nin de traballos presentados por min para a obtención doutros títulos. En Santiago de Compostela, a 16 de maio de 2023 Asdo. Sandra González Rodríguez AUTORIZACIÓN DAS DIRECTORAS DA TESE Production strategies and environmental applications of fungal enzymes Prof. María Teresa Moreira Vilar Dra. Gemma María Eibes González INFORMAN: Que a presente tese, correspóndese co traballo realizado por Dna. Sandra González Rodríguez, baixo a nosa dirección, e autorizamos a súa presentación, considerando que reúne os requisitos esixidos no Regulamento de Estudos de Doutoramento da USC, e que como directoras desta non incorre nas causas de abstención establecidas na Lei 40/2015. De acordo co indicado no Regulamento de Estudos de Doutoramento, declaran tamén que a presente tese de doutoramento é idónea para ser defendida en base á modalidade de Monográfica con reprodución de publicacións, nas que a participación da doutoranda foi decisiva para a súa elaboración e as publicacións se axustan ao Plan de Investigación. En Santiago de Compostela, 16 de maio de 2023 Asdo. María Teresa Moreira Vilar Asdo. Gemma M. Eibes González Agradecimientos En primer lugar, quiero agradecer al Ministerio de Ciencia, Innovación y Universidades por la financiación aportada a través de la ayuda para contratos predoctorales para la formación de doctores 2017 (BES-2017-081677) y los proyectos MODENA (CTQ2016-79461-R), WooBAdh (PCI2018-092866), Bioflav-OH (RTI2018-094482-J-I00) y HP-NANOBIO (PID2019-111163RB-I00), que ha permitido el desarrollo de la investigación recogida en esta tesis. Además, me gustaría agradecer a CRETUS (ED431E 2018/01), por proporcionarme el apoyo institucional para la realización de este trabajo. A mis directoras de tesis, la Dra. Gemma Eibes y la Prof. María Teresa Moreira, por su supervisión, su dedicación y por haberme dado la oportunidad de realizar esta tesis doctoral. Asimismo, quiero expresar mi gratitud al Dr. Thelmo A. Lu-Chau, por toda su ayuda y por las ideas aportadas durante el transcurso de esta tesis. También quiero agradecer al Prof. Andreas Schaeffer del Institute for Environmental Research de la RWTH Aachen University, por haberme acogido con su mejor predisposición durante mi estancia de investigación. Agradecer a todos los miembros del Biogroup (compañeros de laboratorio, profesores y personal técnico y administrativo); especialmente a los compañeros del laboratorio 2.3 y del instituto, por haber soportado mi “monopolio” de shakers y espectrofotómetro a lo largo de estos años y por tantas charlas productivas y reconfortantes acerca de los reveses experimentales y los “pequeños dramas” del doctorado. Gracias a mi familia, especialmente a mis padres y a mi tío, por su apoyo incondicional y por los valores que me han inculcado, que tan fundamentales han sido a lo largo de esta tesis. A mis amigos, por su apoyo y ánimos, y por ayudarme siempre a relativizar. En especial, gracias a Juan, por tantísimos años aguantándome y confiando en mí. Por último, y no por ello menos importante, gracias a Damián, por todo su apoyo, cariño, paciencia y comprensión a lo largo de esta etapa. Grazas por seguir aturando a este toxiño. 1.5.1. Agriculture residues 37 1.5.2. Industrial wastes 39 1.6. Biotechnological applications of ligninolytic enzymes 41 1.6.1. Biomaterial precursors 42 1.6.2. Degradation of emerging pollutants 44 1.6.2.1. Pharmaceutical and personal care products 44 1.6.2.2. Polycyclic aromatic hydrocarbons 46 1.7. Factors controlling the enzyme activity 47 1.7.1. Substrate, enzyme and product concentrations 47 1.7.2. Temperature 49 1.7.3. pH 50 1.7.4. Solvents 51 1.7.5. Mediators 52 1.7.6. Co-factors 53 1.7.7. Inhibitors 53 1.8. Objectives and structure of this Thesis 54 1.8.1. Main objective 54 1.8.2. Research questions 55 1.8.3. Research objectives 55 1.8.4. Thesis structure 55 CHAPTER 2. VALORISATION OF BLACK LIQUOR FROM WOOD ORGANOSOLV PROCESS FOR THE PRODUCTION OF LIGNINOLYTIC ENZYMES 57 2.1. Introduction 61 2.1.1. Ligninolytic enzymes production and wood industry streams valorisation 61 2.1.2. Objectives of this chapter 62 2.2.1. Chemicals, raw material and microorganism 62 2.2.2. Fermentations 63 2.2.2.1. Inoculum preparation 63 2.2.2.2. Solid state fermentation at flask scale 63 2.2.2.3. Submerged fermentation at flask scale 64 2.2.2.4. Submerged fermentation at reactor scale 64 2.2.3. Recovery and concentration of the enzymatic cocktail 65 2.2.4. Shotgun proteomic analysis by mass spectrometry 65 2.2.5. Analytical protocols 66 2.3. Results and discussion 66 2.3.1. Solid state fermentation at flask scale 66 2.3.2. Submerged fermentation at flask scale 68 2.3.3. Scale-up of submerged fermentation 69 2.3.4. Identification of enzyme cocktail proteins 71 2.4. Conclusions 74 2.5. Annexes Chapter 2 75 CHAPTER 3. LIGNINOLYTIC ENZYMES PRODUCTION THROUGH THE VALORISATION OF WHEAT STRAW 77 3.1. Introduction 81 3.1.1. Peroxidases production by Irpex lacteus and valorisation of waste streams 81 3.1.2. Objectives of this chapter 82 3.2. Materials and methods 82 3.2.1. Chemicals, raw material and microorganism 82 3.2.2. Fermentations 83 3.2.2.1. Inoculum preparation 83 3.2.2.2. Solid state fermentations at flask scale 83 3.2.2.3. Submerged fermentations at flask scale 83 3.2.2.4. Submerged fermentation at reactor scale 84 3.2.3. Recovery and concentration of the enzymatic cocktail 85 3.2.4. Protein identification 85 3.2.4.1. Protein gel electrophoresis 85 3.2.4.2. Shotgun proteomic analysis by mass spectrometry 85 3.2.5. Analytical protocols 86 3.2.6. Life Cycle Assessment 86 3.3. Results and discussion 88 3.3.1. Solid state fermentation at flask scale 88 3.3.2. Submerged fermentation at flask scale 89 3.3.3. Scale-up of submerged fermentation 93 3.3.4. Identification of enzyme cocktail proteins 94 3.3.5. Environmental profile associated to enzyme production 96 3.4. Conclusions 100 3.5. Annexes Chapter 3 101 CHAPTER 4. VALORISATION OF BIOETHANOL BYPRODUCTS TO PRODUCE UNSPECIFIC PEROXYGENASE 107 4.1. Introduction 111 4.1.1. Unspecific peroxygenase potential and the importance of medium composition on its expression 111 4.1.2. Objectives of this chapter 112 4.2. Materials and methods 112 4.2.1. Materials 112 4.2.1.1. Chemicals, raw material and microorganism 112 4.2.2. Fermentations 113 4.2.2.1. Inoculum preparation 113 4.2.2.2. Solid state fermentation at flask scale 113 4.2.2.3. Submerged fermentation at flask scale 114 4.2.2.4. Submerged fermentation at reactor scale 114 4.2.3. Protein identification and shotgun proteomic analysis 114 4.2.4. Analytical protocols 116 4.2.5. Life Cycle Assessment 116 4.2.6. Statistical analysis 117 4.3. Results and discussion 117 4.3.1. Solid state fermentation at flask scale 117 4.3.2. Submerged fermentation at flask scale 119 4.3.3. Scale-up of submerged fermentation 124 4.3.4. Identification of enzyme cocktail proteins 126 4.3.5. Environmental profile associated to enzyme production 132 4.4. Conclusions 136 4.5. Annexes Chapter 4 137 CHAPTER 5. FUNCTIONALISATION OF ORGANOSOLV LIGNIN BY ENZYMATIC DEMETHYLATION FOR BIOADHESIVE FORMULATION 141 5.1. Introduction 145 5.1.1. Lignin as promising candidate for bioadhesive formulation 145 5.1.2. Objectives of this chapter 146 5.2. Materials and methods 146 5.2.1. Chemicals, raw material and enzymes 146 5.2.2. Enzymatic modification of lignin in test tubes 147 5.2.3. Enzymatic modification of lignin in 150-mL reactor scale 148 5.2.4. Analytical protocols 148 5.2.4.1. Measurement of laccase activity and methanol content 148 5.2.4.2. Indirect measurement of lignin solubility 149 5.2.4.3. Fourier Transform Infrared Spectroscopy 149 5.2.4.4. Size Exclusion Chromatography analysis 150 5.2.4.5. Matrix-Assisted Laser Desorption/Ionization analysis 150 5.2.5. Statistical analysis 151 5.3. Results and discussion 151 5.3.1. Selection of laccase for lignin demethylation 151 5.3.2. Influence of pH and temperature on lignin demethylation 153 5.3.3. Improvement of lignin solubility 156 5.3.4. Laccase mediator system 159 5.3.5. Aeration 161 5.3.6. Optimal conditions 163 5.4. Conclusions 167 5.5. Annexes Chapter 5 169 CHAPTER 6. ENZYMATIC ABATEMENT OF EMERGING CONTAMINANTS 181 6.1. Introduction 185 6.1.1. Emerging contaminants and the role of ligninolytic enzymes as green biocatalysts in their abatement 185 6.1.2. Objectives of this chapter 186 6.2. Materials and methods 186 6.2.1. Chemicals 186 6.2.2. Experimental design 187 6.2.3. Emerging contaminants degradation 188 6.2.3.1. Removal with I. lacteus enzyme cocktail 188 6.2.3.2. Removal with unspecific peroxygenase 188 6.2.3.3. Analytical protocols 189 6.2.4. Metabolites analysis 190 6.2.4.1. Radiochemical analysis 190 6.2.4.2. Liquid chromatography coupled to mass spectrometry 191 6.3. Results and discussion 192 6.3.1. Pharmaceutical and personal care products degradation 192 6.3.1.1. Removal with manganese peroxidase cocktail 192 6.3.1.2. Removal with unspecific peroxygenase 196 6.3.1.3. Metabolites identification 207 6.3.2. Degradation of a model polycyclic aromatic hydrocarbon 208 6.3.2.1. Removal with manganese peroxidase 208 6.3.2.2. Removal with unspecific peroxygenase 210 6.3.2.3. Metabolites identification 212 6.4. Conclusions 217 6.5. Annexes Chapter 6 218 CHAPTER 7. GENERAL DISCUSSION AND CONCLUSIONS 220 7.1. Detailed findings of the Thesis 221 7.1.1. Production of ligninolytic enzymes through the valorisation of agroindustrial wastes 222 7.1.2. Application of ligninolytic enzymes to environmental purposes 223 7.2. Research gaps and future perspectives 224 7.3. General outcome of the Thesis 226 MANUSCRIPTS CONSIDERED IN THIS THESIS: RIGHTS, CONTRIBUTIONS AND QUALITY INDICATORS 227 COPYRIGHT PERMISSIONS 233 REFERENCES 239 LIST OF PUBLICATIONS 289 FUNDING 293 ABBREVIATIONS AND ACRONYMS Abbreviations and acronyms 1 AaP Agrocybe aegerita peroxygenase ABTS 2,2′- azino-bis-3-ethylbenzothiazoline-6-sulfonicacid APO Aromatic peroxygenase ANOVA Analysis of variance AO Alcohol oxidase ATP Adenosine triphosphate ATR Attenuated total reflectance BLASTP Basic local alignment search tool for proteins BPA Bisphenol A BRF Brown rot fungi CBZ Carbamazepine CDL Chemiluminescence detector CID Charge-injection device CPO Chloroperoxidase CrP Coprinellus radians peroxygenase DBH 2,5-dihydroxy benzoic acid DDGS Distillers’s dry grain solubles ddH2O2 Doubled distilled water DMP 2,6-dimethoxyphenol DyP Dye-decolorizing peroxidase EC Emerging contaminant Resumo 8 Unha vez definido o contexto actual global da tese, xorden dúas cuestións fundamentais: • Cal é o modo de fermentación máis axeitado para a produción de encimas ligninolíticas mediante a valorización de residuos agroindustriais? • Presentan os cócteles encimáticos producidos a través da valorización de residuos un potencial catalítico suficiente para ser empregados no campo da biotecnoloxía ambiental en comparación coas encimas comerciais? Capítulo 2. Valorización dos licores negros procedentes do proceso organosolv de madeira para a produción de encimas ligninolíticas. Tal e como se indica con anterioridade, a necesidade de reducir os custes de produción das encimas é crucial para que estas sexan competitivas fronte a outros tipos de catalizadores. Ademais, débese ter presente o contexto actual, no que se fomenta unha economía circular que promova a boa xestión dos residuos, xa sexa mediante a súa redución ou mediante estratexias de valorización. Tendo en conta estas dúas premisas, neste capítulo abórdase o uso de correntes residuais xeradas na industria madeireira como substrato principal para a produción de lacasa co fungo Ganoderma lucidum. Concretamente, empregouse un residuo denominado licor negro, cun elevado contido en hemicelulosa e compostos fenólicos, sendo estes fundamentais para inducir a produción de encimas ligninolíticas. Empregándose este residuo como fonte de carbono para o crecemento de fungo, evítase o emprego de compostos de alto valor engadido como pode ser a glucosa, polo que se reduce o custo global do proceso de produción. Así mesmo, o alto contido en compostos fenólicos pode inhibir o crecemento doutros microorganismos competidores, atallando deste xeito un dos maiores retos presentes nas fermentacións con fungos, que é a posible proliferación de bacterias ou levaduras cun crecemento máis rápido cos fungos. A estratexia abordada neste capítulo baseouse na produción dun cóctel encimático cunha alta presenza de actividade lacasa mediante o emprego de fermentacións en estado somerxido e estado sólido. Para o primeiro tipo de fermentación empregouse unha dilución do licor negro suplementada con peptona como fonte de nitróxeno e trazas de cobre (Cu) e manganeso (Mn) para inducir a expresión das encimas ligninolíticas, mentres que para a Resumo 9 fermentación en estado sólido empregáronse como soporte espumas de poliuretano embebidas na dilución de licor negro empregado para as fermentación en estado somerxido. Dada a maior produción de lacasa na fermentación en estado somerxido, esta escalouse a un reactor de 4 L para a obtención de maiores cantidades de encima para unha posterior aplicación do cóctel encimático como biocatalizador. Capítulo 3. Produción de encimas ligninolíticas a través da valorización de palla de trigo. Continuando coa estratexia de uso de residuos como substrato principal para a produción de encimas, este capítulo enfócase no emprego de palla de trigo como principal fonte de carbono para o crecemento do fungo Irpex lacteus e a produción dun cóctel encimático con actividade peroxidasa dependente e independente da presenza de manganeso (MnP e MiP, respectivamente). Este substrato foi seleccionado por ser un dos materiais lignocelulósicos máis estendidos a escala global. A palla de trigo foi empregada como soporte para o crecemento do fungo en estado sólido, estudando se por si soa é suficiente para o correcto crecemento microbiano e produción de encimas, ou se, pola contra, é preciso un aporte externo de carbono e nitróxeno. Por outra banda, estudouse a produción encimática de I. lacteus en estado somerxido empregando un extracto da palla de trigo obtido mediante tratamento térmico. Avaliáronse distintas dilucións do extracto e tamén o efecto do tipo de fonte de nitróxeno suplementaria empregada (orgánica e inorgánica). Os mellores resultados acadados nas fermentacións en estado somerxido levaron a un escalado do proceso nun reactor de 30 L nas condicións óptimas (extracto diluído e suplementado con peptona e glicosa). Este escalado permitiu acadar valores de actividade encimática similares aos obtidos a escala matraz, pero cunha redución do tempo de produción. O cóctel encimático producido foi concentrado e analizado mediante análise proteómica e electroforese en xel, co fin de obter máis información sobre a súa composición e, polo tanto, do seu potencial catalítico. Esta análise permitiu detectar a presenza doutras encimas presentes en menor proporción como proteasas, celobiohidrolasas e dehidroxenasas. Ademais, o contraste dos resultados obtidos na electroforese coa bibliografía existente sobre as encimas producidas con fungo I. lacteus permitiu relacionar a actividade MnP cunha peroxidasa dependente do manganeso de cadea corta e a actividade Resumo 10 independente de Mn cunha peroxidasa descolorante (DyP, siglas procedentes do termo en inglés dye-decolorizing peroxidase). Tamén, co obxectivo de estudar a viabilidade medioambiental do proceso de produción de encimas co fungo I. lacteus a escala laboratorio, empregouse a metodoloxía de Análise de Ciclo de Vida (ACV), a cal puxo de manifesto que os impactos derivados da produción están ligados principalmente ao consumo enerxético. Capítulo 4. Valorización de produtos secundarios da síntese de bioetanol para a produción de peroxixenasa inespecífica. Este capítulo ten como obxectivo a produción dunha encima recentemente descuberta: a peroxixenasa inespecífica (UPO, siglas procedentes do termo en inglés unspecific peroxygenase). Para a súa obtención empregouse o fungo Agrocybe aegerita cultivado nun medio baseado en residuos xerados na industria de síntese de bioetanol. Estes residuos teñen un alto contido proteico, que con anterioridade se relacionou ca indución da expresión da peroxixenasa inespecífica con A. aegerita. Continuouse coa estratexia de produción seguida nos Capítulos 3 e 4, empregando como substrato para a fermentación en estado somerxido a corrente líquida obtida na fermentación de xeración de bioetanol, denominada vinaza. Por outra banda, para a produción encimática en estado sólido empregouse o residuo derivado do secado da vinaza, que son os grans solubles de destilería (DDGS, siglas procedentes do termo en inglés distillers’s dry grain soluble). Na fermentación en estado sólido empregouse o DDGS como soporte e única fonte de nutrientes para o crecemento de A. aegerita, obténdose produción de UPO e lacasa, sendo a de UPO a maior produción en estado sólido publicada ata a data. En estado somerxido, os nutrientes do medio de fermentación foron aportados exclusivamente pola vinaza, da cal se avaliaron distintas dilucións para estudar a súa repercusión sobre a indución da produción de UPO. Asemade, avaliáronse inóculos de distintas idades, xa que diversos estudos constatan que a idade do inóculo pode ter un impacto substancial na indución da expresión encimática e no tempo requirido para a obtención dos valores máximos de actividade. A optimización en estado somerxido levou á escolla das condicións óptimas de produción empregando unha dilución de vinaza do 50 % (v:v) e un inóculo Resumo 11 de 14 días. Baixo estas condición escalouse a produción a un reactor de 4 L, no cal se reduciu o tempo de produción e se acadaron valores similares de actividade encimática. Dado que segue sen estar claro que compostos ou condicións inducen a expresión da UPO, recorreuse á análise proteómica para estudar a expresión encimática antes e despois da detección da actividade UPO no medio de fermentación, con fin de relacionar a activación de certas rutas metabólicas coa expresión da UPO. Esta análise permitiu identificar encimas como a UPO, lacasa e DyP, e diversas proteínas relacionadas con funcións de almacenamento, defensa frente a toxinas e produción e control de radicais libres trala detección de actividade UPO no medio. Asemade, tamén se detectaron cambios no metabolismo de azúcares complexos e fontes nitroxenadas durante a fermentación en estado somerxido. Ao igual que no capítulo 3, realizouse unha avaliación do impacto ambiental xerado polo proceso de produción do cóctel encimático producido por A. aegerita, poñéndose novamente de manifesto que o requirimento enerxético está detrás da maioría de impactos xerados. Capítulo 5. Funcionalización da lignina organosolv mediante desmetilación encimática para a formulación de bioadhesivos. Debido á necesidade de encontrar opcións máis sostibles para a síntese de bioadhesivos, nos últimos anos está en auxe a busca de novos precursores destes compostos. Neste sentido, as materias primas renovables como a lignina sitúanse no punto de mira. Non obstante, este polímero natural presenta unha baixa reactividade, polo que é necesario unha modificación previa de parte dos seus grupos funcionais para aumentar a súa viabilidade como precursor de bioadhesivos. Neste capítulo téntase demostrar que o uso de encimas ligninolíticas como a lacasa pode lograr un aumento da reactividade da lignina. Concretamente, abordouse a modificación de lignina organosolv con lacasas comerciais e co cóctel encimático obtido no Capítulo 2 a través dun proceso de desmetilación, que foi avaliado medindo a liberación de metanol. Tras unha análise inicial na que a lacasa comercial do fungo Trametes versicolor resultou lograr un maior grado de desmetilación, continuouse a optimización de distintos factores cruciais para a efectividade da desmetilación encimática da lignina. Deste xeito, avaliáronse parámetros fundamentais para a estabilidade encimática, Resumo 12 como son a temperatura e o pH, estudando rangos destes factores nos que as lacasas reportan as actividades máis elevadas. Posteriormente, dado que a lignina é un substrato cunha baixa solubilidade en auga, tentouse mellorar a súa solubilidade no medio de reacción a través da adición de distintos axentes tensioactivos coma o PEG-600 e o Tween-80, obténdose unha mellora considerable da solubilidade co emprego de Tween-80. Trala optimización destes factores, o seguinte paso centrouse en intentar mellorar a eficiencia da reacción mediante o emprego dun mediador que mellorase o contacto encimasubstrato. Consideráronse dous dos mediadores máis habituais nas reaccións con lacasa, un mediador natural (vainillina) e un artificial (1hidroxibenzotriazol, HBT), acadando os mellores valores de desmetilación co uso de HBT. Finalmente, o proceso de modificación encimática trasladouse a un biorreactor de 150 mL para poder estudar o efecto da aireación e avaliar a transformación encimática da lignina co conxunto de variables optimizadas. Nestes experimentos corroborouse que o suministro de aire favorecía lixeiramente a desmetilación da lignina, pero que os parámetros cun efecto máis significativo sobre o grado de desmetilación acadado foron a mellora da solubilidade da lignina ca adición de Tween-80 e o emprego do HBT como mediador. Tendo en conta toda a batería de factores optimizados, logrouse mellorar o grado de desmetilación inicial nun 239 %. A modificación da lignina organosolv tratada encimaticamente verificouse a través da análise con distintas técnicas analíticas como a espectroscopía infraroxa por transformada de Fourier, (FT-IR), a desorción/ionización láser asistida por matriz (MALDI) e a cromatografía de exclusión de tamaño (SEC). Estas análises permitiron constatar o aumento do peso molecular na lignina modificada e o descenso da sinal ligada os grupos metilo/metoxilo, o cal é indicativo dunha maior reactividade e de que se acadou certo de grado de oligomerización que axuda a reforzar á estrutura polimérica. Capítulo 6. Degradación encimática de contaminantes emerxentes. A presenza de concentracións significativas de certos contaminantes emerxentes (ECs) nas augas residuais e masas de auga naturais levou á busca de métodos de degradación destes compostos co obxectivo de reducir a súa toxicidade. Resumo 13 Este capítulo aborda a biotransformación de produtos farmacéuticos e de coidado persoal (bisfenol A, hormonas e carbamazepina) e tamén dun composto modelo de hidrocarburo policíclico aromático (pireno), para avaliar a capacidade dos proceso encimáticos na eliminación de contaminantes emerxentes. A eliminación dos contaminantes estudouse co cóctel encimático con actividade MnP e MiP obtido no Capítulo 3, co cóctel enzimático con actividade UPO obtido no Capítulo 4 e cunha UPO comercial. Con ambas encimas se avaliaron distintos parámetros condicionantes da actividade encimática como a dose de encima, o pH ou a concentración de H2O2 (cofactor necesario para o ciclo catalítico de ambas encimas). En todas as condicións estudadas conseguiuse a completa degradación dos ECs, agás no caso da carbamazepina, da cal só se conseguiu unha eliminación do 30 % tras 24 h de reacción coa MnP debido ao seu carácter recalcitrante. Non obstante, esta eliminación é a máis alta reportada ata a data co emprego dun método encimático. No caso do bisfenol A e hormonas, logrouse a degradación total en menos dunha hora nas mellores condicións, namentres que para o pireno foron necesarias 3,5 h e observouse que a acción da UPO foi máis eficiente que a da MnP para a súa degradación. Para as hormonas e bisfenol A as condicións establecidas como óptimas para a UPO comercial empregáronse tamén co cóctel encimático obtido no Capítulo 4, obtendo resultados de degradación similares aos acadados coa encima comercial. En xeral, os maiores valores de adición de H2O2 ensaiados resultaron nunha redución do tempo necesario para a completa eliminación dos contaminantes, aínda que as doses deste composto empregadas para a reacción encimática coa MnP e UPO varían considerablemente. Para complementar o estudo da degradación de contaminantes, tentouse identificar os metabolitos xerados na degradación dalgúns dos compostos estudados. Para isto empregáronse bisfenol A, 17α-ethynylestradiol (EE2) e pireno marcados co isótopo 14C, o cal permite o rastrexo da radioactividade xerada por estes átomos e a súa relación con posibles metabolitos. No caso do pireno, logrouse identificar que os produtos de degradación son resultado dun proceso de polimerización, namentres que para o bisfenol A e EE2, constatouse que as encimas empregadas son capaces de mineralizar ditos compostos, chegando a mineralizar case un 4 % de bisfenol A tras 6,5 h de reacción co emprego do cóctel enzimático con actividade MnP. Resumo 14 Capítulo 7. Discusión xeral e conclusións. Neste capítulo esbózanse as principais conclusións derivadas da investigación levada a cabo nos distintos capítulos desta tese, tentando dar resposta ás distintas cuestións expostas no capítulo de introdución. Asemade, estas conclusións tamén dan pé a que xurdan novas propostas que pretenden ampliar o coñecemento xerado nesta tese. Respecto a se é posible a produción de encimas a partir da valorización de residuos e cal é o método óptimo de produción, nos Capítulos 2, 3 e 4 expóñense distintas estratexias de emprego de residuos agroindustriais para a produción exitosa de distintas encimas (lacasa, MnP e UPO). A escolla do residuo a empregar dependerá maiormente do tipo de microorganismo produtor e da encima que se pretende producir. Deste xeito, para a produción de lacasa e manganeso peroxidasa, a selección dun residuo cunha pequena proporción de lignina é fundamental para a indución da expresión destas encimas, que ademais están estimuladas pola presenza doutros elementos traza no medio, como poden ser o Mn ou o Cu. No caso da produción da peroxixenasa inespecífica, conclúese que é necesario que o medio estea composto por residuos cun alto contido proteico, xa que a análise proteómica constatou que a síntese desta encima dáse paralelamente á activación de rutas metabólicas de degradación de fontes complexas de nitróxeno. Non obstante, aínda que a vinaza resultou ser un substrato adecuado para a produción de UPO, implicou o emprego de condicións de esterilización do medio máis agresivas, polo que nesta liña sería interesante a busca de residuos de composición similar derivados de procesos que non empreguen microorganismos. Da mesma maneira que a escolla do tipo de residuo a empregar como substrato inflúe na produción das encimas, tamén o fai o tipo de fermentación que se empregue para o crecemento do fungo. Para todas as encimas producidas acadáronse valores máis altos de produción e nun período máis curto no estado somerxido, aínda que o estado sólido segue a presentar certas vantaxes, como son a menor probabilidade de contaminación causada por microorganismos competidores e un menor gasto enerxético. Aínda que o uso de residuos agroindustriais resultou óptimo para a produción das distintas encimas, atendendo aos resultados obtidos no análise de ciclo de vida dos procesos de produción de MnP e UPO, queda patente que de cara a reducir o impacto medioambiental derivado destes procesos deben Resumo 15 de adoptarse medidas enfocadas á redución do consumo enerxético. Neste sentido, a avaliación de distintas configuracións dos reactores ou a avaliación de factores que non se tiveron en conta nesta tese podería de ser de gran relevancia. Asemade, o estudo dos impactos medioambientais asociados aos procesos de produción a maior escala, resultaría na obtención de datos máis realistas de cara a implantación a nivel industrial. Na mesma liña, a realización dun estudo económico tamén sería de interese para poder avaliar a viabilidade do emprego de cócteles encimáticos como biocatalizadores. En relación á segunda cuestión abordada nesta tese, enfocada á aplicación das encimas a distintos procesos encimáticos, nos Capítulos 5 e 6 demóstrase que estes biocatalizadores teñen o potencial necesario para a modificación de substratos naturais en prol dunha mellora da súa reactividade, e que tamén son capaces de degradar distintos contaminantes emerxentes. O emprego de lacasas para a mellora da reactividade da lignina demostrou que estas encimas son capaces de oxidar os grupos metilo/metoxilo e de reducir o contido en grupos hidroxilo, características que se atribúen a unha mellora da reactividade. Tamén se constatou que a acción destas encimas depende en gran medida de factores que inflúen notoriamente na súa estabilidade ou actividade, ou de compostos que facilitan a dispoñibilidade do substrato no medio de reacción. Aínda que o seguimento da desmetilación da lignina se fixo analizando a cantidade de metanol liberado, as técnicas de FT-IR, MALDI e SEC resultaron ser de gran utilidade para a caracterización da lignina modificada e a verificación dos cambios estruturais ligados ó aumento da reactividade. Neste sentido, o produto obtido na transformación encimática da lignina organosolv semella ser un candidato óptimo para a síntese de bioadhesivos, aínda que un futuro traballo a continuar sería a avaliación experimental da aplicación deste polímero modificado nunha formulación real. Por último, a aplicación dos cócteles encimáticos con actividade MnP e UPO acadou a completa biotransformación de todos os contaminantes emerxentes estudados, coa excepción da carbamazepina, que só se conseguiu degradar parcialmente coa MnP. Aínda que o aumento do tempo de reacción ou a dose de encima empregada xunto co uso de mediadores podería levar a un incremento da porcentaxe de degradación. Demostrouse que os parámetros estudados (dose de encima, concentración de cofactores e pH) inflúen en gran medida no rendemento da eliminación dos Resumo 16 contaminantes, o cal parece estar máis ligado á actividade encimática que á natureza do contaminante diana. As encimas estudadas acadaron resultados de degradación semellantes no caso dos PPCPs, namentres que a UPO resultou ser a mellor candidata para a degradación de pireno ao requirir un menor tempo de reacción. A análise mediante cromatografía líquida e espectrometría de masas dos metabolitos xerados na transformación encimática do pireno reflicte que o mecanismo de reacción catalizado polas encimas foi a polimerización da molécula. Ademais, demóstrase que o emprego de cócteles encimáticos pode ser tan eficiente coma o uso de encimas purificadas que implican un maior custo de produción. Aínda que os ensaios de degradación de contaminantes emerxentes se realizaron considerando as concentracións habituais destes compostos en augas residuais e masas de auga naturais, sería interesante o estudo da degradación en concentracións maiores, para avaliar como afecta este incremento á actividade encimática, e se pode existir unha inhibición da reacción encimática por interacción cos metabolitos xerados. Asemade, resulta de interese un estudo máis exhaustivo da mineralización de BPA e EE2 coa MnP, xa que incrementando o tempo de reacción se poderían acadar maiores porcentaxes de mineralización. En relación a degradación de pireno, o uso de neutralizadores de radicais libres como o ácido ascórbico durante a reacción encimática podería dar lugar a un mecanismo de hidroxilación evitando os procesos de polimerización. CHAPTER 1 INTRODUCTION AND CONTEXT This chapter reproduces content already published in “González-Rodríguez S, LuChau TA, Trueba-Santiso A, Eibes G, Moreira MT. Bundling the removal of emerging contaminants with the production of ligninolytic enzymes from residual streams. Appl Microbiol Biotechnol. 2022; 106(3):1299-1311.https://doi.org/10.1007/s00253-022-117767”, “González-Rodríguez S, Lu-Chau TA, Chen. X, Eibes G, Pizzi A, Feijoo G and Moreira MT. Functionalisation of organosolv lignin by enzymatic demethylation for bioadhesive formulation. Ind. Crops Prod. 2022; 186, 115253. https://doi.org/10.1016/j.indcrop.2022.115253”, “González-Rodríguez S, TruebaSantiso A, Lu-Chau TA, Moreira MT, Eibes G. Valorization of bioethanol by-products to produce unspecific peroxygenase with Agrocybe aegerita: Technological and proteomic perspectives. N Biotechnol. 2023; 76:63-71. https://doi.org/10.1016/j.nbt.2023.05.001” Detailed information available at “Manuscripts considered in this thesis: rights, contributions and quality indicators”, page 227. Chapter 1 24 Figure 1.2. General catalytic cycle of laccases (adapted with permission from Rodríguez-Delgado et al (2017). Copyright 2017, Springer Nature). 1.2.2. Manganese peroxidase MnP (EC 1.11.1.13) is a H2O2-dependent heme enzyme belonging to the oxidoreductases family (Kumar and Arora, 2022). It was discovered in the fungus P. chrysosporium (Glenn and Gold, 1985) and is found in many microorganisms such as bacteria and fungi. In WRFs, which are the main producers of this enzyme, it is considered to be expressed as a primary enzyme during lignin catabolism, sharing 43 % identity with LiP sequences, which is another major enzyme involved in the fungal enzyme system developed for lignin degradation (Hofrichter, 2002). Due to its versatile catalytic performance, MnP has been used in different processes such as biobleaching, pollutant removal or dye decolorisation (Eibes et al., 2006; Moreira et al., 2003; Qin et al., 2014). The molecular structure of this enzyme, with a molecular weight between 25 and 68 kDa, contains two Ca2+ ions and five disulfide-bridging elements, which have the function of maintaining the structure of the MnP active site. This active site consists of several amino acids (AA) such as the proximal histidine ligand, H-bonded to an aspartic acid residue and a distal peroxidasebinding side pocket containing catalytic histidine and arginine residues (Kumar and Chandra, 2020). In relation to their C-terminal tail, MnPs of WRF are classified into short, long or extra-long types. Short MnPs are 20–30 AA shorter in comparison to long and extra-long MnPs, and the fifth cysteine disulphide bond near the Mn2+ 2 H 2 O O 2 + 4 H + Cu (T2) Cu (T3) Cu (T3) Cu (T1) His His His His His His OH His Phe His Cys Substrate Oxidized substrate HOH His His 4 e - Introduction and context 25 binding site present in long and extra-long MnPs is not present in these enzymes. Furthermore, short MnPs have a higher genetic similarity to LiPs and VPs than long or extra-long MnPs (Hofrichter et al., 2010) and some of them (e.g. Pleurotus ostreatus MnP, and Pleurotus eryngii MnP) are capable of oxidising aromatic substrates in the absence of Mn2+ (Heinfling et al., 1998; Sarkar et al., 1997). Nevertheless, the mechanism responsible for the Mn2+- independent oxidation of aromatic substrates by short MnPs is still unknown (Li et al., 2019). Fortunately, the mechanism of long and extra-long MnPs is well known. Their catalytic cycle starts with the transfer of two electrons from the heme group to H2O2 in the resting state, leading to the production of Compound I and water. Compound I then undergoes oxidation of the substrate with the corresponding formation of Compound II and free radicals. Finally, Compound II is able to catalyse the oxidation of Mn2+ to Mn3+, and this cation performs the oxidation of compounds containing aromatic rings. It should be noted that Compound I can oxidize Mn2+ or a substrate molecule, but Compound II requires the presence of Mn2+ for its reaction. When Mn3+ is formed, then it reacts non-specifically with phenolic compounds by removing an electron and a proton from the substrate molecule. Mn2+ promotes the reversion of MnP to its original form (Hofrichter, 2002; Wong, 2009). Figure 1.3 summarises a general catalytic cycle of long and extralong MnPs. Figure 1.3. General catalytic cycle of long and extralong MnPs (adapted with permission from Chowdhary et al. (2019). Copyright 2019, Springer Nature). OMn 2+ MnP MnP II-OMn 2+ MnP I MnP II OMn 2+ OMn 3+ OMn 3+ OMn 2+ H 2 O 2 Chapter 1 26 1.2.3. Unspecific peroxygenase UPOs (EC. 1.11.2.1) are peroxygenases belonging to the heme-thiolate family with peroxygenase (insertion of one O atom) and peroxidase activity (subtraction of one electron) (Kinner et al., 2021). It was discovered almost 20 years ago by Ullrich et al. (2004) in the fungus Agrocybe aegerita. However, in the first years after its discovery, this enzyme was designated as haloperoxidase or aromatic peroxygenase due to the AA sequence similarities and hydroxylation activity on halides and aromatic compounds characteristic of these enzymes, and it was not until 2011 that it was renamed as UPO. What is most remarkable about UPOs is that in addition to the common catalytic cycle of hemoperoxidases, these enzymes are also capable of performing different non-specific reactions attributed to cytochrome P450 monooxygenases, but with the advantages of being more stable and requiring only H2O2 as a co-substrate, which acts as an electron acceptor and oxygen donor (Kinner et al., 2021; Ullrich and Hofrichter, 2005). This enzyme has been secreted by several fungi, such as Agrocybe (Cyclocybe) aegerita, Coprinellus (Coprinus) radians, Marasmius rotula, Chaetomiun globosum, Coprinus verticillatus, Leptoxyphium fumago, Marasmius wettsteinii and Psathyrella aberdarensis (Kinner et al., 2021). The function of these enzymes in nature is still unclear, but it has been suggested that they are probably related to different physiological roles in metabolite synthesis, detoxification processes and interaction with antimicrobial peptides released by host plants (Hofrichter et al., 2015). Despite this unknown role in the fungal enzyme system, their ability to oxidise a wide range of compounds such as alkenes, chlorinated benzenes, halogenated biphenyl ethers, nitroaromatics, polycyclic aromatic hydrocarbons (PAHs), phthalates, steroids, etc. has been explored by different authors (Karich et al., 2017; Peter et al., 2013; Ullrich et al., 2018). UPOs can be classified according to their structural characteristics and affinity for substrates into two main families: Family I of short UPOs and Family II of long UPOs. Long UPOs have a molecular weight around 44 kDa, whereas short UPOs are between 32 and 29 kDa in size and usually do not have a signal peptide (Hofrichter et al., 2020). Furthermore, long UPOs have been detected only in basidiomycetes and ascomycetes, whereas short UPOs are more widespread within fungal phyla (Hofrichter et al., 2015; Pecyna et al., 2009). Introduction and context 27 The structure of both enzymes is different. Long UPOs are monomeric proteins, whereas short UPOs are mostly dimeric. In addition, the former has internal disulphide bridges and arginine as a charge stabiliser and in the case of short UPOs, these bridges are external and stabilised by histidine. Although both families have similar conserved structural motifs, there are relevant differences in the organisation of the heme channel (in topology and dimension), leading to different affinity for the substrates. For example, short UPOs accept large substrates, whereas long UPOs are more active with smaller compounds (Monterrey et al., 2023). Only a few crystal structures of UPOs are available in Protein Data Bank (PDB): one from A. aegerita (PDB: 2YOR for wild-type and 5OXU for the PaDa-I secretion variant) (Piontek et al., 2010; Ramirez-Escudero et al., 2018) and another from Marasmius rotula (PDB: 5FUK; unpublished, available at PDB). From the cited structures, it is elucidated that the core is mostly composed of α-helices that organise the active site around the heme group. The active site is mainly covered by aromatic residues, implying a preference for slightly hydrophobic substrates (Rotilio et al., 2021). The catalytic mechanism of UPOs is based on a combination of the catalytic cycle of hemo-peroxidases and cytochrome P450 monooxygenases (Dunford 1999, Montellano y De Voss, 2005). They are able to oxygenate C-H bonds in a similar way to P450 through their peroxide “shunt” pathway and also have the ability to oxidise phenols like heme peroxidases. UPOs can transfer an oxygen atom contained in the peroxide (H-O-O-R) to various organic substrates. The target substrates can undergo hydroxylation, epoxidation and heteroatom oxygenation reactions and, in turn, spontaneous dealkylation, deacylation or (re)aromatization (Hofrichter and Ullrich, 2014). In addition, UPO also catalyses single-electron oxidations, such as the abstraction of single electrons from phenolic hydroxyl groups, acting similarly to conventional peroxidases (Figure 1.4). Chapter 1 28 Figure 1.4. Catalytic cycles of unspecific peroxygenase using three different substrates. Left: benzylic hydroxylation of p-cresol to 4-hydroxybenzyl alcohol (inner green arrows), O-dealkylation of 1,4-dimethoxybenzene to p-hydroxyanisole (outer blue arrows) via an unstable hemiacetal intermediate (in square brackets); right: epoxidation of styrene to styrene oxide (outer red arrows) and oxidation of p-cresole to two corresponding phenoxy radicals (inner black arrows) (reproduced with permission from Hofrichter et al. (2022). Copyright 2022, The authors, published by MDPI). 1.3. Production of ligninolytic enzymes Interest in LEs production has increased in recent decades due to the need to find new catalysts that are high-performing, biocompatible and biodegradable. The main consideration to carry out the production of an enzyme is the evaluation of different microorganisms that are able to produce the enzyme of interest. These microorganisms are wild strains that are isolated and studied to understand the set of enzymes that make up their enzyme system. The use of wild strains is related to traditional enzyme production, where fermentations with these microorganisms are carried out by studying and optimizing various factors affecting cell growth and enzyme expression. However, in recent years, the use of genetically modified microorganisms is becoming more relevant, as the use of heterologous hosts allows the improvement of enzyme titers and the reduction of production costs, as well as the synthesis of enzymes “à la carte”. Introduction and context 29 1.3.1. Production of wild-type enzymes Species that can produce LEs without any genetic modification are designed as natural or homologous hosts and are responsible for the production of the wild-type version of these enzymes. The most commonly used microorganisms for the production of LEs are WRF, due to its capability to express one or various LEs. The use of natural or synthetic media similar in composition to the medium in which these fungi grow in nature is required, and various factors involved in cell growth and enzyme induction (e.g., pH, aeration, temperature) must be optimised to achieve high titers of activity. There are two typical strategies for enzyme production with native strains based on the conditions under which fermentations are carried out: solid state fermentation (SSF) and submerged state fermentation (SmF). The former uses solid substrates and fungal growth occurs under static conditions, replicating the natural conditions in which the fungus grows. This fermentation strategy is simple and only requires ensuring optimal humidity and temperature for the development of the selected species. Different authors have evaluated SSF with WRF such as P. chrysosporium, Pleurotus ostreatus, T. versicolor, Irpex lacteus or Ganoderma lucidum for LEs production (Gupte et al., 2007; Rodrigues et al., 2019). For other hand, SmF require a liquid medium in which the fungus grows under controlled temperature and agitation (Elisashvili et al., 2010; Olajuyigbe et al., 2018). Although the use of SmF is more widespread at the industrial level due to the easier management of the fermenter, better control over aseptic conditions and shorter times to obtain the enzymes, SSF is still being investigated due to higher process yields and cost-effective production compared to SmF (Musoni et al., 2015). Commonly, the production of wild-type enzymes requires a preliminary flask-scale optimisation step and then, the process is developed to reactor scale. In the case of fungal LEs, most of them are extracellular enzymes, so the downstream process for obtaining the enzymes is simpler than for intracellular enzymes. The downstream process requires the removal of biosolids from the enzymatic crude. If isolation of the enzyme is required, a purification process is necessary and is usually carried out by chromatographic techniques. However, the level of purification depends on Chapter 1 30 the final application of the product and is in many cases unnecessary due to the possibility to apply the crude enzyme. 1.3.2. Production of recombinant-type enzymes In recent years, the use of recombinant microorganisms with WRF genes has gained special attention in the field of LEs production due to the advantage of increased productivity, reduced time and lower costs (Debnath and Saha, 2020). Recombinant protein expression, also called heterologous expression, involves the use of a host microorganism into which a gene from a native enzyme producer is inserted, encoding the necessary instructions for enzyme expression. In general, the procedure followed involves the following steps: cloning of the gene of interest into an expression vector, transformation of the host microorganism (expression system), selection process of the transformants, growth of the microorganism and expression in bioreactors, and finally recovery and purification of the protein (Ongley et al., 2013). Host microorganisms commonly used to produce recombinant-type LEs are different types of fast-growing yeasts, bacteria or filamentous fungi (e.g. Aspergillus niger, Aspergillus oryzae and Trichoderma reesei), due to their faster growth compared to fungi expressing the enzymes of interest. Specifically, Escherichia coli is probably the most studied candidate due to its faster growth and the extended knowledge about it (Zelena et al., 2014). However, in the case of LEs expression its use is less common due to the inability of bacteria to perform post-translational modifications typical in eukaryotes, such as glycosylation on peroxidases secreted by basidiomycota that confer them a functional structure (Demain and Vaishnav, 2009). Moreover, recombinant proteins expressed by this system accumulate in the cytoplasm or in the periplasmic space with the corresponding formation of aggregates that can inactivate the enzyme and decrease its solubility (Fischer et al., 1992). For this reason, although there are other bacterial hosts that can reduce these obstacles (Terpe, 2006), yeasts are the best candidates for expressing LE, in addition to their status as GRAS (generally regarded as safe) microorganisms (Baghban et al., 2019). The most commonly used yeast strains for heterologous expression are Pichia pastoris and Saccharomyces cerevisae (Demain and Vaishnav, 2009). For example, yeasts such as P. pastoris have been widely used for the expression of different LEs, reaching production levels that can also be higher Introduction and context 31 than those obtained with natural hosts (Table 1.1). In addition, genetic engineering also allows the introduction of several modifications in the genetic code that are able to visibly change the enzyme structure to make it more suitable for specific substrates. Another advantage is the option to introduce genes that confer resistance to a specific antibiotic compound, which can be used during fermentation to avoid contamination by external microorganisms and thus reduced production yield (Nordén et al., 2011). Table 1.1. Production of ligninolytic enzymes by heterologous expression using P. pastoris as host. Enzyme Origin microorganism Scale (L) Activity (U/L) Reference Lignin peroxidase Phanerochaete chrysosporium 10 4480 (Majeke et al., 2020) Manganese peroxidase Phanerochaete chrysosporium 2 2500 (Jiang et al., 2008) Laccase Trametes versicolor 5 18,123 (Li et al., 2014) Unspecific peroxygenase Agrocybe aegerita 2500 30,000 (Tonin et al., 2021) However, there are also several bottlenecks in the use of this technology. One of them is that heterologous expression focuses mainly on the production of a specific enzyme, without taking into account that other enzymes must be present in the medium for the proper functioning of the enzyme. For example, H2O2-dependent enzymes requires the presence of enzymes that produce H2O2. In fermentations with wild-type strains, the set of enzymes produced by the microorganism ensures the optimal functioning of the enzyme system, so that the enzymes can act as catalysts while being excreted. In heterologous expression, it is possible to overcome this shortcoming by including more genes to ensure the expression of different enzymes or by using tandem systems such as S. cerevisae and P. pastoris, but this leads to an increase in the complexity of expression. 1.4. Factors involved in the expression of ligninolytic enzymes Environmental conditions have a direct impact on the regulation of fungal metabolism in nature, so the control of different parameters during fungal fermentation is critical to induce LEs expression. In the production of LEs on Chapter 1 32 a laboratory or industrial scale, these parameters can be divided into those related to the composition of the medium and those concerning the operating conditions of the fermenters. 1.4.1. Medium composition 1.4.1.1. Carbon source In general, filamentous fungi are able to use carbon (C) provided by monosaccharides, disaccharides and oligosaccharides. The preference for one source or another depends on the type of C compounds present in their natural environment. Although there are several studies assessing the influence of the type of C source on LEs production (Raghuwanshi et al., 2022; Wang et al., 2016), it is unclear how fungi detect complex carbohydrates in biomass and how this detection can be translated into an intracellular metabolic response that induces the expression of one enzyme or another (Wu et al., 2020). The preferred source of C for growth and enzyme production differs for each type of fungus. For example, Bettin et al. (2009) found that laccase activity in Pleurotus sajor-caju was higher when the main source of C was glucose compared to lactose. However, lactose proved to be the best C source for laccase production by Pseudotrametes gibbose (Elisashvili and Kachlishvili, 2009). On the other hand, the expression of LEs seems to be related to low concentrations of C in the medium (Galhaup et al., 2002; Lú-Chau et al., 2018; Saparrat et al., 2002). Lu-Chau et al. (2018) suggested that the boosting of LEs production with G. lucidum and I. lacteus at low glucose concentration could be related to physiological changes involved in the change from primary to secondary metabolism. 1.4.1.2. Nitrogen source Nitrogen (N) limitation can be determinant in the induction of the secondary metabolism and therefore, in the enzyme expression. The most studied WRF, P. chrysosporium, only produces LiP and MnP under nitrogen-limiting conditions (Reddy and D’Souza, 1994). Although some WRFs such as Bjerkandera sp. BOS55 do not show this correlation between nitrogen limitation and LEs expression, and rich-N medium are required for optimal LEs expression (Kaal et al., 1993; Moreira et al., 2000). This difference may be Introduction and context 33 the result of LEs production in some species being carried out by metabolic pathways different from those traditionally studied for WRFs. Moreover, the type of N source also has a relevant impact on LEs expression. Several authors have evaluated the impact of different N sources, including organic and inorganic compounds (Kanwal and Reddy, 2011; Levin et al., 2010; Reddy and Kanwal, 2022). The use of organic compounds such as yeast extract or casein as nitrogen source reported the highest laccase production for Morchella spongiola (Reddy and Kanwal, 2022). In addition, Levin et al. (2010) also reported higher levels of MnP production by Trametes sp using organic nitrogen compounds such as glutamic acid and supplementation with vitamins such as thiamine. 1.4.1.3. Inducers The inducer is a particular molecule that stimulates the synthesis of the relevant enzyme and is typically a substrate for the enzyme (Vrsanska et al., 2015). Commonly, inductors are classified into natural and synthetic inducers. In the case of WRF lignocellulosic substrates can act as inducers, as they have a small concentration of lignin that can stimulate LEs production. In this regard, different lignocellulosic residues have gained special attention, as several studies have demonstrated the performance of these residues as optimal substrates for fungal growth and expression of enzymes such as laccases and/or peroxidases (Kanwal and Reddy, 2011; Wan and Li, 2012). Other inducers of LEs expression are metals, which are required in trace amounts by fungi, although they can be toxic in excess (Baldrian, 2003). Metals essential for fungal growth include Cu, iron, zinc, nickel, manganese and molybdenum (Gadd, 1993). Once metals enter the fungal cell, they have the ability to affect metabolic reactions and thus the production of extracellular enzymes (Baldrian, 2003). Specifically, for WRF there are two essential metals: Mn and Cu (Vrsanska et al., 2015), which can also act as co-factors in the catalytic cycle of some LEs. Several studies have reported the effect of supplementing the fermentation medium with Cu on laccase production, which can regulate enzyme expression at the transcriptional level (Collins and Dobson, 1997; Soden and Dobson, 2001). In addition, MnP production has also been increased by adding traces of Mn to the fermentation medium (Lueangjaroenkit et al., 2019). Chapter 1 40 2021). Moreover, once LEs production takes place, the enzyme system is able to reduce the toxicity of these wastes through the transformation of phenolic compounds, decolorisation, and reduction of N content (Bohacz and Korniłłowicz-Kowalska, 2020; Pant and Adholeya, 2007; Raghukumar et al., 2004). Different residues from agricultural industrial processes have been evaluated for LEs production (Table 1.4). For example, vinasse, a liquid waste stream from bioethanol synthesis industry, has been used as a sole source of nutrients and supplemented with different C sources to produce laccase with Trametes sp in SSF (Ahmed et al., 2022). In this study, Ahmed et al. (2022) use an SSF technology in which the fungus immobilised on polyurethane foam cubes was grown with different dilutions of vinasse. It was found that after the end of fermentation, replacing the liquid stillage fraction with a fresh stillage fraction could lead to even higher enzyme yields, reaching a laccase production of 361 U/L. In addition, this study also reports a decolorisation of the waste stream and the decrease of phenolic, chemical oxygen demand and biological oxygen demand concentrations, confirming that LEs production is also linked to the detoxification of the waste stream. Gassara et al. (2010) evaluated fish and brewery wastes, pulp and paper sludge and apple waste (pomace) for LEs production with P. chrysosporium, achieving MnP production with all tested wastes. For laccase production, it was observed that the use of copper sulfate as an inducer can trigger enzyme production in all waste-based media, whereas LiP was only found in fermentations with pomace and pulp industry sludge as substrates and with the addition of veratryl alcohol as an inducer of enzyme expression. A maximum MnP activity of 631 U/g was reached with pomace and veratryl alcohol as inducer and a maximum laccase activity of 739 U/g with brewery waste and copper sulfate as inducer, after 8 and 14 d, respectively. Introduction and context 41 Table 1.4. Production of ligninolytic enzymes using agrowastes. Microorganism Substrate Enzyme Activity Reference Trametes sp Sugarcane vinasse Laccase 361 U/L (Ahmed et al., 2022) Phanerochaete chrysosporium Apple pomace MnP 631 U/g (Gassara et al., 2010) Phanerochaete chrysosporium Brewery waste Laccase 739 U/g (Gassara et al., 2010) Phanerochaete chrysosporium Apple pomace Laccase 720 U/g (Gassara et al., 2010) Phanerochaete chrysosporium Pulp and paper sludge MnP 488 U/g (Gassara et al., 2010) Pleurotus eryngii Cherry waste MnP 2263 U/L (Akpinar and Ozturk Urek, 2020) Pleurotus eryngii Cherry waste Laccase 4677 U/L (Akpinar and Ozturk Urek, 2020) Pleurotus eryngii Cherry waste LiP 70 U/L (Akpinar and Ozturk Urek, 2020) 1.6. Biotechnological applications of ligninolytic enzymes The need to apply biocatalysts as LEs in industry and biotechnology is growing rapidly due to their potential use in a wide range of processes (Maciel et al., 2010). Potential applications of LEs include numerous fields such as chemical, pulp and paper, food, paper, textile and cosmetic industries and bioremediation processes (Figure 1.5). The LEs complex is involved in the degradation of various xenobiotic compounds and dyes. This ability to transform xenobiotic substances into polymeric products makes these enzymes an effective biocatalyst for bioremediation purposes (Maciel et al., 2010; Yadav and Yadav, 2015). Chapter 1 42 Figure 1.5. Biotechnological applications of ligninolytic enzymes. In addition, the use of enzymes as biocatalysts can be more effective than other conventional treatments, although it is important the evaluation of the environmental impacts associated to enzymatic treatment to confirm their suitability for environmental purposes. 1.6.1. Biomaterial precursors In recent years, the need to find alternatives to the current synthesis of different products involving the use of non-renewable resources or the generation of negative environmental impact is in the spotlight (DelgadoSánchez et al., 2022). LEs are considered as promising candidates to replace conventional chemical processes in several industries (Maciel et al., 2010). Some studies have reported the transformation of natural polymers by LEs to improve their structure and make them more suitable for different industrial applications (Chen et al., 2019; Huber et al., 2016; Zhu et al., 2021). For example, related to the synthesis of bioadhesives, enzymatic modification of materials such as starch or lignin has been evaluated to obtain transformed polymers with characteristics that increase their reactivity or obtain a more homogeneous network. Enzymatic treatment of these polymers can promote their oxidative activation through a polymerisation initiated via radical mechanism (Pizzi, 2014). Huber et al. (2016) evaluated the polymerisation of kraft lignin and lignosulfonate by a laccase from Myceliophthora thermophila immobilised on polypropylene beads. The results of this study show that the molecular Biotechnological applications Textile industry Food industry Pulp and paper industry Bioremediation Organic synthesis, pharmaceutical and cosmetics applications Introduction and context 43 weight (MW) of lignosulfonate increased 12-fold after 24 h of enzymatic treatment, whereas in the case of kraft lignin the increase in MW was considerably lower (1.7-fold). This increase in MW could improve the dispersion properties of these materials, which is key for their use as plasticizers. Another field where enzymatic modification of polymers is becoming more relevant is the food industry, where this technique is used to facilitate the formation of network structures towards improved properties (Chen et al., 2019; Zhu et al., 2021). Zhu et al. (2021) studied the transformation of potato flour by laccase and peroxidase, achieving a more ordered protein structure and greater stability after enzymatic treatment, which favor the formation of protein-protein and/or protein-starch cross-linking networks, leading to gelation. On the other hand, Chen et al. (2019) studied the combination of heat treatment with laccase-catalysis for the synthesis of double network gels using soy protein isolate and sugar beet pectin as raw materials. This study reported that increasing laccase concentrations led to a reduction of the time required for gel formation, linked to an improvement of its viscosity. In recent years, the use of laccase mediator systems (LMS) has also gained special attention due to their ability to directly generate polymers that could not be produced by conventional chemical synthesis (Aktaş and Tanyolaç, 2003). The use of mediators has shown an enhancement of the catalytic action of laccase, achieving strong oxidation of the treated polymers. The evaluation of the treatment of an industrial kraft pine lignin with a partially purified laccase from Fusarium proliferatum in the absence and presence of 2,2′- azinobis-3-ethylbenzothiazoline-6-sulfonic-acid (ABTS) as mediator was carried out by González Arzola et al. (2006). The modification with LMS led to a higher degree of oxidation and depolymerisation than transformation with laccase alone, demonstrating that the use of mediators can improve enzymatic treatment to facilitate the biotechnological use of industrial lignin. Moreover, Qiu et al. (2023) studied the regioselective C6-OH oxidation of corn starch using the laccase-TEMPO system with the aim of improving the functional properties of this natural polymer, such as water absorption capacity. Specifically, using TEMPO as a mediator in the enzymatic oxidation, an increase in the carboxyl content was obtained, which resulted in an improvement of 34.1-110.1 % in the water absorption properties of oxidised corn starch relative to the unmodified raw material. This improvement in Chapter 1 44 water absorption properties increases the potential of this material for use in the adhesive, paper and textile industries. 1.6.2. Degradation of emerging pollutants Emerging contaminants (ECs) are defined by the US EPA (United States – Environmental Protection Agency) as new chemicals without regulatory status and whose impact on environment and human health is unclear. Due to the industrialisation and other human anthropogenic activities, the presence of these compounds in the environment is rising significantly (Deblonde et al., 2011). Therefore, the evaluation of environmentally friendly technologies is essential to remove these contaminants. The degradation of ECs present in the environment can be addressed by enzymes, which through their catalytic cycle have the capability of transform these compounds into non-toxic forms. In addition, the enzymatic treatment allows a more specific degradation in comparison with other processes because the removal mechanism completely depends on the enzyme-substrate interaction (Saravanan et al., 2021). 1.6.2.1. Pharmaceutical and personal care products Pharmaceuticals and personal care products (PPCPs) include a wide range of organic compounds, including pharmaceutical drugs and compounds used in different personal care products (PCPs) such as lotions, fragrances, sunscreens, etc. Since the use of these compounds is expanded globally, these contaminants and their metabolites can enter the aquatic environment because they are not removed in conventional wastewater treatment (Daughton and Ternes, 1999; Liu and Wong, 2013). Some of these compounds are classified as endocrine disruptors (EDCs) due its similitude with natural steroid hormones and consequently to produce negative health effects in the organisms (Grelska and Noszczyńska, 2020; Vandenberg et al., 2009). These EDCs include compounds such as estrogens, which are sex hormones that can be classified as natural or synthetic hormones and which in recent years have been detected in significant concentrations in the environment due to their use in contraceptive drugs. The hormones most typically found in wastewater includes estrone (E1) and 17βestradiol (E2) (natural hormones) and 17α-ethinylestradiol (EE2) (synthetic hormone) (Ying et al., 2002). Moreover, other compounds such as bisphenol A (BPA), that is typically used to make polycarbonate plastics and epoxy Introduction and context 45 resins for packaging of personal care product or foods, can act as EDC (Vandenberg et al., 2009). The molecular structure and characteristics of these compounds are shown in Table 1.5. Table 1.5. Characteristics of different emerging contaminants studied in this Thesis. Compound Molecular weight (Da) Water solubility (mg/L) Structure Estrone 270.4 30 17β-estradiol 272.4 3.6 17α-ethinylestradiol 296.4 11.3 Bisphenol A 228.3 12 Carbamazepine 236.3 17.7 Pyrene 202.25 0.135* *Extracted from Mackay and Shiu (1977). Several studies have been focused on the enzymatic treatment of EDCs, mainly using LEs such as laccase, MnP or LiP. The efficacy of enzymatic treatment lies in the phenolic structure of EDCs and the low substrate specificity of LEs (Cajthaml et al., 2009). Several studies have reported elimination rates higher than 80 % for different EDCs using commercial laccases from T. versicolor and M. thermophila. (Becker et al., 2017; Onaizi and Alshabib, 2021). However, most of these studies were performed with commercial enzymes. In this regard, although a few authors have evaluated the use of unpurified N O NH 2 HO O H H H HO OH H H H HO HO H H H HO OH Chapter 1 46 enzymes or crude enzyme extracts, their use as biocatalysts is less extended (de Freitas et al., 2017; Eibes et al., 2011; Taboada-Puig et al., 2016). Further research with these types of biocatalysts is needed to make enzymatic treatment a suitable option for EDC removal. Another type of PPCPs that have received special attention are those with a recalcitrant nature, such as carbamazepine (CBZ, Table 1.5) and trimethoprim. In order to consider enzymatic treatment as a suitable option for the removal of these compounds it is important to elucidate which type of enzymes are most involved in the degradation of these contaminants, since the removal depends on the binding efficiency of the contaminants in the catalytic region (Bilal et al., 2022). Although the use of laccase have reported significant degradations of CBZ (Alharbi et al., 2019), other types of LE, such as VP or LiP, have degradation rates of less than 10 % (Eibes et al., 2011; Zhang and Geißen, 2010). In addition, the combined use of LEs to tertiary wastewater treatment technologies, such as UV radiation, can also increase the removal performance of these compounds (Tufail et al., 2021). However, such combined systems require high energy consumption and can also result in the generation of toxic by-products (Mohapatra et al., 2014). 1.6.2.2. Polycyclic aromatic hydrocarbons PAHs are compounds that can be generated in the combustion processes associated with the oil, coal and gas industries. These chemicals are highly toxic and are considered persistent pollutants due to the negative impact of their complex mixtures on the environment and the long periods required for their natural degradation (Anyanwu et al., 2020; Patel et al., 2020). Its structure is composed by two or more fused benzene rings, without presence of heteroatoms and substituents on the polycyclic rings. Attending to the number of rings that integrate its structure, PAHs can be classified into two main categories: light PAHs (containing up to four rings) and heavy PAHs (containing more than 4 rings). In general, a higher molecular weight and angularity of PAHs is associated with an increase in the hydrophobicity and electrochemical stability (Sahoo et al., 2020). Several technologies have been evaluated for their removal from the environment, including strong oxidising agents such as Fenton chemicals or ozone (Jonsson et al., 2006). Due to the ability of LEs to destabilise the bonds that make up the PAH structure (Chang et al., 2002), enzymatic treatment is a Introduction and context 47 suitable method to remove these compounds in an environmentally friendly way, although its effectiveness is highly dependent on the PAH structure, among other factors (Nzila, 2018). Most PAHs degradation studies focus on their removal from soils or solid substrates. In this regard, several studies have employed in vivo assays with WRF to degrade these contaminants (Jove et al., 2016; Novotný et al., 2004). PAHs degradation in aqueous media has received less attention, but some studies have reported the degradation of PAHs with LEs in aqueous media containing organic solvents. Under these conditions, avoiding enzyme deactivation becomes crucial (Eibes et al., 2005). Eibes et al. (2006) studied the degradation of anthracene, dibenzothiophene and pyrene (Table 1.5) by MnP in presence of acetone in order to increase the solubility of PAHs. Different enzyme doses and reaction times were tested, reaching degradation rates close to 100 % with MnP doses between 550 and 1340 U/L in 7 h for anthracene and 24 h for pyrene and dibenzothiophene. In addition, the transformation products were analysed, verifying that the enzymatic transformation of anthracene and pyrene was achieved by an oxidation process involving OH∙. Other studies evaluated the enzymatic degradation of PAHs using surfactants such as Tween-80 to improve the solubility of these compounds and immobilising the enzyme on magnetic particles. This allows for a reusable biocatalyst that can be used in several cycles, although the removal efficiency of PAHs decreased after several cycles (Deng et al., 2022). 1.7. Factors controlling the enzyme activity Considering that LEs are involved in different metabolic processes, their activity can be modulated by different factors including the concentration of the compounds involved in the enzymatic reactions, temperature, pH, the nature of the reaction medium and the presence of compounds that can act as activators or inhibitors. Therefore, the study of these factors is essential to ensure optimal functionality and reaction yields. 1.7.1. Substrate, enzyme and product concentrations The kinetics of enzyme reactions is highly influenced by the concentrations of substrate, enzyme and products. Modifications in their concentrations can lead to a change in the enzymatic reaction, which is schematically described according to a multi-step mechanism (Equation 1.1) (Olkiewicz et al., 2022): Chapter 1 48 Eq. 1.1 In this mechanism, first the enzyme (E) and substrate (S) bind to each other forming the enzyme-substrate complex (ES). Then, the substrate is transformed into the product(s), giving an enzyme-product complex (EP). Finally, the product(s) detach from the enzyme, releasing the enzyme to react again. Kf, kr, kcat, kcatr, kdis, kasoc are forward rate constant, reverse rate constant, catalytic rate constant, catalytic reverse rate constant, dissociation constant and association constant (Olkiewicz et al., 2022). Nevertheless, this equation can be simplified to Equation 1.2 (known as Michaelis-Menten equation), that describes reversible binding of E and S into the ES complex, and irreversible conversion of the complex into the product (P) and the free E. Eq. 1.2 Michaelis-Menten equation is explained by the mathematical model indicated in Equation 1.3, where KM = kr + kcat/kf is the Michaelis constant, roughly the dissociation constant of the complex and Vmax = kcat [E]0 is the maximum reaction velocity and [E]0 is the initial enzyme concentration. Eq. 1.3 In this kinetic model, which can only be considered when the substrate concentration greatly exceeds the enzyme concentration or when the energy released during the reaction is high, the influence of substrate and enzyme concentration on the efficiency of the enzymatic reaction is noteworthy. However, if the assumptions required for the use of the Michaelis-Menten equation are not met, the use of more complex models is necessary (Tummler et al., 2014; Wong et al., 2015), and also, in the case of the presence of inhibitors it is required the inclusion of inhibitor effects in the mathematic model. A common enzymatic inhibition may be that produced by the products generated during the enzymatic reaction, which commonly have similarities with the original substrate and may interact with the active site reducing the amount of free active sites for binding with substrate molecules, with a Introduction and context 49 consequent decrease in the efficiency of the enzymatic reaction. In addition, the reaction products obtained in the oxidation of typical substrates of multicopper oxidases, such as ABTS, can react with organic sulfhydryl compounds and form complexes with the Cu atoms of the enzyme, leading to enzymatic inhibition (Valles et al., 2020). Pamidipati and Ahmed (2020) also observed a decrease in laccase activity using different concentrations of common intermediates generated during lignin degradation. Therefore, intermediates may also cause competitive inhibition that decreases the yield of the enzymatic reaction. 1.7.2. Temperature The study of the optimum temperature of an enzyme is crucial for its application as a biocatalyst. In general, chemical reaction rate and enzyme activity increase with increasing temperature. At moderate temperatures, the rate of enzyme deactivation is negligible and can be ignored, but at higher temperatures the deactivation is noticeable due to the compromise of the enzyme structure. If the enzyme reaction is carried out at the optimum temperature, a balance between reaction yield and enzyme activation/deactivation is established (Wojcik and Miłek, 2016). The effect of temperature in reaction rate is explained by the Arrhenius equation (Equation 1.4) that was proposed in the 19th century based on empirical observations. In this equation the rate constant (k) is expressed in function of a pre-exponential factor (A), the activation energy (Ea), the universal gas constant (R) and the temperature in kelvin (T). Ea is defined as the energy barrier which has to be overcome by the relative translational motion of the reactants in order for the reaction to occur (Arcus et al., 2016; Menzinger and Wolfgang, 1969). In the practice, the increase in temperature allows a higher initial level of energy, which will be translated in a reduction of Ea, facilitating the reaction performance (Grahame et al., 2015). 𝑘𝑘=𝐴𝐴 ∙𝑒𝑒−�𝐸𝐸𝑎𝑎 𝑅𝑅𝑅𝑅� Eq. 1.4 On the other hand, the effect of high temperatures in the structure of the enzyme is responsible of the decrease in the enzyme activity. In general, fungal LEs can show higher activities at high temperatures, but this activity is not maintained during a long period of time, so optimal temperatures for their use as biocatalysts are in the range of 20 and 40 °C (Preethi et al., 2013; Thoa Chapter 1 56 Figure 1.6. Structure of the PhD Thesis. Chapters 2, 3 and 4 focused on the production of different LEs cocktails through the valorisation of wastes streams from different origin. Chapter 2 aims the production of laccase with G. lucidum using the residual liquid fraction (black liquor) of the organosolv process, Chapter 3 focuses on the production of MnP by I. lacteus from fermentation media based on raw wheat straw and extracts obtained from this material and Chapter 4 pursues the production of UPO by A. aegerita using by-products generated in the bioethanol industry, including vinasse and dried distillers’s grains with solubles (DDGS). In Chapters 5 and 6, an evaluation of the enzyme cocktails produced in the previous chapters as catalysts in different biotechnological processes was carried out. Chapter 5 deals with the modification of organosolv lignin with laccases to increase its reactivity and transform it into a viable precursor for the synthesis of bioadhesives, while Chapter 6 studies the removal of different emerging contaminants with the enzyme cocktails with MnP and UPO activity. Finally, Chapter 7 summarises the main conclusions derived from the Thesis and discusses new issues that may arise from the results obtained. CHAPTER 2 Valorisation of black liquor from wood organosolv process for the production of ligninolytic enzymes CHAPTER 3 Ligninolytic enzyme production through the valorisation of wheat straw CHAPTER 4 Valorisation of bioethanol by-products to produce unspecific peroxygenase CHAPTER 1 Introduction and context CHAPTER 5 Functionalisation of organosolv lignin by enzymatic demethylation for bioadhesive formulation CHAPTER 6 Enzymatic abatement of emerging contaminants CHAPTER 7 General discussion and conclusions RQ1 Which type of fermentation is most suitable for the production of ligninolytic enzymes through the valorisation of agro-industrial wastes? RQ2 Do enzyme cocktails produced from the valorisation of waste streams have sufficient catalytic potential to be applied in the field of environmental biotechnology compared to commercial enzymes? MAIN OBJECTIVE CHAPTER 2 VALORISATION OF BLACK LIQUOR FROM WOOD ORGANOSOLV PROCESS FOR THE PRODUCTION OF LIGNINOLYTIC ENZYMES Valorisation of black liquor from wood organosolv process for the production of ligninolytic enzymes 59 SUMMARY White rot fungi (WRF) are characterised by the production of extracellular ligninolytic enzymes (LEs), which are capable of degrading a polymer as complex as lignin due to their high oxidation potential and non-specific character. Although ligninolytic fungi have been the topic of numerous studies in recent years, the complexity of the production of these enzymes, typically linked to secondary metabolism and their application at large-scale requires further research and development. Therefore, the search for new substrates to formulate the culture media that allow the production of enzymes in an efficient way at low cost is especially relevant. The valorisation of industrial waste streams rich in lignin is here presented as a promising alternative to foster LEs production. In this sense, the work presented in this chapter focuses on the production of LEs (laccase and manganese peroxidase) by Ganoderma lucidum in a fermentation medium based on black liquor, a byproduct of the organosolv process of beechwood. Fermentations were carried out in solid (SSF) and submerged (SmF) state. High titers of laccase activity were achieved in the SmF while manganese peroxidase (MnP) activity was less relevant. The enzyme production was scaled-up to a 4 L reactor, achieving a laccase production of 2417 U/L, which is an 18.7 % decrease in enzyme production with respect to the flask scale, but it allowed a reduction in the time required to achieve peak activity. Finally, a proteomic analysis of the enzyme cocktail was performed in order to evaluate the main enzymes present at the end of the fermentation. Valorisation of black liquor from wood organosolv process for the production of ligninolytic enzymes 61 2.1. Introduction 2.1.1. Ligninolytic enzymes production and wood industry streams valorisation The performance in the production of LEs is highly dependent on the microorganism, substrate and cultivation method (Elisashvili et al., 2008). Although several microorganisms are capable of producing LE, the most efficient producers of these proteins are fungi belonging to the WRF class (Plácido and Capareda, 2015). Among this class of fungi one that has gained special relevance in recent years has been G. lucidum. This fungus, which has been extensively studied for its medicinal and nutritional properties (El Sheikha, 2022), is also known to be a LE producer. The main lignin-modifying enzymes expressed by G. lucidum are laccases and heme peroxidases (Zhou et al., 2018). Although several studies have evaluated the use of G. lucidum to produce LEs, some of them are focused on the utilisation of high-value substrates rather than the use of waste streams (Arora and Gill, 2001; Kuhar and Papinutti, 2014). The use of industrial wastes can lead to a dual purpose: enzyme production and waste valorisation. In this regard, lignin-rich waste streams can stimulate LEs production in G. lucidum. Furthermore, it is relevant to search for residues that are produced in large quantities on a global scale, such as waste derived from wood industry (Adhikari and Ozarska, 2018). Specifically, the black liquor associated with chemical pulping of cellulose is a promising candidate to boost LEs production. In particular, black liquor from the organosolv process, although much less abundant than Kraft liquor, has chemical oxygen demand (COD) values above those allowed for discharge and with low biodegradability levels, due to the presence of lignin and their derivatives. In addition the high hemicellulose content in the stream ensures the presence of a C source to ensure fungal growth, and the high phenolic content can prevent the proliferation of other competing microorganisms, such as several bacteria species (Pacheco-Ordaz et al., 2018). Moreover, the fungal treatment of this liquid fraction from the organosolv process can detoxify this stream increasing its biodegradability (García-Torreiro et al., 2018). Chapter 2 62 2.1.2. Objectives of this chapter Despite the suitable composition of black liquor to enhance enzyme production, the potential applications of this hemicellulose-rich waste stream were only explored in a few studies (García-Torreiro et al., 2018; Lú-Chau et al., 2018; Narra et al., 2020). The integral valorisation of organic waste streams through different biotechnological processes constitutes a challenge that pursues a double benefit: efficient waste management and the production/recovery of high value-added compounds. In addition, this allows adding an economic value to waste streams that are initially of no interest. Among these high value-added compounds, LEs are of particular interest for their potential application in different biocatalytic processes. This is one of the reasons why the formulation of fermentation media incorporating waste streams such as black liquor offers a possibility that needs to be evaluated for possible large-scale development. A complete source of nutrients can be provided by these wastes, in which it may be necessary to add additional nutrients but in much lower concentration than in synthetic media. In this sense, the cost of enzyme production can be reduced, making enzymatic processes more competitive in comparison with other conventional technologies. As a summary, it should be noted that the research carried out in this chapter has as its main objective the production of LEs, focusing mainly on the production of laccase. In addition, as a parallel objective, the valorisation of a residual stream such as black liquor is sought. 2.2. Materials and methods 2.2.1. Chemicals, raw material and microorganism MnSO4, H2O2, 2,6-dimethoxyphenol (DMP), 2,2'-azino-bis(3ethylbenzothiazoline-6-sulfonic acid) (ABTS), CuSO4 and Antifoam 204 (A6426) were acquired from Sigma-Aldrich. Glucose, KH2PO4 and NaC2H3O2 were purchased from Panreac, meal peptone from Cultimed, yeast extract from iNtRON Biotechnology and MgSO4∙7H2O from Fluka. Black liquor from the organosolv process of beechwood was used to prepare the fermentation medium. This stream was provided by the Fraunhofer Center for Chemical-Biotechnological Processes (CBP) (Germany). The characterisation of this waste fraction is shown in Table 2.1. Valorisation of black liquor from wood organosolv process for the production of ligninolytic enzymes 63 Table 2.1. Characteristics of the concentrated stream from the hemicellulosic fraction resulting from the organosolv treatment of beech wood. pH 5.5 Total reducing sugars (g/L) 225 Total nitrogen (mg/L) 748 Glucose (g/L) 10.8 Xylose (g/L) 144.3 Rhamnose (g/L) 44.1 Xylose oligomer (g/L) 75.7 Acetic acid (g/L) 17.1 The fungus G. lucidum was isolated from mushroom spent substrate, kindly provided by Hifas da Terra S.L. (Pontevedra, Spain). The strain was stored in Petri dishes with a Kimura medium below -4 °C. 2.2.2. Fermentations 2.2.2.1. Inoculum preparation For the fungal culture under static conditions, three plugs of active mycelia were transferred from Petri dishes to Fernbach flasks with 100 mL of Kimura medium (glucose 20 g/L, peptone 5 g/L, yeast extract 2 g/L, KH2PO4 1 g/L, MgSO4∙7H2O 0.5 mg/L) and maintained at 30 °C for 7 d. After this step, the contents of the Fernbach flasks were crushed in a blender and used as inoculum for the Erlenmeyer flasks. Regarding the operation in the reactor, the content of the crushed Fernbach (10 mL) was transferred to Erlenmeyer flasks with Kimura medium (90 mL) previously autoclaved at 110 °C for 30 min. Erlenmeyer flasks were incubated at 30 °C for 7 d, and after that the culture broth from the flasks was used as inoculum for reactor operation (10 % v:v). 2.2.2.2. Solid state fermentation at flask scale Polyurethane foam cubes were used as support for G. lucidum growth in SSF. Each flask was filled with 0.5 g of polyurethane foam cubes with a volume of approximately of 1 cm3 (1 cm × 1 cm × 1cm). To ensure the required supply of nutrients for fungal growth in each flask, a volume of 18 ml of a black liquor dilution (1:37) supplemented with meat peptone (2 g/L), MnSO4 (0.5 mM) and Chapter 2 64 CuSO4 (0.15 mM) at pH 4.5 was added, ensuring that the foam cubes were thoroughly soaked. Erlenmeyer flasks with the soaked cubes were sterilised in an autoclave (Raypa AES-7) for 30 min at 110 °C and inoculated with 2 mL of inoculum (10 % v:v). The Erlenmeyer flasks were incubated for 21 d at 30 °C in a humiditysaturated environment under static conditions. All experiments were conducted in duplicate. For enzyme harvesting, the content of each Erlenmeyer flask was transferred to a 100 mL syringe and the fermentation medium absorbed into the polyurethane foam cubes was recovered. After that, the recovered medium was centrifuged at 4500 rpm for 5 min and the supernatant was withdrawn to analyse the enzyme activity and pH. 2.2.2.3. Submerged fermentation at flask scale SmF was carried out in 250 mL-Erlenmeyer flasks, with a working volume of 100 mL. The supplemented black liquor dilution used in SSF was used as fermentation medium. Erlenmeyer flasks containing 90 mL of the final medium were autoclaved at 110 °C for 30 min and inoculated with 10 mL of inoculum. The Erlenmeyer flasks were incubated on an orbital shaker (COMECTA 100D Incubation Shaker) at 30 °C and 150 rpm for 16 d. Fermentations were carried out in triplicate with an initial pH of 4.5. Enzyme activity, pH and total nitrogen were determined in the supernatant of the samples taken periodically during the fermentation. For this purpose, 3 mL samples were taken and centrifuged at 4500 rpm for 5 min to remove the biomass. 2.2.2.4. Submerged fermentation at reactor scale The enzyme production was performed in a 4 L stirred tank bioreactor (Biostat Bplus, Sartorius Stedim Biotech S.A.). The fungus grown in Erlenmeyer flasks (see section 2.2.2.3) was used as inoculum at 10 % (v:v). The fermentation medium was prepared as described for SSF at flask scale (see section 2.2.2.2). The conditions for medium sterilisation were 100 °C and 60 min. The bioreactor was operated for 2 d at controlled temperature (30 °C), under mechanical agitation (100-150 rpm), air supply (1-2 L/min) and pH monitoring. Foaming was automatically controlled by the addition of antifoam through a peristaltic pump. Valorisation of black liquor from wood organosolv process for the production of ligninolytic enzymes 65 2.2.3. Recovery and concentration of the enzymatic cocktail The culture broth collected at maximum laccase activity in the 4 L reactor was filtered through filter paper (Whatman No.1, Maidstone). Afterwards, it was passed through a microfiltration membrane (Filtron Minisette System, Pall Corporation; 0.2 µm cut-off) and finally concentrated by ultrafiltration with a 10 kDa membrane (Filtron Minisette System, Pall Corporation). The cell-free concentrated crude, designated as enzymatic cocktail, was stored at -20 °C. 2.2.4. Shotgun proteomic analysis by mass spectrometry The concentrated enzyme cocktail was processed in solution by trypsin digestion, reduction-alkylation and finally desalted using ZipTip-µC18 material (Merck Millipore). The obtained sample was analysed using the nanoUHPLC-TIMS-QTOF technique. Peptide samples (0.3 µg of protein) were injected onto a timsTOF Pro (Bruker) equipped with a nano-electrospray source (CaptiveSpray) and a TIMS-QTOF analyser. The chromatographic analysis was performed using a nanoELUTE chromatograph (Bruker) with a ReproSil C18 column (50 × 0.075 mm, 1.9 µm, 120 Å, Bruker). The nHPLC was set up with binary mobile phases that included solvent A (0.1 % formic acid in ddH2O), and solvent B (0.1 % formic acid in acetonitrile). The analysis time was 20 min, in which B/A solvent ratio was gradually increased. For MS acquisition, a CID fragmentation and nanoESI positive ionization mode was applied. The PASEF-MSMS scan mode was set for an acquisition range of 100-1700 m/z. MS/MS spectra was processed with PEAKS Studio (Bioinformatics Solutions) software for protein identifications using a database with all protein sequences available in NCBI protein database for G. lucidum taxonomy. The label-free module from PEAKS Studio was used for protein semi-quantification. The parameters analyzed from this analysis were the accession number (Accession number of the protein from NCBI database), area (the area under the curve of the peptide feature found at the same m/z and retention time as the MS/MS scan), n° peptides (peptides identified for each protein), n° unique peptides (number of peptide sequences unique to a protein group), avg. mass (promedium molecular weight) and coverage (calculated by dividing the number of amino acids in all peptides found by the total number of amino acids in the whole protein sequence). The Spec value is based on peptide spectrum matches (PSM) and was used as indicator for the relative abundance of the proteins in each sample (Ma et al., 2003). Chapter 2 72 laccases from Ganoderma sp belong to middle redox potential laccases, as its redox potential is between 430 and 710 mV (Sharma et al., 2013). Beta-actins, the proteins detected in higher proportion after laccases, belong to a cytoskeletal filament protein class, which are involved in different cellular functions, including cell motility. In relation to beta-glucosidases, their functions and expression have been widely studied in fungi (Korotkova et al., 2009). The main function of these enzymes in WRF is the hydrolysis of different polysaccharides present in lignocellulosic materials, leading to the release of glucose (Sørensen et al., 2013). Therefore, the expression of this enzyme is expected in fermentation by G. lucidum in black liquor-based medium. Finally, polyubiquitin, an enzyme also present in the enzyme cocktail, was shown to play an important role in fungal development, stress tolerance, an virulence (Wang et al., 2019). In fact, several studies suggests that it can be involved in proteasome degradation (Gilkerson et al., 2015; Gutierrez et al., 2018). Valorisation of black liquor from wood organosolv process for the production of ligninolytic enzymes 73 Table 2.2. List of proteins identified in the shotgun proteomics analysis of the enzyme cocktail from G. lucidum. Proteins are ranked by their Spec number, calculated with Peaks software and which can be correlated with their abundance in the sample. NCBI Accession Description Area Nº peptides Nº unique peptides Avg. Mass (kDa) Coverage (%) gi|117959694 laccase 5.00∙10 3 8 3 56.29 19 gi|117959692 laccase 5.00∙10 3 8 3 56.26 19 gi|558633451 laccase partial 2.86∙10 4 7 2 56.61 18 gi|564733409 beta-actin 0 3 3 41.52 12 gi|558633473 laccase partial 0 2 1 54.94 4 gi|1701710131 beta-glucosidase 3 0 1 1 85.40 1 gi|2103683182 transcription factor bHLH7 0 1 1 105.75 1 gi|545693750 polyubiquitin 0 1 1 18.20 6 gi|564733421 polyubiquitin 0 1 1 18.20 6 gi|564733417 polyubiquitin 0 1 1 35.66 3 gi|564733419 polyubiquitin 0 1 1 42.74 2 Chapter 2 74 2.4. Conclusions Black liquor from the organosolv process of wood industry, has been shown to be a suitable nutrient source for G. lucidum growth and LEs expression. The type of fermentation has a significant impact on enzyme production, especially laccase activity. SmF resulted in higher laccase production than SSF, while in relation to MnP production, both fermentation types achieve similar enzyme production results. Scaling up the fermentation to a 4 L reactor resulted in a reduction of the maximum enzyme activity for both enzymes, probably due to the addition of antifoam, and/or the mechanical agitation which can negatively affect fungal growth. However, the time required to reach the maximum enzyme activities was significantly reduced. Finally, the presence of laccase in the concentrated enzyme cocktail obtained in the reactor could be verified by proteomic analysis and could match with G. lucidum laccases previously described in the literature. Valorisation of black liquor from wood organosolv process for the production of ligninolytic enzymes 75 2.5. Annexes Chapter 2 Figure A2.1. Aspect of fermentation medium of SmF by G. lucidum in 4 L reactor with diluted black liquor at (a) day 0 and (b) day 2. (a) (b) CHAPTER 3 LIGNINOLYTIC ENZYMES PRODUCTION THROUGH THE VALORISATION OF WHEAT STRAW Part of the results of this chapter have already been published as “González-Rodríguez S, Lu-Chau TA, Trueba-Santiso A, Eibes G, Moreira MT. Bundling the removal of emerging contaminants with the production of ligninolytic enzymes from residual streams. Appl Microbiol Biotechnol. 2022; 106(3):1299-1311. https://doi.org/10.1007/s00253-022-11776-7”. Detailed information available at “Manuscripts considered in this thesis: rights, contributions and quality indicators”, page 227. Ligninolytic enzymes production through the valorisation of wheat straw 79 SUMMARY Enzymes offer interesting features as biological catalysts for industry: high specificity, activity under mild conditions, accessibility and environmental friendliness. Being able to produce enzymes in large quantities and having them available in a stable and reusable form reduces the production costs of any enzyme-based process. Agricultural residues have recently demonstrated their potential as substrates to produce LEs by different white rot fungi (WRF). In this chapter, the biotechnological production of manganese peroxidase (MnP) by Irpex lacteus was conducted through solid state fermentation (SSF) with wheat straw as substrate and submerged fermentation (SmF) using wheat straw extract (WSE). The enzyme cocktail produced also showed manganese-independent activity (MiP), related to the presence of a short MnP and a dye-decolorizing peroxidase (DyP) which was confirmed by shotgun proteomic analyses. In view of the enhanced production of LEs in SmF, different parameters such as WSE concentration and nitrogen source were optimized. The highest enzyme titers were obtained with a medium formulated with glucose and peptone (339 U/L MnP and 15 U/L MiP). The scale-up to a 30 L reactor achieved similar activities, demonstrating the feasibility of enzyme production from the residual substrate at different production scales. Moreover, the Life Cycle Assessment (LCA) methodology was applied in order to study the environmental impact of the enzyme production process at 30 L reactor scale. Ligninolytic enzymes production through the valorisation of wheat straw 81 3.1. Introduction 3.1.1. Peroxidases production by Irpex lacteus and valorisation of waste streams WRF include a broad spectrum of fungal species that produce LEs such as laccases, lignin peroxidase (LiP), MnP and other peroxidases, whose main function in nature is lignin mineralisation (Yadav and Yadav, 2015). Among WRF, I. lacteus has shown a significant role in different biotechnological fields due to the production of extracellular oxidative and hydrolytic enzymes (Novotný et al., 2009). Genome analysis reveals that the main ligninolytic enzyme of I. lacteus is MnP, with a major role in lignin degradation (Yao et al., 2017). However, depending on the substrates used and fermentation conditions, other ligninolytic enzymes, such as versatile peroxidase (VP) and laccase are also produced (Rothschild et al., 2002). For example, Salvachúa et al. (2013b) described the joint production of peroxidases with Mn(II)- dependent and Mn(II)-independent activities in fermentations with I. lacteus, which increases the range of use of its enzyme cocktail in different biodegradation processes. Among the different fermentation media that allow the correct growth of the fungus and enzyme expression, several studies have focused on the valorisation of waste streams for the production of fungal LEs in recent years (Gupte et al., 2007; Pena et al., 2012; Xu et al., 2009). From the point of view of the fungal culture, two options can be considered for enzyme production: SmF or SSF. The former has been applied to a wider range of processes since the design, operation and control of the fermenter is relatively simple (Musoni et al., 2015). However, SSFs also offer some advantages, such as less complexity in downstream processing, low costs and energy, lower sterility demands and culture conditions similar to natural fungal habitat; but, with the negative aspects of heterogeneity, limited mixing and oxygen transfer (Couto and Sanromán, 2006; Hölker et al., 2004). Although the technical aspects of fungal fermentations have been extensively studied, only a few studies focus on the environmental impact associated with the enzyme production process. Therefore, further research on the environmental impact implications of enzyme production processes is required. Chapter 3 88 3.3. Results and discussion 3.3.1. Solid state fermentation at flask scale Wheat straw was used as substrate and support for the SSF of I. lacteus. The additional supply of glucose (4 g/L) and peptone (1 g/L) as carbon and nitrogen sources was evaluated in terms of MnP production (Figure 3.2). MnP activity was higher in the SSF without supplementation (NS-SSF, 176.0 ± 8.6 U/L), compared with those of the SSF supplemented with glucose and peptone (GP-SSF, 53.6 ± 11.8 U/L). Several studies have demonstrated that low N and C concentrations in SSF can stimulate secondary metabolism in WRF, which is responsible of LEs production (Elisashvili et al., 2008; Iqbal et al., 2011). More specifically, Elisashvili et al. (2008) have established that nitrogen supplementation in SSF with wheat straw had a negative impact on enzyme production by Pleurotus ostreatus. It is likely that the fungus first consumes easily metabolizable nutrients, and then initiates the secretion of LEs as a strategy to access the C source present in wheat straw (Elisashvili et al., 2008). This fungus presents a high capacity to degrade acid-insoluble lignin (Zuo et al., 2018), which is the most abundant type of lignin present in the wheat straw used in this study (Table A3.1). This ability to degrade lignocellulosic material present in the deeper layers of wheat straw is directly related to a higher production of LEs. It is also noteworthy that the time required to reach the maximum MnP production was similar in both SSF, being 9 d in the NS-SSF and 7 d in the GPSSF. Once the maximum activity was reached, a decrease in MnP activity was observed. SSF studies using non-pretreated wheat straw showed different fermentation periods to reach maximum MnP activity, varying from 14 to 23 d (Dias et al., 2010; Salvachúa et al., 2013b). Nevertheless, SSF studies using other agro-industrial wastes, such as olive biomass, have shown longer fermentation times (30 d) to reach maximum enzyme production (MartínezPatiño et al., 2018). Ligninolytic enzymes production through the valorisation of wheat straw 89 Figure 3.2. Enzyme activities during SSFs: (a) MnP (♦) and MiP (▲) activities in the NS-SSF with wheat straw as only C and N source; (b) MnP (♦) and MiP (▲) activities in the GP-SSF with wheat straw, glucose and peptone. Regarding MiP activity, its profile in NS-SSF shows production peaks similar to the maximum in earlier stages of the fermentation, whereas in GP-SSF the highest activity is reached in the initial phase of the fermentation, followed by a decrease of the activity. The maximum MiP activities (26.6 ± 2.8 and 12.1 ± 2.3 U/L) were reached on days 3 and 9 for the SSF without and with extra nutrient addition, respectively. The pretreatment of wheat straw used in SSF can influence enzyme production. Despite the potential of enzyme production in SSF, the period of time required to obtain maximum production is too long, which was one of the reasons why the production of LEs in SmF was addressed. 3.3.2. Submerged fermentation at flask scale Submerged fermentation was conducted using WSE supplemented with glucose and peptone, to ensure a nutrient-balanced medium for an optimal fungal growth. In addition, to evaluate whether any of the substances present in the extract could affect the growth of the microorganism, SmF was performed using the crude WSE and the diluted extract (25 % WSE in distilled water). Enzyme production was higher for fermentations with diluted extract (Figure 3.3-b), which could be related to the lower glucose consumption in the medium with the raw extract. The presence of possible inhibitory compounds in WSE may affect fungal growth and thus glucose consumption. 0 50 100 150 200 250 04812 16 20 24 28 MnP and MiP activity (U/L) Time (days) 0 20 40 60 04812 16 20 MnP and MiP activity (U/L) Time (days) (a) (b) Chapter 3 90 Figure 3.3. Evolution of MnP activity (♦), MiP activity (▲) and glucose concentration (○) in the SmF with (a) raw WSE and (b) diluted WSE both supplemented with glucose and peptone. Maximum activities were reached on day 4 in the fermentation with diluted extract (339.4 ± 11.6 U/L of MnP; 14.8 ± 13.9 U/L of MiP). In the fermentation with raw extract (Figure 3.3-a) the maximum MiP activity was 10.7 ± 6.1 U/L, and the maximum MnP activity 249.0 ± 51.0 U/L. Interestingly, the production of enzymes when using 100 % WSE was faster (activity started on day 2 compared to day 4 in diluted extract), which could be due to dissolved lignin derived compounds acting as inducers of LEs production. The effect of aromatic compounds acting as inducers has been previously described, and it was suggested that LEs synthesis may function as a defense mechanism against chemical stress (Elisashvili et al., 2010). Although the production of 0 1 2 3 4 5 6 7 0 100 200 300 400 0 2 4 6 Glucose (g/L) MnP and 10*MiP activity (U/L) Time (days) (a) 0 1 2 3 4 5 6 0 100 200 300 400 0 2 4 6 Glucose (g/L) MnP and 10*MiP activity (U/L) Time (days) (b) Ligninolytic enzymes production through the valorisation of wheat straw 91 enzymes was faster in the raw extract and maintained for a longer period, the maximum production was achieved in the diluted extract after 4 d. It was also evaluated whether a change in the type of N source could affect enzyme production. For this, casein hydrolysate was used as N source instead of peptone, in addition to the nutrients provided by WSE. As in the previous fermentations with peptone, both raw and diluted WSE were used. Figure 3.4 shows that the highest MnP activity was achieved with the medium with the raw WSE (227.9 ± 21.9 U/L at day 6), and the activity in the diluted medium was 96.7 ± 6.3 U/L, also at day 6. For MiP activity, the diluted WSE performed similarly to raw extract, reaching maximum activities of 18.0 ± 9.5 and 12.2 ± 3.5 U/L after 6 and 3 d, respectively. Figure 3.4. Evolution of MnP activity (♦), MiP activity (▲) and glucose concentration (○) in the SmF with (a) raw and (b) diluted WSE, both supplemented with glucose and casein hydrolysate. 0 1 2 3 4 5 6 7 0 100 200 300 0 2 4 6 Glucose (g/L) MnP and 10*MiP activity (U/L) Time (days) (a) 0 1 2 3 4 5 6 0 100 200 300 0 2 4 6 Glucose (g/L) MnP and 10*MiP activity (U/L) Time (days) (b) Chapter 3 92 The choice of the type of nitrogen sources can have an important impact in fungus development. Previous studies reported that organic compounds perform better than inorganic compounds in fungal SmF, due to their ability to stimulate fungal growth (Juwon and Emmanuel, 2012). The use of peptone was reported as booster of enzyme production in several studies (Lú-Chau et al., 2018; Pinheiro et al., 2020). Moreover, it is noteworthy that for all tested conditions MiP was produced in lower values than MnP, in agreement with other reports on I. lacteus (Salvachúa et al., 2013b). Specifically, the maximum levels of MnP activity were approximately 10 times higher than the maximum levels of MiP activities. In addition, to have a reference of the enzyme production of I. lacteus and to evaluate the impact of the use of WSE, SmF with I. lacteus was carried out using two of the most common conventional media for fungal culture: Kirk and Kimura (Kimura et al., 1990; Tien and Kirk, 1988). In these experiments only the Mn(II)-dependent activity was studied. Table 3.2 shows the MnP production throughout the 14-day fermentation. The highest MnP activity (100.3 ± 26.5 U/L) was reached in Kimura medium on day 8, while in the case of Kirk medium the maximum production peak (60.4 ± 6.7 U/L) was reached on day 14. Table 3.2. MnP activity (U/L) during SmF on conventional media. Day 2 Day 4 Day 6 Day 8 Day 10 Day 12 Day 14 Kirk 18.5 ± 0.5 22.6 ± 2.0 46.0 ± 11.8 45.4 ± 14.4 51.7 ± 15.2 51.7 ± 2.5 60.4 ± 6.7 Kimura 17.3 ± 2.2 38.2 ± 2.2 57.2 ± 8.0 100.3 ± 26.5 98.4 ± 18.4 31.0 ± 8.7 22.3 ± 1.9 It should be noted that all SmFs with WSE-based medium showed similar or higher MnP production than SmFs in conventional media, which makes WSE a good alternative for the production of LE, with the advantage that it is possible to valorise an agricultural waste in the context of a circular economy approach. The fact that wheat straw contains significant levels of manganese (50 mg/L) (Hofrichter et al., 1999) leads to more suitable conditions that favor MnP production. Laccase activity was also measured in all fermentations with wheat straw-based media, but no laccase activity was detected in any of the trials. Ligninolytic enzymes production through the valorisation of wheat straw 93 3.3.3. Scale-up of submerged fermentation The SmF with maximum enzyme production was scaled up to a total volume of 30 L in a stirred tank reactor, obtaining a maximum MnP production of 345 U/L. During the operation of the reactor, different parameters were measured online (Figure 3.5). The analysis of the redox potential and pH profile allows to determine an indicator of increased enzyme production. Regarding the pH, before the onset of enzyme production, the pH value decreased to around 4.4 and after the peak of MnP production, pH increased again till 4.7. On the other hand, the redox potential underwent an increase as more enzyme was produced, reaching its maximum value (500 mV) in parallel to the peak of enzyme production (Lú-Chau et al., 2018). Figure 3.5. Evolution of pH (- -), redox potential (-), MnP activity (♦), MiP activity (▲) and glucose concentration (○) during the fermentation in a 30 L fermenter. Another remarkable fact is that enzyme production started once the glucose concentration was below 0.8 g/L, similar to the trend observed in Erlenmeyer flask experiments. This is mainly because low glucose levels induce a phase shift from primary to secondary metabolism, a phase in which the fungus begins to synthesize LEs (Iqbal et al., 2011). At the morphological level, it was observed that the morphology of the fungus varied during the fermentation. Initially, the fungal pellets had a well-defined spherical shape (Figure A3.1-a) because the addition of Tween-80 favours their agglomeration (Q. Li et al., 2018). As fermentation progresses, due to the 0 1 2 3 4 5 6 0 100 200 300 400 500 600 0123 pH, glucose (g/L) MnP and 10*MiP activity (U/L), redox (mV) Time (days) Chapter 3 94 shear stress that the fungus undergoes by continuous agitation and aeration, the mycelium fragmented into smaller amorphous pieces (Figure A3.1-b) reaching a point where it started to form spores (Figure A3.1-c,d,e), trying to conserve its genetic material. WRF sporulation only occurs under certain conditions of metabolic and mechanical stress associated with agitation, conditions in which the fungus uses spore formation as a survival mechanism (Su et al., 2012). 3.3.4. Identification of enzyme cocktail proteins Due to the detection of Mn(II)-dependent and Mn(II)-independent activities, the different proteins present in the concentrated enzyme cocktail were separated using SDS-PAGE, as a tool to differentiate the enzymes responsible for these activities. Figure 3.6 shows the migration pattern of triplicate cocktail aliquots. The band with the highest intensity corresponds to a molecular weight of 53 kDa, while also other bands with molecular weights of 89, 59 and 44 kDa are visible. Figure 3.6. SDS-PAGE of triplicate aliquots from the concentrated enzyme cocktail obtained in the SmF with I. lacteus (lanes 2-4). Molecular weight marker is shown in lane 1. By shotgun proteomic analyses, 18 enzymes were identified in the enzyme cocktail as shown in Table A3.3 (considering only the proteins with >2 unique Ligninolytic enzymes production through the valorisation of wheat straw 95 peptides). Most of them correspond to peroxidases, and among them, interestingly, long-chain MnPs, one short-chain MnP and one DyP. Other enzymes present in the enzyme cocktail included proteinases, cellobiohydrolases and dehydrogenases. The production of enzyme cocktails with MnP and MiP activities has been documented for different WRF species (Chen et al., 2015; Duan et al., 2018). However, very few articles analyze whether this activity is due to a single enzyme or to a set of enzymes. The expression of a short MnP with both MnP and MiP activity has already been described for I. lacteus (Li et al., 2019). Therefore, it is essential to determine its presence in the obtained enzyme cocktail, as it could offer some advantages due to its shorter C-terminal length that can increase the catalytic properties by a better interaction with the substrate molecule (Li et al., 2019). Considering the SDS-PAGE electrophoresis results, it is clear that the proteins present in highest abundance correspond to a molecular weight (MW) of 53 kDa. However, in shotgun proteomic analyses the most abundant proteins were MnPs with MWs ranging from 37.25 – 40.25 kDa. This is in agreement to Shin et al. (2005), who reported a purified MnP from I. lacteus with a MW of 38.3 according to their MALDI-TOF analyses, while in SDS-PAGE its band was located in 53 kDa. Also, in Wang et al. (2002), an isolated MnP from Bjerkandera adusta showed an apparent MW in SDS-PAGE of 43 kDa in SDSPAGE and 36.6 kDa by MALDI-TOF. These differences can be explained by the fact that MnPs are glycosylated proteins, and high carbohydrate contents can affect migration in SDS-PAGE. Another band observed in the gel at 44 kDa might correspond to a short MnP. Its MW is in agreement with other values reported in the literature for short MnPs of I. lacteus in SDS-PAGE (4345 kDa) (Chen et al., 2015; Duan et al., 2018). By mass spectrometry one enzyme identified with 5 unique peptides corresponds with a short MnP of I. lacteus previously characterised by Chen et al. (2015) (GenBank: AGO86670.2). On the other hand, the presence of a band at the MW of 59 kDa, could correspond to a DyP. Salvachúa et al. (2013a) have already described a DyP produced with I. lacteus on wheat straw with a molecular weight of 57 kDa in SDS-PAGE. The presence of this enzyme together with short MnP would justify the MiP activity measured in the enzyme cocktail. Accordingly, proteomic analysis detected a DyP with 17 unique peptides. Chapter 3 96 Finally, a weak band was observed in SDS-PAGE at a corresponding MW of 89 kDa which could not be explained by any of the proteins identified by shotgun proteomics. One possibility is that this band is composed by the choline dehydrogenases detected in shotgun with MW of 65.87 kDa. Nonetheless it cannot be discarded that this might be result of the artificial dimerization of an enzyme during the sample processing for SDS-PAGE, which is rare but have already been described, especially for highly hydrophobic proteins (Rath et al., 2009). Regarding the abovementioned, the proteomic analysis confirmed the presence of different peroxidases in the enzyme cocktail and is a more precise tool for the identification of enzymes and their corresponding MW in mixed cocktails. 3.3.5. Environmental profile associated to enzyme production In order to evaluate the components of the life-cycle inventory that contributes the most to the environmental impacts, the LCA methodology was used to the complete process of MnP production. Figure 3.7 shows the environmental profile of the different stages of the process, in which it is possible see that stage II (fermentation) is the largest contributor to the impacts estimated for all the categories selected. Nevertheless, stage I (inoculum preparation) also had a significant contribution to environmental burdens of the complete process of MnP production. Table A3.5 shows the average values by impact category and process stage. Figure 3.7. Environmental impact contributions per process stage in MnP production with I. lacteus in 30 L reactor. 0% 20% 40% 60% 80% 100% GW IR TE FET MET HN-CT FRS Relative contribution (%) Stage I Stage II Stage III Ligninolytic enzymes production through the valorisation of wheat straw 97 To try to determine why these stages contribute the most to the overall environmental impact of the process, the environmental profile of each stage was analyzed separately. Figure 3.8 presents the contributions to the environmental impact at the different stages of MnP production. It shows the breakdown distribution of environmental impacts for each category in terms of energy required, water consumption, chemicals and treatment of the output streams. Energy consumption was the main issue in all categories evaluated. For the correct growth of the fungus and the expression of enzymes, a series of environmental conditions such as suitable temperature, agitation and aeration are necessary, which imply the use of equipment with a considerable energy demand for several days. Nevertheless, for stage III, in which it is only required energy for the operation of the peristaltic pumps that allows the flow of the enzyme cocktail to the membranes of micro and ultrafiltration, electricity had less weight in the environmental impacts than in previous stages. The second main contributor to the impacts in stage III was the use of chemicals (except for terrestrial ecotoxicity category) due to the addition of NaOH for the cleaning of the filtration membranes. Chapter 3 104 Table A3.5. Midpoint values per impact category and process stage. Impact category Acronym Unit Total Stage I Stage II Stage III Global warming GW kg CO 2 eq 2.41 ∙10-3 7.14∙10-4 1.55∙10-3 1.41∙10-4 Stratospheric ozone depletion SOD kg CFC11 eq 2.38∙10 -9 4.99∙10 -10 1.78∙10 -9 9.94∙10 -11 Ionizing radiation IR kBq Co -60 eq 1.16 ∙10-3 3.68∙10-4 7.45∙10-4 4.86∙10-5 Ozone formation, Human health OF kg NO x eq 4.44∙10 -6 1.34∙10 -6 2.79∙10 -6 3.12∙10 -7 Fine particulate matter formation FPM kg PM2.5 eq 3.73∙10 -6 1.13∙10 -6 2.34∙10 -6 2.54∙10 -7 Ozone formation, Terrestrial ecosystems OF kg NO x eq 4.49 ∙10-6 1.36∙10-6 2.82∙10-6 3.16∙10-7 Terrestrial acidification TA kg SO 2 eq 9.60∙10 -6 2.92∙10 -6 6.11∙10 -6 5.69∙10 -7 Freshwater eutrophication FE kg P eq 2.92 ∙10-6 8.49∙10-7 1.93∙10-6 1.43∙10-7 Marine eutrophication ME kg N eq 6.85 ∙10-7 1.08∙10-7 5.27∙10-7 4.87∙10-8 Terrestrial ecotoxicity TE kg 1,4-DCB 7.23∙10 -3 2.17∙10 -3 4.55∙10 -3 5.10∙10 -4 Freshwater ecotoxicity FET kg 1,4 -DCB 2.50 ∙10-4 7.77∙10-5 1.61∙10-4 1.14∙10-5 Marine ecotoxicity MET kg 1,4-DCB 3.13∙10 -4 9.79∙10 -5 2.00∙10 -4 1.48∙10 -5 Human carcinogenic toxicity HCT kg 1,4 -DCB 2.03 ∙10-4 5.53∙10-5 1.14∙10-4 3.39∙10-5 Human noncarcinogenic toxicity HN-CT kg 1,4 -DCB 3.80 ∙10-3 1.16∙10-3 2.42∙10-3 2.15∙10-4 Land use LU m 2 a crop eq 3.46∙10 -4 5.53∙10 -5 2.86∙10 -4 4.71∙10 -6 Mineral resource scarcity MRS kg Cu eq 6.27 ∙10-6 1.79∙10-6 3.76∙10-6 7.20∙10-7 Fossil resource scarcity FRS kg oil eq 6.21∙10 -4 1.93∙10 -4 3.91∙10 -4 3.59∙10 -5 Water consumption WC m3 1.41 ∙10-4 1.50∙10-5 3.33∙10-5 9.24∙10-5 Ligninolytic enzymes production through the valorisation of wheat straw 105 Figure A3.1. Microscopy images of fermentation samples taken from the 30 L reactor at: (a) day 0 (10x), (b) day 1 (10x), (c) day 2 (10x), (d) day 3 (10x), and (e) day 3 (40x). (a) (b) (c) (d) (e) CHAPTER 4 VALORISATION OF BIOETHANOL BY-PRODUCTS TO PRODUCE UNSPECIFIC PEROXYGENASE Part of the results of this chapter have already been published as “González-Rodríguez S, Trueba-Santiso A, Lu-Chau TA, Moreira MT, Eibes G. Valorization of bioethanol byproducts to produce unspecific peroxygenase with Agrocybe aegerita: Technological and proteomic perspectives. N Biotechnol. 2023; 76:63-71. https://doi.org/10.1016/j.nbt.2023.05.001”. Detailed information available at “Manuscripts considered in this thesis: rights, contributions and quality indicators”, page 227. Valorisation of bioethanol by-products to produce unspecific peroxygenase 109 SUMMARY Unspecific peroxygenases (UPOs) (EC 1.11.2.1) represent an interesting subclass of oxidoreductases with peroxygenase and peroxidase activity. These characteristics allow their use as industrial biocatalysts on a wide range of applications. In this chapter, the biotechnological production of UPO by Agrocybe aegerita using bioethanol by-products as substrate is addressed. Solid-state and submerged fermentations (SSF and SmF) were evaluated, achieving the highest titers of UPO and laccase activities in SmF using vinasse as nutrient source. Optimal UPO production of 331 U/L was achieved in 50 % (v:v) vinasse medium with an inoculum grown for 14 d. These conditions were applied for the operation of a 4 L reactor, reaching an UPO activity of 265 U/L. Proteome expression before and after UPO production was analyzed by shotgun proteomic analysis to identify potential inducers of UPO expression. UPO and laccase, as well as proteins related to the production of reactive oxygen species (ROS) and the defense against excess of ROS were detected. Interestingly, metabolism of complex nitrogen sources was activated once UPO was expressed, supporting that media with high nitrogen content could act as inducers of UPO. Valorisation of bioethanol by-products to produce unspecific peroxygenase 111 4.1. Introduction 4.1.1. Unspecific peroxygenase potential and the importance of medium composition on its expression The discovery of UPO family is relatively recent, being described for the first time in the basidiomycetous fungus Agrocybe aegerita in 2004 (René Ullrich et al., 2004). Although it was initially cataloged as haloperoxidase or aromatic peroxygenase (APO) due to the partial similitude of its amino acid sequence with the heme chloroperoxidase (CPO) from the ascomycete Caldariomyces fumago and due to the hydroxylation activity found on halides and aromatic compounds, some years later this class of enzymes was renamed as unspecific peroxygenases (UPOs, E.C. 1.11.2) (Hofrichter et al., 2015; Hofrichter and Ullrich, 2006; René Ullrich et al., 2004). These hemo-thiolate enzymes are particularly interesting for their combination of the catalytic cycle of heme peroxidases with the ability to perform some nonspecific reactions commonly attributed to cytochromes P450 (Kinner et al., 2021). Nevertheless, in contrast to cytochromes P450, UPOs only need H2O2 as co-substrate, which act as both electron acceptor and oxygen donor (Linde et al., 2020; Ullrich and Hofrichter, 2005). Since this peroxygenase is produced during secondary metabolism, its production requires a long time, from 2 to 4 weeks of cultivation (Kinner et al., 2021). The composition of the culture medium has a direct impact on the accomplishment of UPO production. Previous reports suggest that a high nitrogen content, which can be provided by plant-derived compounds such as soybean meal or soybean peptone, may trigger UPO expression (Gröbe et al., 2011; René Ullrich et al., 2004), although this relationship has not yet been elucidated. Therefore, it seems essential to investigate the effects of new substrates with high nitrogen content. Accordingly, the liquid side-stream (vinasse) from the bioethanol production process and the solid fraction, which mainly correspond to distillers's dried grains with solubles (DDGS), are interesting candidates as nitrogen source in the fermentation medium. In addition, it is important to analyse the factors involved in UPO expression in order to identify the most suitable culture medium conditions to induce the expression of this enzyme. The physiological role of UPO in nature is still uncertain, different activities having been proposed, including metabolite synthesis, detoxification processes of lignin degradation and interaction with Chapter 4 112 antimicrobial peptides released by host plants (Hofrichter et al., 2015). Bormann et al. (2022) suggested that, in addition to external culture conditions, a type of fungal autoregulation may influence UPO synthesis and secretion. 4.1.2. Objectives of this chapter The most studied fermentation media to produce UPO are based on highvalue compounds such as soybean meal or glucose, so it is important to give more preference to fermentations that are developed from residues or low value compounds rich in C and/or N that will be used as substrates in the formulation of the culture medium. This work proposes an alternative use of by-products from the bioethanol industry in the formulation of the fermentation media suitable for UPO production by A. aegerita, demonstrating that different types of fermentation (SSF and SmF) are optimal for fungal growth and UPO expression. In addition, due to the lack of information about the factors that induce the production of this enzyme, it is especially important to use techniques that allow to better understand the metabolic mechanisms that lead to the expression of the target enzyme. In this sense, proteomic analysis provides valuable information on the changes in proteome expression at different times of the fungal culture with the aim of relating other cellular activities to the induction of these enzymes. 4.2. Materials and methods 4.2.1. Materials 4.2.1.1. Chemicals, raw material and microorganism H2O2, veratryl alcohol, 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) and MgCl2 were purchased from Sigma-Aldrich, malt extract from Biolife italiana, KH2PO4 and ethylenediaminetetraacetic acid (EDTA) from Panreac, yeast extract from iNtRON Biotechnology, MgSO4∙7H2O from Fluka and sodium dodecyl sulfate (SDS) from Invitrogen. Vinasse and DDGS were provided by Bioetanol Galicia S.A. (Teixeiro-Curtis, Spain). The vinasse was stored at -18 °C, whereas DDGS was stored at room temperature until use. The characterisation of DDGS kindly provided by Bioetanol Galicia S.A. is shown in Table 4.1. Valorisation of bioethanol by-products to produce unspecific peroxygenase 113 Table 4.1. Composition of DDGS provided by Bioetanol Galicia S.A.*. Starch and solubles (% owb**) 3.9 Ash (% owb**) 4.3 Crude fibre (% owb**) 7.4 Fat (% owb**) 12.3 Humidity (%) 9.9 Protein (% owb**) 27.7 *Personal communication. **owb: on wet basis. The fungus A. aegerita, strain DSM 22459, was obtained from the German Collection of Microorganisms and Cell Cultures GmbH, Braunschweig. 4.2.2. Fermentations 4.2.2.1. Inoculum preparation For mycelium production in static cultures, three plugs of active mycelia were transferred from Petri dishes to Fernbach flasks with 100 mL of malt extractbased medium (malt extract 7.5 g/L, yeast extract 2 g/L, KH2PO4 1 g/L, MgSO4∙7H2O 0.5 mg/L) and maintained at 28 °C for 14-28 d. After this step, the content of the Fernbach flasks was crushed and used as inoculum for the Erlenmeyer flasks. 4.2.2.2. Solid state fermentation at flask scale DDGS was used as support and substrate for SSF of A. aegerita. The DDGS pellets were crashed using a pestle to increase the surface area of the substrate. Each 100 mL Erlenmeyer flask containing 3 g of DDGS and 7.5 mL of distilled water was sterilized for 20 min at 121 °C (RAYPA AES-75) and inoculated with 750 µL of inoculum. The Erlenmeyer flasks were incubated for 42 d at 28 °C in a humidity-saturated environment under static conditions. The moisture content was replenished weekly to ensure optimal humidity conditions for fungal growth. All experiments were conducted in duplicate. For enzyme extraction, 30 mL of distilled water was added to each Erlenmeyer flask and incubated for one hour at 175 rpm on an orbital shaker (C24 Incubator Shaker, New Brunswick Scientific) to promote separation of the Chapter 4 120 It is noteworthy that the time required to obtain maximum production (19 days) in 25 % (v:v) was shorter than in the other dilutions tested. In the case of the 75 % (v:v) experiment, the maximum UPO production of 167 ± 191 U/L was reached on day 32. The large difference between the replicate values can be expected given the long fermentation periods. Previous works where fermentation times exceed 30 d have also shown significant differences between replicates (Reina et al., 2017). Figure 4.3. Evolution of (a) UPO and (b) laccase activity in the SmF with 25 % (•), 50 % (•) and 75 % (•) vinasse medium. With reference to laccase production (Figure 4.3-b), a similar trend was observed. The time required to reach maximum activity increased with vinasse concentration. The highest production was obtained in 50 % (v:v) medium, reaching 11,252 ± 4501 U/L on day 21. This activity was much higher than those previously achieved in our laboratory also using vinasse but with another basidiomycetous fungus (Pena et al., 2012) and with different 0 100 200 300 400 500 600 0 5 10 15 20 25 30 35 UPO (U/L) Time (d) (a) 0 3000 6000 9000 12000 15000 0 5 10 15 20 25 30 35 Laccase (U/L) Time (d) (b) Valorisation of bioethanol by-products to produce unspecific peroxygenase 121 substrates such as a residual stream from the organosolv process (GarcíaTorreiro et al., 2018). Production reached in 25 and 75 % (v:v) media were also significant (6848 ± 2062 and 3499 ± 3574 U/L on days 13 and 28, respectively), but notably lower than that reached in the 50 % (v:v) medium. The high laccase activity can also be consequence of the residual ethanol remaining in the vinasse, which has been suggested to act as inducer of laccase production (Manavalan et al., 2013). In this regard, Hernández et al. (2015) reported that ethanol only functions as an inducer of laccase when simple nitrogen sources are not available in the culture medium, but in the presence of complex nitrogen sources, such as those present in vinasse, ethanol can have a positive synergistic effect on laccase production. Other studies focusing on UPO production with different strains of A. aegerita in conventional medium have reported higher UPO expression (2021 U/L) but lower laccase production, supporting the hypothesis of laccase induction by ethanol from vinasse (René Ullrich et al., 2004). As for the pH trend (Figure A4.2), UPO production started at pH values close to 8, with a maximum activity around pH 8.5. This pH profile is similar to that reported by Ullrich et al. (2004) in fermentations with A. aegerita in soybean meal-based medium. After optimising the percentage of stillage used to maximise UPO production, the effect of inoculum age on UPO production was studied. Inoculum age can play a crucial role in fungal metabolic activity and growth (Shah et al., 2014). In order to evaluate its potential effect on UPO expression, inocula from 2 to 4 weeks were used to cultivate A. aegerita under submerged conditions. Additionally, H2O2 concentration was monitored since its production may be related to UPO secretion. As depicted in Figure 4.4 and Figure 4.5, inoculum age seems not to have a very strong effect on the onset of UPO and laccase production in the vinasse based medium. Chapter 4 122 Figure 4.4. Evolution of UPO activity (•), pH (--) and H2O2 (∙∙) in SmF in vinasse-based medium 50 % v:v with inocula of different age: (a) 14 days, (b) 21 days and (c) 28 days. 0 100 200 300 400 0 5 10 15 20 25 010 20 30 40 50 UPO activity (U/L) pH, H 2 O 2 (mg/L) Time (d) (a) 0 100 200 300 400 0 5 10 15 20 25 010 20 30 40 UPO activity (U/L) pH, H 2 O 2 (mg/L) Time (d) (b) 0 100 200 300 400 0 5 10 15 20 25 010 20 30 UPO activity (U/L) pH, H 2 O 2 (mg/L) Time (d) (c) Valorisation of bioethanol by-products to produce unspecific peroxygenase 123 Figure 4.5. Evolution of laccase activity (♦) and pH (--) in SmF in vinasse-based medium 50 % v:v employing inocula of different age: (a) 14 days, (b) 21 days and (c) 28 days. 0 3000 6000 9000 12000 15000 5 6 7 8 9 010 20 30 40 50 Laccase activity (U/L) pH Time (d) (a) 0 2000 4000 6000 8000 10000 5 6 7 8 9 010 20 30 40 Laccase activity (U/L) pH Time (d) (b) 0 3000 6000 9000 12000 15000 5 6 7 8 9 010 20 30 Laccase activity (U/L) pH Time (d) (c) Chapter 4 124 As observed in SSF, laccase production in SmF (Figure 4.5) started earlier in the culture (day 3), whereas UPO production (Figure 4.4) started between days 11 and 16. Although production values using inocula of different ages do not show significant differences (statistical analysis in Figure A4.1, p-value 0.88) the time required to reach maximum UPO production was shorter in the fermentation with 14-day-old inoculum, where a production of 223 ± 137 U/L was obtained on day 28. In terms of laccase activity, younger and older inocula led to similar titers (8030 ± 3124 and 8219 ± 3148 U/L on day 23, respectively). On the other hand, the intermediate-aged inoculum required a shorter period to obtain the highest laccase activity (18 d), but only an activity of 5899 ± 2176 U/L was achieved. The presence of H2O2 was detected before the onset of UPO production (Figure 4.4), increasing to a maximum before the peak of UPO production. This trend in the concentration is probably related to the consumption of this compound by UPO and/or other enzymes (e.g. catalases) in different fungal metabolic pathways, leading to a decrease in the concentration of available H2O2 as enzymatic production increases. In fact, the maximum levels of UPO coincided with the minimum concentration of H2O2, suggesting that enzyme production could be affected by the lack of H2O2 required for its catalytic cycle. Inactivation of UPO due to high H2O2 concentration could be ruled out because a maximum concentration of 0.74 mM (25 mg/L) was detected, which is lower than the concentration reported by Karich et al. (2016) to completely inactivate UPO from A. aegerita at pH 7. On the other hand, H2O2 concentration could affect laccase activity, as 1 mM hydrogen peroxide was reported to inactivate 76 % of laccase from Pleurotus pulmonarius (Marques De Souza and Peralta, 2003). 4.3.3. Scale-up of submerged fermentation The SmF with the medium based on a dilution of 50 % v:v of vinasse was successfully scaled-up to a total volume of 4 L. After several episodes of yeast contamination, probably from the stillage, medium sterilisation was intensified, and inoculum concentration doubled to reduce the possibility of contamination. Fermentation was monitored for 17 d. Both enzymes were expressed, obtaining maximum values of 265 U/L for UPO on day 16 of fermentation, and 9639 U/L for laccase on day 11 (Figure 4.6). As observed in Valorisation of bioethanol by-products to produce unspecific peroxygenase 125 the Erlenmeyer flask cultures, pH increased throughout the fermentation, with the greatest increase coinciding with the maximum UPO activities. The maximum UPO activity was obtained at pH 8.6, which was similar to the pH reached in the Erlenmeyer scale SmF (8.4), so pH could be used as an optimal indicator of the enzymatic production stage. Figure 4.6. Evolution of pH (- -), UPO activity (⧫) and laccase activity (•) during the fermentation in a 4 L bioreactor. The UPO and laccase activity values achieved in the reactor were similar to those obtained with Erlenmeyer flasks. Therefore, mechanical stress due to agitation, which affects fungal morphology by causing pellet breakage, does not seem to affect enzyme production (Jafari et al., 2007; Lopez-Ramirez et al., 2018). Interestingly, these maximum titers were achieved in shorter time than at Erlenmeyer flask scale, which could be related to the higher inoculum concentration or to the more efficient aeration system enhancing mass transfer in the bioreactors (Lú-Chau et al., 2018). UPO production from A. aegerita achieved by Ullrich et al. (2004) was higher and required shorter fermentation periods (maximum UPO activity of 1550 U/L, about 10 mg/L, at day 10 in a 5 L bioreactor), but the laccase production was significantly lower, reaching only 290 U/L compared to 9639 U/L achieved with a vinasse-based medium. Chapter 4 126 Heterologous expression has allowed higher UPO production values. For example, Tonin et al. (2021) reached a maximum UPO production of 300 mg/L after 7 d (activity of 30,000 U/L by ABTS assay) using Pichia pastoris as host microorganism. This suggests that heterologous expression seems the most suitable option to produce UPO. However, it should be noted that obtaining enzyme cocktails with a predominance of several enzyme activities, as laccase and UPO in this work, can result in a synergistic effect when applying these cocktails as biocatalysts in different reactions. Furthermore, homologous expression may be useful in the study of the natural functions of UPO, which remain unclear. The elucidation of its role in nature is not merely a scientific question but is also important for biotechnological development and ecophysiological considerations of the enzyme (Bormann et al., 2022). 4.3.4. Identification of enzyme cocktail proteins To explore the enzymatic set expressed by the fungus and its cellular activities in different moments of the biotechnological process, shotgun proteomic analysis was performed at two different periods of A. aegerita growth in Erlenmeyer flasks: on day 7 of the culture (no UPO activity had yet been detected) and on day 16, when UPO activity was spectrophotometrically confirmed in the supernatant. A total of 328 proteins were identified in this study (after considering only those with a minimum of 2 peptides detected). Table 4.2 summarises the selected identified proteins. The AA sequences from the detected peptides were analysed by Unipept and grouped into Interpro protein categories. Those categories activated, suppressed, or upregulated at day 16 compared to day 7 are shown in Figure A4.3. Valorisation of bioethanol by-products to produce unspecific peroxygenase 127 Table 4.2. List of selected proteins identified in the shotgun proteomic analysis of Erlenmeyer SmF of A. aegerita grown with vinasse and their relative abundances on the proteome before and after UPO activity was spectrophotometrically confirmed. NCBI Accession Description No UPO activity in the medium UPO activity in the medium Spec NPT NPU Spec NPT NPU CAA7262553.1 Catalase 43 21 21 25 14 14 CAA7263457.1 SOD 10 7 6 10 8 0 pdb|2YP1|B UPO 0 0 0 13 8 0 CAA7265189.1 Laccase 0 0 0 11 5 0 CAA7270351.1 Trx-DP 0 0 0 7 5 0 CAA7269993.1 Lectin 2 2 2 5 3 0 CAA7266156.1 SOD 3 3 2 2 2 0 CAA7268902.1 Lectin 2 2 2 2 2 0 CAA7262403.1 UPO 0 0 0 2 2 0 CAA7264511.1 Lectin 2 2 2 0 0 0 CAA7264490.1 DyP 2 2 2 0 0 0 CAA7260134.1 Peptidase 5 4 4 7 4 0 CAA7264546.1 Peptidase 3 1 1 8 2 0 CAA7265101.1 Peptidase 2 2 2 3 2 0 CAA7266636.1 Peptidase 1 1 1 4 2 0 CAA7265216.1 Peptidase 0 0 0 2 2 0 CAA7264859.1 Oxidase 0 0 0 2 0 2 CAA7260317.1 PS 0 0 0 9 5 0 NPT: number of total peptides identified in the sample and corresponding to that protein; NPU: number of unique peptides (i.e. high-confidence supporting peptides) that are mapped to only one protein group identified in the sample and corresponding to this protein; SOD: superoxidase dismutase; Trx-DP: thioredoxin dependent peroxidase; PS: phosphate shynthase. First, shotgun proteomics served to corroborate the production of unspecific peroxygenases (accession numbers: pdb|2YP1|B and CAA7262403.1) and laccase (CAA7265189.1) by A. aegerita. According to the specific activities measured on site, the expression of unspecific peroxygenases was detected at day 16 whereas at day 7 no UPO activity was detected. Laccase activity was already detected on day 7 of fermentation, although the values were lower than those obtained on day 16. Interestingly, also a DyP-type peroxidase (CAA7264490.1) was found on day 6, which is known to reduce H2O2 to water and oxidize phenolic compounds as well as non-phenolic lignin model dimers and veratryl alcohol (Mattila et al., 2022). Chapter 4 128 A catalase (CAA7262553.1), responsible for the degradation of H2O2 to H2O and O2, was detected and its expression was negatively correlated with both UPO expression and the H2O2 concentration in the medium. It was found to be more abundant before the UPO activity was detected (when the H2O2 concentration had not yet been detected) than in the second period (H2O2 concentration detected). This observation might suggest that UPO and catalase follow different induction patterns. Nevertheless, at stage in which catalase was detected, the production of laccase has already started. According to Romanholo Ferreira et al. (2020), catalase can be expressed as response to the presence of recalcitrant coloured compounds derived from vinasse degradation and also to the degraded products produced by ligninolytic enzymes, such as laccase. NADP-dependent oxidoreductases were overexpressed when UPO was active. These enzymes are involved in providing reducing power for the decomposition of ROS species. Furthermore, two superoxide dismutases (CAA7266156.1 and CAA7263457.1) involved in the intracellular ROS control (Mattila et al., 2022) catalysing the reduction of superoxide (O−2) or molecular oxygen (O2) into intracellular hydrogen peroxide (H2O2), were also detected. However, in this case no significant change in their expression was found among the two conditions studied, suggesting they expression was not related to UPO activity. Regarding Interpro protein categories: thioredoxin-like family (which contains UPO) was overexpressed at day 16, according to the results abovementioned. Interestingly, other proteins involved in ROS defense were only found at day 16, when UPO activity was detected. Among them, alkyl hydroperoxide reductases, cupredoxins, peroxiredoxins and redoxins were detected. Peroxiredoxins are thiol-dependent redox enzymes responsible for quenching of intracellular ROS and building up the effective cellular defense system in living organisms against oxidative stress. The expression of these enzymes is induced by stressful conditions resulting from the presence of ROS from vinasse degradation, coinciding with the described by Romanholo Ferreira et al. (2020). Other interesting finding was the presence of lectins at the time in which UPO production starts (sp|Q6WY08.1|ATLE_AGRAE and CAA7264511.1). This type of proteins is proposed as protein reserve in mushrooms and also as a defense mechanism (Sun et al., 2003). Moreover, in the last years these Valorisation of bioethanol by-products to produce unspecific peroxygenase 129 proteins have been increasingly studied due to its antitumour and antimicrobial activity (Heim et al., 2015; Yang et al., 2009). Previous literature reports have shown the relevance of nutrient concentrations in the environment to activate UPO production (Gröbe et al., 2011; René Ullrich et al., 2004). Interestingly, those enzymes involved in the transformation of complex sugars, such as beta-galactosidase and glycoside hydrolase were only activated at day 16 (when UPO was active). Pyruvate kinase and ATP synthase activities were, however, suppressed, indicating that glycolysis and growth may be compromised at this stage, which could be attributed to total reducing sugars depletion in the fermentation medium. In addition, different proteins involved in the metabolism of nitrogen compounds, such as those belonging to the Interpro protein categories arginase (IPR014033) and ureohydrolase (IPR006035; IPR023696; IPR020855) were active only in the second condition (when UPO is active). This suggests that complex nitrogen compounds present in vinasse are decomposed into more simple molecules like urea or ammonia, that can be responsible of the higher pH value of the medium in the stage where UPO is produced. It is remarkable that arginine, the substrate for arginase, is the amino acid with the highest nitrogen content (32 %). Results from AA profiling of fermentation samples from SmF (Figure 4.7) showed a decrease of 71.8 % on the arginine content at the end of the experiment. COPYRIGHT PERMISSIONS Copyright permissions 235 Figure 1.2. General catalytic cycle of laccases (Chapter 1) Adapted from: Rodríguez-Delgado, M., Ornelas-Soto, N., 2017. Laccases: A blue enzyme for greener alternative technologies in the detection and treatment of emerging pollutants, in: Singh, R., Kumar, S. (Eds.), Green Technologies and Environmental Sustainability. Springer International Publishing, Cham, pp. 45–65. https://doi.org/10.1007/978-3-319-50654-8_2 Section reproduced: Figure 2.1 Authorization to use this figure on the current PhD thesis: Adapted with permission from Springer Nature through the license number 5531400533964 provided on April 17, 2023 for its use in Thesis/Dissertation in print and electronic format. Copyright permissions 236 Figure 1.3. General catalytic cycle of long and extralong MnPs (Chapter 1) Adapted from: Chowdhary, P., Shukla, G., Raj, G., Ferreira, L.F.R., Bharagava, R.N., 2019. Microbial manganese peroxidase: a ligninolytic enzyme and its ample opportunities in research. SN Appl. Sci. 1, 45. https://doi.org/10.1007/s42452018-0046-3 Section reproduced: Figure 1 Authorization to use this figure on the current PhD thesis: Adapted with permission from Springer Nature through the license number 5531400533964 provided on April 17, 2023 for its use in Thesis/Dissertation in print and electronic format. Copyright permissions 237 Figure 1.4. Catalytic cycles of unspecific peroxygenase using three different substrates (Chapter 1) Reproduced from: Hofrichter, M., Kellner, H., Herzog, R., Karich, A., Kiebist, J., Scheibner, K., Ullrich, R., 2022. Peroxide-mediated oxygenation of organic compounds by fungal peroxygenases. Antioxidants 11, 163. https://doi.org/10.3390/antiox11010163 Section reproduced: Figure 4 Authorization to use this figure on the current PhD thesis: Reproduced with permission from MDPI. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/), which allows copy and redistribute the material in any medium or format. REFERENCES References 241 Aaronson, L.R., Johnston, A.M., Martin, C.E., 1982. The effects of temperature acclimation on membrane sterols and phospholipids of Neurospora crassa. Biochim. Biophys. Acta - Lipids Lipid Metab. 713, 456– 462. https://doi.org/10.1016/0005-2760(82)90265-X Adhikari, S., Ozarska, B., 2018. Minimizing environmental impacts of timber products through the production process “From Sawmill to Final Products.” Environ. Syst. Res. 7. https://doi.org/10.1186/s40068-0180109-x Agustí-Brisach, C., Armengol, J., 2012. Effects of temperature, pH and water potential on mycelial growth, sporulation and chlamydospore production in culture of Cylindrocarpon spp. Associated with black foot of grapevines. Phytopathol. Mediterr. 51, 37–50. Ahmed, P.M., Nieto-Peñalver, C.G., de Figueroa, L.I.C., Pajot, H.F., 2022. Vinasse odyssey: sugarcane vinasse remediation and laccase production by Trametes sp. immobilized in polyurethane foam. Biodegradation 33, 333–348. https://doi.org/10.1007/s10532-022-09985-y Akpinar, M., Ozturk Urek, R., 2020. Decolorization and degradation potential of enhanced lignocellulolytic enzymes production by Pleurotus eryngii using cherry waste from industry. Biotechnol. Appl. Biochem. 67, 760– 773. https://doi.org/10.1002/bab.1846 Aktaş, N., Tanyolaç, A., 2003. Reaction conditions for laccase catalyzed polymerization of catechol. Bioresour. Technol. 87, 209–214. https://doi.org/10.1016/S0960-8524(02)00254-7 Alcalde, M., 2007. Laccases: Biological functions, molecular structure and industrial applications. Ind. Enzym. Struct. Funct. Appl. 461–476. https://doi.org/10.1007/1-4020-5377-0_26 Alharbi, S.K., Nghiem, L.D., van de Merwe, J.P., Leusch, F.D.L., Asif, M.B., Hai, F.I., Price, W.E., 2019. Degradation of diclofenac, trimethoprim, carbamazepine, and sulfamethoxazole by laccase from Trametes versicolor: Transformation products and toxicity of treated effluent. Biocatal. Biotransformation 37, 399–408. https://doi.org/10.1080/10242422.2019.1580268 Ali, S., Fradi, A., Al-araji, A., 2017. Effect of some physical factors on growth References 248 Daughton, C.G., Ternes, T.A., 1999. Pharmaceuticals and personal care products in the environment: Agents of subtle change? Environ. Health Perspect. 107, 907–938. https://doi.org/10.1289/ehp.99107s6907 de Freitas, E.N., Bubna, G.A., Brugnari, T., Kato, C.G., Nolli, M., Rauen, T.G., Peralta Muniz Moreira, R. de F., Peralta, R.A., Bracht, A., de Souza, C.G.M., Peralta, R.M., 2017. Removal of bisphenol A by laccases from Pleurotus ostreatus and Pleurotus pulmonarius and evaluation of ecotoxicity of degradation products. Chem. Eng. J. 330, 1361–1369. https://doi.org/10.1016/j.cej.2017.08.051 Deblonde, T., Cossu-Leguille, C., Hartemann, P., 2011. Emerging pollutants in wastewater: A review of the literature. Int. J. Hyg. Environ. Health 214, 442–448. https://doi.org/10.1016/j.ijheh.2011.08.002 Debnath, R., Saha, T., 2020. An insight into the production strategies and applications of the ligninolytic enzyme laccase from bacteria and fungi. Biocatal. Agric. Biotechnol. 26, 101645. https://doi.org/10.1016/j.bcab.2020.101645 del Cerro, C., Erickson, E., Dong, T., Wong, A.R., Eder, E.K., Purvine, S.O., Mitchell, H.D., Weitz, K.K., Markillie, L.M., Burnet, M.C., Hoyt, D.W., Chu, R.K., Cheng, J.F., Ramirez, K.J., Katahira, R., Xiong, W., Himmel, M.E., Subramanian, V., Linger, J.G., Salvachúa, D., 2021. Intracellular pathways for lignin catabolism in white-rot fungi. Proc. Natl. Acad. Sci. U. S. A. 118, 1–10. https://doi.org/10.1073/pnas.2017381118 Delgado-Sánchez, C., Tenorio-Alfonso, A., Cortés-Triviño, E., Borrero-López, A.M., Valencia, C., 2022. Development of bio-based materials: Synthesis, characterization and applications. Polymers (Basel). 14, 3599. https://doi.org/10.3390/polym14173599 Demain, A.L., Vaishnav, P., 2009. Production of recombinant proteins by microbes and higher organisms. Biotechnol. Adv. 27, 297–306. https://doi.org/10.1016/j.biotechadv.2009.01.008 Deng, J., Wang, H., Zhan, H., Wu, C., Huang, Y., Yang, B., Mosa, A., Ling, W., 2022. Catalyzed degradation of polycyclic aromatic hydrocarbons by recoverable magnetic chitosan immobilized laccase from Trametes versicolor. Chemosphere 301, 134753. https://doi.org/10.1016/j.chemosphere.2022.134753 References 249 Di Cera, E., 2006. A structural perspective on enzymes activated by monovalent cations. J. Biol. Chem. 281, 1305–1308. https://doi.org/10.1074/jbc.R500023200 Dias, A.A., Freitas, G.S., Marques, G.S.M., Sampaio, A., Fraga, I.S., Rodrigues, M.A.M., Evtuguin, D. V., Bezerra, R.M.F., 2010. Enzymatic saccharification of biologically pre-treated wheat straw with white-rot fungi. Bioresour. Technol. 101, 6045–6050. https://doi.org/10.1016/j.biortech.2010.02.110 Dinte, E., Sylvester, B., 2018. Adhesives: Applications and recent advances, in: Özer, H. (Ed.), Applied Adhesive Bonding in Science and Technology. InTech, London. https://doi.org/10.5772/intechopen.71854 Domínguez-Robles, J., Tarrés, Q., Delgado-Aguilar, M., Rodríguez, A., Espinach, F.X., Mutjé, P., 2018. Approaching a new generation of fiberboards taking advantage of self lignin as green adhesive. Int. J. Biol. Macromol. 108, 927–935. https://doi.org/10.1016/j.ijbiomac.2017.11.005 Duan, Z., Shen, R., Liu, B., Yao, M., Jia, R., 2018. Comprehensive investigation of a dye-decolorizing peroxidase and a manganese peroxidase from Irpex lacteus F17, a lignin-degrading basidiomycete. AMB Express 8. https://doi.org/10.1186/s13568-018-0648-6 Edokpayi, J.N., Odiyo, J.O., Popoola, O.E., Msagati, T.A.M., 2016. Determination and distribution of polycyclic aromatic hydrocarbons in rivers, sediments and wastewater effluents in Vhembe District, South Africa. Int. J. Environ. Res. Public Health 13. https://doi.org/10.3390/ijerph13040387 Eibes, G., Cajthaml, T., Moreira, M.T., Feijoo, G., Lema, J.M., 2006. Enzymatic degradation of anthracene, dibenzothiophene and pyrene by manganese peroxidase in media containing acetone. Chemosphere 64, 408–414. https://doi.org/10.1016/j.chemosphere.2005.11.075 Eibes, G., Debernardi, G., Feijoo, G., Moreira, M.T., Lema, J.M., 2011. Oxidation of pharmaceutically active compounds by a ligninolytic fungal peroxidase. Biodegradation 22, 539–550. https://doi.org/10.1007/s10532-010-9426-0 Eibes, G., Lú-Chau, T., Feijoo, G., Moreira, M.T., Lema, J.M., 2005. Complete References 250 degradation of anthracene by manganese peroxidase in organic solvent mixtures. Enzyme Microb. Technol. 37, 365–372. https://doi.org/10.1016/j.enzmictec.2004.02.010 El Mansouri, N.E., Yuan, Q., Huang, F., 2011. Synthesis and characterization of kraft lignin-based epoxy resins. BioResources 6, 2492–2503. https://doi.org/10.15376/biores.6.3.2492-2503 El Sheikha, A.F. El, 2022. Nutritional profile and health benefits of Ganoderma lucidum “Lingzhi, Reishi, or Mannentake” as functional foods: Current scenario and future perspectives. Foods 11, 1030. https://doi.org/10.3390/foods11071030 Elegir, G., Daina, S., Zoia, L., Bestetti, G., Orlandi, M., 2005. Laccase mediator system: Oxidation of recalcitrant lignin model structures present in residual kraft lignin. Enzyme Microb. Technol. 37, 340–346. https://doi.org/10.1016/j.enzmictec.2005.02.017 Elisashvili, V., Kachlishvili, E., 2009. Physiological regulation of laccase and manganese peroxidase production by white-rot Basidiomycetes. J. Biotechnol. 144, 37–42. https://doi.org/10.1016/j.jbiotec.2009.06.020 Elisashvili, V., Kachlishvili, E., Khardziani, T., Agathos, S.N., 2010. Effect of aromatic compounds on the production of laccase and manganese peroxidase by white-rot basidiomycetes. J. Ind. Microbiol. Biotechnol. 37, 1091–1096. https://doi.org/10.1007/s10295-010-0757-y Elisashvili, V., Kachlishvili, E., Penninckx, M., 2008. Effect of growth substrate, method of fermentation, and nitrogen source on lignocellulose-degrading enzymes production by white-rot basidiomycetes. J. Ind. Microbiol. Biotechnol. 35, 1531–1538. https://doi.org/10.1007/s10295-008-0454-2 Ellouze, M., Sayadi, S., 2016. White-rot fungi and their enzymes as a biotechnological tool for xenobiotic bioremediation, in: Management of Hazardous Wastes. InTech, pp. 103–120. https://doi.org/10.5772/64145 Evstigneev, E.I., 2011. Factors affecting lignin solubility. Russ. J. Appl. Chem. 84, 1040–1045. https://doi.org/10.1134/S1070427211060243 Fernández-Fueyo, E., Linde, D., Almendral, D., López-Lucendo, M.F., Ruiz- References 251 Dueñas, F.J., Martínez, A.T., 2015. Description of the first fungal dyedecolorizing peroxidase oxidizing manganese(II). Appl. Microbiol. Biotechnol. 99, 8927–8942. https://doi.org/10.1007/s00253-015-6665-3 Fischer, B., Sumner, I., Goodenough, P., 1992. Isolation and renaturation of bio-active proteins expressed in Escherichia coli as inclusion bodies. Arzneimittel-Forschung/Drug Res. 42, 1512–1515. Freakley, S.J., Kochius, S., van Marwijk, J., Fenner, C., Lewis, R.J., Baldenius, K., Marais, S.S., Opperman, D.J., Harrison, S.T.L., Alcalde, M., Smit, M.S., Hutchings, G.J., 2019. A chemo-enzymatic oxidation cascade to activate C–H bonds with in situ generated H2O2. Nat. Commun. 10. https://doi.org/10.1038/s41467-019-12120-w Fukasawa, Y., Osono, T., Takeda, H., 2011. Wood decomposing abilities of diverse lignicolous fungi on nondecayed and decayed beech wood. Mycologia 103, 474–482. https://doi.org/10.3852/10-246 Gadd, G.M., 1993. Interactions of fungi with toxic metals. New Phytol. 124, 25–60. https://doi.org/10.1111/j.1469-8137.1993.tb03796.x Galhaup, C., Goller, S., Peterbauer, C.K., Strauss, J., Haltrich, D., 2002. Characterization of the major laccase isoenzyme from Trametes pubescens and regulation of its synthesis by metal ions. Microbiology 148, 2159– 2169. https://doi.org/10.1099/00221287-148-7-2159 Gálvez, F., Maggiolino, A., Domínguez, R., Pateiro, M., Gil, S., De Palo, P., Carballo, J., Franco, D., Lorenzo, J.M., 2019. Nutritional and meat quality characteristics of seven primal cuts from 9-month-old female veal calves: a preliminary study. J. Sci. Food Agric. 99, 2947–2956. https://doi.org/10.1002/jsfa.9508 García-Torreiro, M., Martínez-Patiño, J.C., Gullón, B., Lú-Chau, T.A., Moreira, M.T., Lema, J.M., Eibes, G., 2018. Simultaneous valorization and detoxification of the hemicellulose rich liquor from the organosolv fractionation. Int. Biodeterior. Biodegrad. 126, 112–118. https://doi.org/10.1016/j.ibiod.2017.10.010 Gassara, F., Ajila, C.M., Brar, S.K., Tyagi, R.D., Verma, M., Valero, J., 2013a. Influence of aeration and agitation modes on solid-state fermentation of apple pomace waste by Phanerochaete chrysosporium to produce References 252 ligninolytic enzymes and co-extract polyphenols. Int. J. Food Sci. Technol. 48, 2119–2126. https://doi.org/10.1111/ijfs.12194 Gassara, F., Brar, S.K., Tyagi, R.D., Verma, M., Surampalli, R.Y., 2010. Screening of agro-industrial wastes to produce ligninolytic enzymes by Phanerochaete chrysosporium. Biochem. Eng. J. 49, 388–394. https://doi.org/10.1016/j.bej.2010.01.015 Gassara, F., Brar, S.K., Verma, M., Tyagi, R.D., 2013b. Bisphenol A degradation in water by ligninolytic enzymes. Chemosphere 92, 1356– 1360. https://doi.org/10.1016/j.chemosphere.2013.02.071 Gavrilaş, S., Dumitru, F., Stǎnescu, M.D., 2012. Commercial laccase oxidation of phenolic compounds. UPB Sci. Bull. Ser. B Chem. Mater. Sci. 74, 3–10. Gessler, N.N., Aver’yanov, A.A., Belozerskaya, T.A., 2007. Reactive oxygen species in regulation of fungal development. Biochem. 72, 1091–1109. https://doi.org/10.1134/S0006297907100070 Gilkerson, J., Kelley, D.R., Tam, R., Estelle, M., Callis, J., 2015. Lysine residues are not required for proteasomemediated proteolysis of the auxin/indole acidic acid protein IAA1. Plant Physiol. 168, 708–720. https://doi.org/10.1104/pp.15.00402 Glenn, J.K., Gold, M.H., 1985. Purification and characterization of an extracellular Mn(II)-dependent peroxidase from the lignin-degrading basidiomycete, Phanerochaete chrysosporium. Arch. Biochem. Biophys. 242, 329–341. https://doi.org/10.1016/0003-9861(85)90217-6 Godswill Awuchi, C., Nyakundi Ondari, E., Josiah Eseoghene, I., Twinomuhwezi, H., Otuosorochi Amagwula, I., Morya, S., 2022. Fungal growth and mycotoxins production: Types, toxicities, control strategies, and detoxification, in: Fungal Reproduction and Growth. IntechOpen. https://doi.org/10.5772/intechopen.100207 Gohara, D.W., Di Cera, E., 2016. Molecular mechanisms of enzyme activation by monovalent cations. J. Biol. Chem. 291, 20840–20848. https://doi.org/10.1074/jbc.R116.737833 González Arzola, K., Polvillo, O., Arias, M.E., Perestelo, F., Carnicero, A., González-Vila, F.J., Falcón, M.A., 2006. Early attack and subsequent changes produced in an industrial lignin by a fungal laccase and a References 253 laccase-mediator system: An analytical approach. Appl. Microbiol. Biotechnol. 73, 141–150. https://doi.org/10.1007/s00253-006-0630-0 Goodell, B., Qian, Y., Jellison, J., 2008. Fungal decay of wood: Soft rot-brown rot-white rot. ACS Symp. Ser. 982, 9–31. https://doi.org/10.1021/bk-20080982.ch002 Gouveia, S., Otero, L., Fernández-Costas, C., Filgueira, D., Sanromán, Á., Moldes, D., 2018. Green binder Bbased on enzymatically polymerized eucalypt kraft lignin for fiberboard manufacturing: A preliminary study. Polymers (Basel). 10, 642. https://doi.org/10.3390/polym10060642 Grahame, D.A.S., Bryksa, B.C., Yada, R.Y., 2015. Factors affecting enzyme activity, in: Improving and tailoring enzymes for food quality and functionality. Elsevier, pp. 11–55. https://doi.org/10.1016/B978-1-78242285-3.00002-8 Grelska, A., Noszczyńska, M., 2020. White rot fungi can be a promising tool for removal of bisphenol A, bisphenol S, and nonylphenol from wastewater. Environ. Sci. Pollut. Res. 27, 39958–39976. https://doi.org/10.1007/s11356-020-10382-2 Gribas, A. V., Zatsepin, T.S., Korolev, S.P., Gottikh, M.B., Sakharov, I.Y., 2016. Suicide inactivation of covalent peroxidase-mimicking DNAzyme with hydrogen peroxide and its protection by a reductant substrate. Talanta 155, 212–215. https://doi.org/10.1016/j.talanta.2016.04.033 Gröbe, G., Ullrich, R., Pecyna, M.J., Kapturska, D., Friedrich, S., Hofrichter, M., Scheibner, K., 2011. High-yield production of aromatic peroxygenase by the agaric fungus Marasmius rotula. AMB Express 1, 1– 11. https://doi.org/10.1186/2191-0855-1-31 Günther, T., Sack, U., Hofrichter, M., Lätz, M., 1998. Oxidation of PAH and PAH-derivatives by fungal and plant oxidoreductases. J. Basic Microbiol. 38, 113–122. https://doi.org/10.1002/(SICI)15214028(199805)38:2<113::AID-JOBM113>3.0.CO;2-D Gupte, A., Gupte, S., Patel, H., 2007. Ligninolytic enzyme production under solid-state fermentation by white rot fungi. J. Sci. Ind. Res. (India). 66, 611–614. Guthi, J.S., Therapeutics, A., 2016. 3H & 14C radiolabeled compounds and their References 254 applications in metabolism studies. Gutierrez, F.M., Simsek, D., Mizrak, A., Deutschbauer, A., Braberg, H., Johnson, J., Xu, J., Shales, M., Nguyen, M., Tamse-Kuehn, R., Palm, C., Steinmetz, L.M., Krogan, N.J., Toczyski, D.P., 2018. Genetic analysis reveals functions of atypical polyubiquitin chains. Elife 7, 1–24. https://doi.org/10.7554/eLife.42955 Hämäläinen, V., Grönroos, T., Suonpää, A., Heikkilä, M.W., Romein, B., Ihalainen, P., Malandra, S., Birikh, K.R., 2018. Enzymatic processes to unlock the lignin value. Front. Bioeng. Biotechnol. 6, 1–10. https://doi.org/10.3389/fbioe.2018.00020 Hansen, B., Kusch, P., Schulze, M., Kamm, B., 2016. Qualitative and quantitative analysis of lignin produced from beech wood by different conditions of the organosolv process. J. Polym. Environ. 24, 85–97. https://doi.org/10.1007/s10924-015-0746-3 Heath, R.S., Turner, N.J., 2022. Recent advances in oxidase biocatalysts: Enzyme discovery, cascade reactions and scale up. Curr. Opin. Green Sustain. Chem. 38, 100693. https://doi.org/10.1016/j.cogsc.2022.100693 Heim, C., Hertzberg, H., Butschi, A., Bleuler-Martinez, S., Aebi, M., Deplazes, P., Künzler, M., Štefanić, S., 2015. Inhibition of Haemonchus contortus larval development by fungal lectins. Parasites and Vectors 8, 1–10. https://doi.org/10.1186/s13071-015-1032-x Heinfling, A., Ruiz-Dueñas, F.J., Martínez, M.J., Bergbauer, M., Szewzyk, U., Martínez, A.T., 1998. A study on reducing substrates of manganeseoxidizing peroxidases from Pleurotus eryngii and Bjerkandera adusta. FEBS Lett. 428, 141–146. https://doi.org/10.1016/S0014-5793(98)00512-2 Hernández, C.A., Sandoval, N., Mallerman, J., García-Pérez, J.A., Farnet, A.M., Perraud-Gaime, I., Alarcón, E., 2015. Ethanol induction of laccase depends on nitrogen conditions of Pycnoporus sanguineus. Electron. J. Biotechnol. 18, 327–332. https://doi.org/10.1016/j.ejbt.2015.05.008 Hofrichter, M., 2002. Review: Lignin conversion by manganese peroxidase (MnP). Enzyme Microb. Technol. 30, 454–466. https://doi.org/10.1016/S0141-0229(01)00528-2 References 255 Hofrichter, M., Kellner, H., Herzog, R., Karich, A., Kiebist, J., Scheibner, K., Ullrich, R., 2022. Peroxide-mediated oxygenation of organic compounds by fungal peroxygenases. Antioxidants 11, 163. https://doi.org/10.3390/antiox11010163 Hofrichter, M., Kellner, H., Herzog, R., Karich, A., Liers, C., Scheibner, K., Kimani, V.W., Ullrich, R., 2020. Fungal peroxygenases: A phylogenetically old superfamily of heme enzymes with promiscuity for oxygen transfer reactions, in: Grand Challenges in Biology and Biotechnology. pp. 369–403. https://doi.org/10.1007/978-3-030-295417_14 Hofrichter, M., Kellner, H., Pecyna, M.J., Ullrich, R., 2015. Fungal unspecific peroxygenases: Heme-thiolate proteins that combine peroxidase and cytochrome P450 properties. Adv. Exp. Med. Biol. 851, 341–368. https://doi.org/10.1007/978-3-319-16009-2_13 Hofrichter, M., Ullrich, R., 2014. Oxidations catalyzed by fungal peroxygenases. Curr. Opin. Chem. Biol. 19, 116–125. https://doi.org/10.1016/j.cbpa.2014.01.015 Hofrichter, M., Ullrich, R., 2006. Heme-thiolate haloperoxidases: versatile biocatalysts with biotechnological and environmental significance. Appl. Microbiol. Biotechnol. 71, 276–288. https://doi.org/10.1007/s00253006-0417-3 Hofrichter, M., Ullrich, R., Pecyna, M.J., Liers, C., Lundell, T., 2010. New and classic families of secreted fungal heme peroxidases. Appl. Microbiol. Biotechnol. 87, 871–897. https://doi.org/10.1007/s00253-010-2633-0 Hofrichter, M., Vares, T., Kalsi, M., Galkin, S., Scheibner, K., Fritsche, W., Hatakka, A., 1999. Production of manganese peroxidase and organic acids and mineralization of 14C-labelled lignin (14C-DHP) during solidstate fermentation of wheat straw with the white rot fungus Nematoloma frowardii. Appl. Environ. Microbiol. 65, 1864–1870. https://doi.org/10.1128/aem.65.5.1864-1870.1999 Hölker, U., Höfer, M., Lenz, J., 2004. Biotechnological advantages of laboratory-scale solid-state fermentation with fungi. Appl. Microbiol. Biotechnol. 64, 175–186. https://doi.org/10.1007/s00253-003-1504-3 Hollenberg, P.F., 2008. Enzyme inhibition, in: Drug Metabolism Handbook. References 256 John Wiley & Sons, Inc., Hoboken, NJ, USA, pp. 341–358. https://doi.org/10.1002/9780470439265.ch14 Hong, J.W., Park, J.Y., Gadd, G.M., 2010. Pyrene degradation and copper and zinc uptake by Fusarium solani and Hypocrea lixii isolated from petrol station soil. J. Appl. Microbiol. 108, 2030–2040. https://doi.org/10.1111/j.1365-2672.2009.04613.x Howard, R.L., Abotsi, E., Van Rensburg, E.L.J., Howard, S., 2003. Lignocellulose biotechnology: Issues of bioconversion and enzyme production. African J. Biotechnol. 2, 702–733. https://doi.org/10.5897/ajb2003.000-1115 Huber, D., Pellis, A., Daxbacher, A., Nyanhongo, G.S., Guebitz, G.M., 2016. Polymerization of various lignins via immobilized Myceliophthora thermophila Laccase (MtL). Polymers (Basel). 8, 1–10. https://doi.org/10.3390/polym8080280 Ibrahim, V., Mendoza, L., Mamo, G., Hatti-Kaul, R., 2011. Blue laccase from Galerina sp.: Properties and potential for Kraft lignin demethylation. Process Biochem. 46, 379–384. https://doi.org/10.1016/j.procbio.2010.07.013 Illman, B.L., Highley, T.L., 1989. Decomposition of wood by brown-rot fungi, in: Biodeterioration Research 2. Springer US, Boston, MA, pp. 465–484. https://doi.org/10.1007/978-1-4684-5670-7_40 Imam, A., Suman, S.K., Vempatapu, B.P., Tripathi, D., Ray, A., Kanaujia, P.K., 2022. Pyrene remediation by Trametes maxima: an insight into secretome response and degradation pathway. Environ. Sci. Pollut. Res. 29, 44135– 44147. https://doi.org/10.1007/s11356-022-18888-7 Iqbal, H.M.N., Asgher, M., Bhatti, H.N., 2011. Optimization of physical and nutritional factors for synthesis of lignin degrading enzymes by a novel strain of Trametes versicolor. BioResources 6, 1273–1287. https://doi.org/10.15376/biores.6.2.1273-1287 Isikhuemhen, O.S., Mikiashvili, N.A., Kelkar, V., 2009. Application of solid waste from anaerobic digestion of poultry litter in Agrocybe aegerita cultivation: Mushroom production, lignocellulolytic enzymes activity and substrate utilization. Biodegradation 20, 351–361. References 257 https://doi.org/10.1007/s10532-008-9226-y Jafari, A.R., Sarrafzadeh, M.H., Alemzadeh, I., Vosoughi, M., 2007. Effect of stitter speed and aeration rate on the production of glucose oxidase by Aspergillus niger. J. Biol. Sci. https://doi.org/10.3923/jbs.2007.270.275 Janusz, G., Pawlik, A., Sulej, J., Świderska-Burek, U., Jarosz-Wilkolazka, A., Paszczyński, A., 2017. Lignin degradation: Microorganisms, enzymes involved, genomes analysis and evolution. FEMS Microbiol. Rev. 41, 941–962. https://doi.org/10.1093/femsre/fux049 Janusz, G., Pawlik, A., Świderska-Burek, U., Polak, J., Sulej, J., JaroszWilkołazka, A., Paszczyński, A., 2020. Laccase properties, physiological functions, and evolution. Int. J. Mol. Sci. 21. https://doi.org/10.3390/ijms21030966 Ji, X., Guo, M., 2018. Preparation and properties of a chitosan-lignin wood adhesive. Int. J. Adhes. Adhes. 82, 8–13. https://doi.org/10.1016/j.ijadhadh.2017.12.005 Jiang, F., Kongsaeree, P., Charron, R., Lajoie, C., Xu, H., Scott, G., Kelly, C., 2008. Production and separation of manganese peroxidase from heme amended yeastc cultures. Biotechnol Bioeng. 99, 540–549. https://doi.org/10.1002/bit.21590 Jin, X., Yu, X., Zhu, G., Zheng, Z., Feng, F., Zhang, Z., 2016. Conditions optimizing and application of laccase-mediator system (LMS) for the laccase-catalyzed pesticide degradation. Sci. Rep. 6, 35787. https://doi.org/10.1038/srep35787 Johannes, C., Majcherczyk, A., 2000. Laccase activity tests and laccase inhibitors. J. Biotechnol. 78, 193–199. https://doi.org/10.1016/S01681656(00)00208-X Jonsson, S., Persson, Y., Frankki, S., Lundstedt, S., van Bavel, B., Haglund, P., Tysklind, M., 2006. Comparison of Fenton’s reagent and ozone oxidation of polycyclic aromatic hydrocarbons in aged contaminated soils. J. Soils Sediments 6, 208–214. https://doi.org/10.1065/jss2006.08.179 Jove, P., Olivella, M.A., Camarero, S., Caixach, J., Planas, C., Cano, L., De Las Heras, F.X., 2016. Fungal biodegradation of anthracene-polluted cork: A References 264 cellulolytic enzymes production by Trichoderma harzianum in a novel bench-scale solid-state fermentation bioreactor. Bioresour. Technol. 265, 291–298. https://doi.org/10.1016/j.biortech.2018.06.015 Lopina, O.D., 2017. Enzyme inhibitors and activators, in: Enzyme Inhibitors and Activators. InTech, p. 13. https://doi.org/10.5772/67248 Lousa, D., Baptista, A.M., Soares, C.M., 2013. A molecular perspective on nonaqueous biocatalysis: contributions from simulation studies. Phys. Chem. Chem. Phys. 15, 13723. https://doi.org/10.1039/c3cp51761f Lozano, P., de Diego, T., Guegan, J., Vaultier, M., Iborra, J.L., 2001. Stabilization of a-chymotrypsin by ionic liquids in transesterification reactions. Biotechnol. Bioeng. 75, 563–569. https://doi.org/10.1002/bit.10089 Lú-Chau, T.A., Martínez-Patiño, J.C., Gullón, B., García-Torreiro, M., Moreira, M.T., Lema, J.M., Eibes, G., 2018. Scale-up and economic analysis of the production of ligninolytic enzymes from a side-stream of the organosolv process. J. Chem. Technol. Biotechnol. 93, 3125–3134. https://doi.org/10.1002/jctb.5664 Lueangjaroenkit, P., Teerapatsakul, C., Chitradon, L., 2018. Morphological characteristic regulation of ligninolytic enzyme produced by Trametes polyzona. Mycobiology 46, 396–406. https://doi.org/10.1080/12298093.2018.1537586 Lueangjaroenkit, P., Teerapatsakul, C., Sakka, K., Sakka, M., Kimura, T., Kunitake, E., Chitradon, L., 2019. Two manganese peroxidases and a laccase of Trametes polyzona KU-RNW027 with novel properties for dye and pharmaceutical product degradation in redox mediator-free system. Mycobiology 47, 217–229. https://doi.org/10.1080/12298093.2019.1589900 Lundquist, K., Kristersson, P., 1985. Exhaustive laccase-catalysed oxidation of a lignin model compound (vanillyl glycol) produces methanol and polymeric quinoid products. Biochem. J. 229, 277–279. https://doi.org/10.1042/bj2290277 Luo, Y., Guo, W., Ngo, H.H., Nghiem, L.D., Hai, F.I., Zhang, J., Liang, S., Wang, X.C., 2014. A review on the occurrence of micropollutants in the aquatic environment and their fate and removal during wastewater References 265 treatment. Sci. Total Environ. 473–474, 619–641. https://doi.org/10.1016/j.scitotenv.2013.12.065 Ma, B., Zhang, K., Hendrie, C., Liang, C., Li, M., Doherty-Kirby, A., Lajoie, G., 2003. PEAKS: Powerful software for peptide de novo sequencing by tandem mass spectrometry. Rapid Commun. Mass Spectrom. 17, 2337– 2342. https://doi.org/10.1002/rcm.1196 Machado, M.L.O., Paz, E.C., Pinheiro, V.S., de Souza, R.A.S., Neto, A.M.P., Gaubeur, I., dos Santos, M.C., 2022. Use of WO2.72 nanoparticles/Vulcan® XC72 GDE electrocatalyst combined with the photoelectro-Fenton process for the degradation of 17α-ethinylestradiol (EE2). Electrocatalysis 13, 457–468. https://doi.org/10.1007/s12678-02200724-8 Maciel, M.J.M., Castro e Silva, A., Ribeiro, H.C.T., 2010. Industrial and biotechnological applications of ligninolytic enzymes of the basidiomycota: A review. Electron. J. Biotechnol. 13, 1–13. https://doi.org/10.2225/vol13-issue6-fulltext-2 Mackay, D., Shiu, W.Y., 1977. Aqueous solubility of polynuclear aromatic hydrocarbons. J. Chem. Eng. Data 22, 399–402. https://doi.org/10.1021/je60075a012 Maganhotto De Souza Silva, C.M., Soares De Melo, I., De Oliveira, P.R., 2005. Ligninolytic enzyme production by Ganoderma spp. Enzyme Microb. Technol. 37, 324–329. https://doi.org/10.1016/j.enzmictec.2004.12.007 Mahindra, A., Bagra, N., Wangoo, N., Khan, S.I., Jacob, M.R., Jain, R., 2014. Discovery of short peptides exhibiting high potency against Cryptococcus neoformans. ACS Med. Chem. Lett. 5, 315–320. https://doi.org/10.1021/ml500011v Majeke, B.M., García-Aparicio, M., Biko, O.., Viljoen-Bloom, M., van Zyl, W.H., Görgens, J.F., 2020. Synergistic codon optimization and bioreactor cultivation toward enhanced secretion of fungal lignin peroxidase in Pichia pastoris: Enzymatic valorization of technical (industrial) lignins. Enzyme Microb. Technol. 139, 109593. https://doi.org/10.1016/j.enzmictec.2020.109593 Mäkelä, M.R., Lundell, T., Hatakka, A., Hildén, K., 2013. Effect of copper, References 266 nutrient nitrogen, and wood-supplement on the production of ligninmodifying enzymes by the white-rot fungus Phlebia radiata. Fungal Biol. 117, 62–70. https://doi.org/10.1016/j.funbio.2012.11.006 Maller, A., Damásio, A.R.L., Silva, T.M. Da, Jorge, J.A., Terenzi, H.F., Polizeli, M. de L.T. de M., 2011. Biotechnological potential of agro-industrial wastes as a carbon cource to thermostable polygalacturonase production in Aspergillus niveus. Enzyme Res. 2011, 1–6. https://doi.org/10.4061/2011/289206 Manavalan, T., Manavalan, A., Thangavelu, K.P., Heese, K., 2013. Characterization of optimized production, purification and application of laccase from Ganoderma lucidum. Biochem. Eng. J. 70, 106–114. https://doi.org/10.1016/j.bej.2012.10.007 Mangos, T.J., Haas, M.J., 1996. Enzymatic determination of methanol with alcohol oxidase, peroxidase, and the chromogen 2,2′-azinobis(3ethylbenzthiazoline-6-sulfonic acid) and its application to the determination of the methyl ester content of pectins. J. Agric. Food Chem. 44, 2977–2981. https://doi.org/10.1021/jf960274z Maniak, H., Talma, M., Matyja, K., Trusek, A., Giurg, M., 2020. Synthesis and structure-activity relationship studies of hydrazide-hydrazones as inhibitors of laccase from Trametes versicolor. Molecules 25, 1255. https://doi.org/10.3390/molecules25051255 Marco-Urrea, E., Pérez-Trujillo, M., Vicent, T., Caminal, G., 2009. Ability of white-rot fungi to remove selected pharmaceuticals and identification of degradation products of ibuprofen by Trametes versicolor. Chemosphere 74, 765–772. https://doi.org/10.1016/j.chemosphere.2008.10.040 Marques De Souza, C.G., Peralta, R.M., 2003. Purification and characterization of the main laccase produced by the white-rot fungus Pleurotus pulmonarius on wheat bran solid state medium. J. Basic Microbiol. 43, 278–286. https://doi.org/10.1002/jobm.200390031 Martínez-Patiño, J.C., Lu-Chau, T.A., Gullón, B., Ruiz, E., Romero, I., Castro, E., Lema, J.M., 2018. Application of a combined fungal and diluted acid pretreatment on olive tree biomass. Ind. Crops Prod. 121, 10–17. https://doi.org/10.1016/j.indcrop.2018.04.078 Martinez, D., Larrondo, L.F., Putnam, N., Sollewijn Gelpke, M.D., Huang, K., References 267 Chapman, J., Helfenbein, K.G., Ramaiya, P., Detter, J.C., Larimer, F., Coutinho, P.M., Henrissat, B., Berka, R., Cullen, D., Rokhsar, D., 2004. Genome sequence of the lignocellulose degrading fungus Phanerochaete chrysosporium strain RP78. Nat. Biotechnol. 22, 695–700. https://doi.org/10.1038/nbt967 Marzo, C., Díaz, A.B., Caro, I., Blandino, A., 2019. Valorization of agroindustrial wastes to produce hydrolytic enzymes by fungal solid-state fermentation. Waste Manag. Res. 37, 149–156. https://doi.org/10.1177/0734242X18798699 Mateljak, I., Rice, A., Yang, K., Tron, T., Alcalde, M., 2019. The generation of thermostable fungal laccase chimeras by SCHEMA-RASPP structureguided recombination in vivo. ACS Synth. Biol. 8, 833–843. https://doi.org/10.1021/acssynbio.8b00509 Mattila, H., Österman-Udd, J., Mali, T., Lundell, T., 2022. Basidiomycota fungi and ROS: Genomic perspective on key enzymes involved in generation and mitigation of reactive oxygen species. Front. Fungal Biol. 3. https://doi.org/10.3389/ffunb.2022.837605 Mauk, M.R., Kishi, K., Gold, M.H., Mauk, A.G., 1998. pH-linked binding of Mn(II) to manganese peroxidase. Biochemistry 37, 6767–6771. https://doi.org/10.1021/bi972932u Mehinto, A.C., Hill, E.M., Tyler, C.R., 2010. Uptake and biological effects of environmentally relevant concentrations of the nonsteroidal antiinflammatory pharmaceutical diclofenac in rainbow trout (oncorhynchus mykiss). Environ. Sci. Technol. 44, 2176–2182. https://doi.org/10.1021/es903702m Mehra, R., Muschiol, J., Meyer, A.S., Kepp, K.P., 2018. A structural-chemical explanation of fungal laccase activity. Sci. Rep. 8, 17285. https://doi.org/10.1038/s41598-018-35633-8 Méndez-Hernández, J.E., Eibes, G., Arca-Ramos, A., Lú-Chau, T.A., Feijoo, G., Moreira, M.T., Lema, J.M., 2015. Continuous removal of nonylphenol by versatile peroxidase in a two-stage membrane bioreactor. Appl. Biochem. Biotechnol. 175, 3038–3047. https://doi.org/10.1007/s12010-0141474-1 Menzinger, M., Wolfgang, R., 1969. The Meaning and use of the Arrhenius References 268 activation energy. Angew. Chemie Int. Ed. English 8, 438–444. https://doi.org/10.1002/anie.196904381 Mikiashvili, N., Wasser, S.P., Nevo, E., Elisashvili, V., 2006. Effects of carbon and nitrogen sources on Pleurotus ostreatus ligninolytic enzyme activity. World J. Microbiol. Biotechnol. 22, 999–1002. https://doi.org/10.1007/s11274-006-9132-6 Mishra, A., Kumar, S., 2007. Cyanobacterial biomass as N-supplement to agro-waste for hyper-production of laccase from Pleurotus ostreatus in solid state fermentation. Process Biochem. 42, 681–685. https://doi.org/10.1016/j.procbio.2006.09.022 Mohapatra, D.P., Brar, S.K., Tyagi, R.D., Picard, P., Surampalli, R.Y., 2014. Analysis and advanced oxidation treatment of a persistent pharmaceutical compound in wastewater and wastewater sludgecarbamazepine. Sci. Total Environ. 470–471, 58–75. https://doi.org/10.1016/j.scitotenv.2013.09.034 Monterrey, D.T., Menés-Rubio, A., Keser, M., Gonzalez-Perez, D., Alcalde, M., 2023. Unspecific peroxygenases: The pot of gold at the end of the oxyfunctionalization rainbow? Curr. Opin. Green Sustain. Chem. 41, 100786. https://doi.org/10.1016/j.cogsc.2023.100786 Moon, D.S., Song, H.G., 2012. Degradation of alkylphenols by white rot fungus Irpex lacteus and its manganese peroxidase. Appl. Biochem. Biotechnol. 168, 542–549. https://doi.org/10.1007/s12010-012-9795-4 Mora-Pale, M., Meli, L., Doherty, T. V, Linhardt, R.J., Dordick, J.S., 2011. Room temperature ionic liquids as emerging solvents for the pretreatment of lignocellulosic biomass. Biotechnol. Bioeng. 108, 1229–1245. https://doi.org/10.1002/bit.23108 Moreira, M.T., Feijoo, G., Canaval, J., Lema, J.M., 2003. Semipilot-scale bleaching of kraft pulp with manganese peroxide. Wood Sci. Technol. 37, 117–123. https://doi.org/10.1007/s00226-003-0175-7 Moreira, M.T., Torrado, A., Feijoo, G., Lema, J.M., 2000. Manganese peroxidase production by Bjerkandera sp. BOS55: 2. Operation in stirred tank reactors. Bioprocess Eng. 23, 663–667. https://doi.org/10.1007/s004490000217 References 269 Morozova, O. V, Shumakovich, G.P., Gorbacheva, M.A., Shleev, S. V, Yaropolov, A.I., 2007. “Blue” laccases. Biochem. 72, 1136–1150. https://doi.org/10.1134/S0006297907100112 Munekata, P.E.S., Pateiro, M., Domínguez, R., Zhou, J., Barba, F.J., Lorenzo, J.M., 2020. Nutritional characterization of sea bass processing byproducts. Biomolecules 10, 1–18. https://doi.org/10.3390/biom10020232 Muñiz-Mouro, A., Oliveira, I.M., Gullón, B., Lú-Chau, T.A., Moreira, M.T., Lema, J.M., Eibes, G., 2017. Comprehensive investigation of the enzymatic oligomerization of esculin by laccase in ethanol: Water mixtures. RSC Adv. 7, 38424–38433. https://doi.org/10.1039/c7ra06972c Murugananthan, M., Yoshihara, S., Rakuma, T., Shirakashi, T., 2008. Mineralization of bisphenol A (BPA) by anodic oxidation with borondoped diamond (BDD) electrode. J. Hazard. Mater. 154, 213–220. https://doi.org/10.1016/j.jhazmat.2007.10.011 Musoni, M., Destain, J., Thonart, P., Bahama, J.B., Delvigne, F., 2015. Bioreactor design and implementation strategies for the cultivation of filamentous fungi and the production of fungal metabolites: From traditional methods to engineered systems. Biotechnol. Agron. Soc. Environ. 19, 430–442. Narra, M., Rudakiya, D.M., Macwan, K., Patel, N., 2020. Black liquor: A potential moistening agent for production of cost-effective hydrolytic enzymes by a newly isolated cellulo-xylano fungal strain Aspergillus tubingensis and its role in higher saccharification efficiency. Bioresour. Technol. 306, 123149. https://doi.org/10.1016/j.biortech.2020.123149 Nejedly, T., Klimes, J., 2017. A model of natural degradation of 17-αethinylestradiol in surface water and identification of degradation products by GC-MS. Environ. Sci. Pollut. Res. 24, 23196–23206. https://doi.org/10.1007/s11356-017-9743-5 Neto, Y.A.A.H., Garzon, N.G. da R., Pedezzi, R., Cabral, H., 2018. Specificity of peptidases secreted by filamentous fungi. Bioengineered 9, 30–37. https://doi.org/10.1080/21655979.2017.1373531 Newman, W.H., Glasser, W.G., 1985. Engineering plastics from lignin - XII. Synthesis and performance of lignin adhesives with isocyanate and References 270 melamine. Holzforschung 39, 345–353. https://doi.org/10.1515/hfsg.1985.39.6.345 Nguyen, L.N., van de Merwe, J.P., Hai, F.I., Leusch, F.D.L., Kang, J., Price, W.E., Roddick, F., Magram, S.F., Nghiem, L.D., 2016. Laccasesyringaldehyde-mediated degradation of trace organic contaminants in an enzymatic membrane reactor: Removal efficiency and effluent toxicity. Bioresour. Technol. 200, 477–484. https://doi.org/10.1016/j.biortech.2015.10.054 Nicolini, L., Volpe, C., Pezzotti, A., Carilli, A., 1993. Changes in in-vitro digestibility of orange peels and distillery grape stalks after solid-state fermentation by higher fungi. Bioresour. Technol. 45, 17–20. https://doi.org/10.1016/0960-8524(93)90137-Z Nielsen, J.E., McCammon, J.A., 2003. Calculating pKa values in enzyme active sites. Protein Sci. 12, 1894–1901. https://doi.org/10.1110/ps.03114903 Nisa, F.T., Asgher, M., Anwar, Z., Afroz, A., Zeeshan, N., Rashid, U., Irshad, M., Mehmood, S., Sughra, K., 2014. Influence of culture conditions on ligninolytic enzymes production from Ganoderma lucidum using agroindustrial residues. Fresenius Environ. Bull. 23, 3441–3447. Nordén, K., Agemark, M., Danielson, J.Å., Alexandersson, E., Kjellbom, P., Johanson, U., 2011. Increasing gene dosage greatly enhances recombinant expression of aquaporins in Pichia pastoris. BMC Biotechnol. 11, 47. https://doi.org/10.1186/1472-6750-11-47 Novotný, Č., Cajthaml, T., Svobodová, K., Šušla, M., Šašek, V., 2009. Irpex lacteus, a white-rot fungus with biotechnological potential - Review. Folia Microbiol. (Praha). 54, 375–390. https://doi.org/10.1007/s12223-0090053-2 Novotný, Č., Svobodová, K., Erbanová, P., Cajthaml, T., Kasinath, A., Lang, E., Šašek, V., 2004. Ligninolytic fungi in bioremediation: Extracellular enzyme production and degradation rate. Soil Biol. Biochem. 36, 1545– 1551. https://doi.org/10.1016/j.soilbio.2004.07.019 Nugroho Prasetyo, E., Kudanga, T., Østergaard, L., Rencoret, J., Gutiérrez, A., del Río, J.C., Ignacio Santos, J., Nieto, L., Jiménez-Barbero, J., Martínez, A.T., Li, J., Gellerstedt, G., Lepifre, S., Silva, C., Kim, S.Y., Cavaco-Paulo, A., Seljebakken Klausen, B., Lutnaes, B.F., Nyanhongo, G.S., Guebitz, References 271 G.M., 2010. Polymerization of lignosulfonates by the laccase-HBT (1hydroxybenzotriazole) system improves dispersibility. Bioresour. Technol. 101, 5054–5062. https://doi.org/10.1016/j.biortech.2010.01.048 Nzila, A., 2018. Biodegradation of high-molecular-weight polycyclic aromatic hydrocarbons under anaerobic conditions: Overview of studies, proposed pathways and future perspectives. Environ. Pollut. 239, 788– 802. https://doi.org/10.1016/j.envpol.2018.04.074 Okumura, R., Nakamura, Y., Sasaki, C., Asada, C., 2021. Effects of Tween series and agar additives on mycelia biomass and β-glucan production by Hericium erinaceus in submerged culture. Biomass Convers. Biorefinery 3135–3141. https://doi.org/10.1007/s13399-021-01342-y Olajuyigbe, F.M., Fatokun, C.O., Oyelere, O.M., 2018. Biodelignification of some agro-residues by Stenotrophomonas sp. CFB-09 and enhanced production of ligninolytic enzymes. Biocatal. Agric. Biotechnol. 15, 120– 130. https://doi.org/10.1016/j.bcab.2018.05.016 Olkiewicz, M., Tylkowski, B., Montornés, J.M., Garcia-Valls, R., Gulaczyk, I., 2022. Modelling of enzyme kinetics: cellulose enzymatic hydrolysis case. Phys. Sci. Rev. 7, 901–921. https://doi.org/10.1515/psr-2020-0039 Onaizi, S.A., Alshabib, M., 2021. The degradation of bisphenol A by laccase: Effect of biosurfactant addition on the reaction kinetics under various conditions. Sep. Purif. Technol. 257, 117785. https://doi.org/10.1016/j.seppur.2020.117785 Ongley, S.E., Bian, X., Neilan, B.A., Müller, R., 2013. Recent advances in the heterologous expression of microbial natural product biosynthetic pathways. Nat. Prod. Rep. 30, 1121–1138. https://doi.org/10.1039/c3np70034h Ooijkaas, L.P., Weber, F.J., Buitelaar, R.M., Tramper, J., Rinzema, A., 2000. Defined media and inert supports: their potential as solid-state fermentation production systems 18, 356–360. https://doi.org/10.1016/S0167-7799(00)01466-9 Ortner, A., Huber, D., Haske-Cornelius, O., Weber, H.K., Hofer, K., Bauer, W., Nyanhongo, G.S., Guebitz, G.M., 2015. Laccase mediated oxidation of industrial lignins: Is oxygen limiting? Process Biochem. 50, 1277–1283. References 272 https://doi.org/10.1016/j.procbio.2015.05.003 Pacheco-Ordaz, R., Wall-Medrano, A., Goñi, M.G., Ramos-ClamontMontfort, G., Ayala-Zavala, J.F., González-Aguilar, G.A., 2018. Effect of phenolic compounds on the growth of selected probiotic and pathogenic bacteria. Lett. Appl. Microbiol. 66, 25–31. https://doi.org/10.1111/lam.12814 Palma, C., Lloret, L., Sepúlveda, L., Contreras, E., 2016. Production of versatile peroxidase from Pleurotus eryngii by solid-state fermentation using agricultural residues and evaluation of its catalytic properties. Prep. Biochem. Biotechnol. 46, 200–207. https://doi.org/10.1080/10826068.2015.1084513 Pamidipati, S., Ahmed, A., 2020. A first report on competitive inhibition of laccase enzyme by lignin degradation intermediates. Folia Microbiol. (Praha). 65, 431–437. https://doi.org/10.1007/s12223-019-00765-5 Pant, D., Adholeya, A., 2007. Identification, ligninolytic enzyme activity and decolorization potential of two fungi isolated from a distillery effluent contaminated site. Water. Air. Soil Pollut. 183, 165–176. https://doi.org/10.1007/s11270-007-9366-4 Papinutti, V.L., Forchiassin, F., 2003. Optimization of manganese peroxidase and laccase production in the South American fungus Fomes sclerodermeus (Lév.) Cke. J. Ind. Microbiol. Biotechnol. 30, 536–541. https://doi.org/10.1007/s10295-003-0077-6 Pardo, E., Marín, S., Ramos, A.J., Sanchis, V., 2006. Ecophysiology of ochratoxigenic Aspergillus ochraceus and Penicillium verrucosum isolates. Predictive models for fungal spoilage prevention - A review. Food Addit. Contam. 23, 398–410. https://doi.org/10.1080/02652030500376102 Parra Guardado, A.L., Belleville, M.-P., Rostro Alanis, M. de J., Parra Saldivar, R., Sanchez-Marcano, J., 2019. Effect of redox mediators in pharmaceuticals degradation by laccase: A comparative study. Process Biochem. 78, 123–131. https://doi.org/10.1016/j.procbio.2018.12.032 Patel, A.B., Shaikh, S., Jain, K.R., Desai, C., Madamwar, D., 2020. Polycyclic aromatic hydrocarbons: Sources, toxicity, and remediation approaches. References 273 Front. Microbiol. 11. https://doi.org/10.3389/fmicb.2020.562813 Pecyna, M.J., 2015. Molekularbiologische Charakterisierung von HämThiolatund DyP-type-Peroxidasen ausgewählter Basidiomyceten. Technische Universität Dresden. https://nbnresolving.org/urn:nbn:de:bsz:14-qucosa-209428 Pecyna, M.J., Ullrich, R., Bittner, B., Clemens, A., Scheibner, K., Schubert, R., Hofrichter, M., 2009. Molecular characterization of aromatic peroxygenase from Agrocybe aegerita. Appl. Microbiol. Biotechnol. 84, 885–897. https://doi.org/10.1007/s00253-009-2000-1 Pena, R., Lú-Chau, T.A., Lema, J.M., 2012. Use of white-rot fungi for valorization of stillage from bioethanol production. Waste and Biomass Valorization 3, 295–303. https://doi.org/10.1007/s12649-012-9130-x Peter, S., Kinne, M., Ullrich, R., Kayser, G., Hofrichter, M., 2013. Epoxidation of linear, branched and cyclic alkenes catalyzed by unspecific peroxygenase. Enzyme Microb. Technol. 52, 370–376. https://doi.org/10.1016/j.enzmictec.2013.02.013 Philippoussis, A., Zervakis, G., Diamantopoulou, P., 2001. Bioconversion of agricultural lignocellulosic wastes through the cultivation of the edible mushrooms Agrocybe aegerita, Volvariella volvacea and Pleurotus spp. World J. Microbiol. Biotechnol. 17, 191–200. https://doi.org/10.1023/A:1016685530312 Pinheiro, V.E., Michelin, M., Vici, A.C., de Almeida, P.Z., Teixeira de Moraes Polizeli, M. de L., 2020. Trametes versicolor laccase production using agricultural wastes: a comparative study in Erlenmeyer flasks, bioreactor and tray. Bioprocess Biosyst. Eng. 43, 507–514. https://doi.org/10.1007/s00449-019-02245-z Piontek, K., Ullrich, R., Liers, C., Diederichs, K., Plattner, D.A., Hofrichter, M., 2010. Crystallization of a 45 kDa peroxygenase/peroxidase from the mushroom Agrocybe aegerita and structure determination by SAD utilizing only the haem iron. Acta Crystallogr. Sect. F Struct. Biol. Cryst. Commun. 66, 693–698. https://doi.org/10.1107/S1744309110013515 Pizzi, A., 2014. Types, processing and properties of bioadhesives for wood and fibers, in: Waldron, K. (Ed.), Advances in Biorefineries. Elsevier, Cambridge, UK, pp. 736–770. References 280 Su, Y.Y., Qi, Y.L., Cai, L., 2012. Induction of sporulation in plant pathogenic fungi. Mycology. https://doi.org/10.1080/21501203.2012.719042 Sun, H., Chen, G.Z., Tong, X., Yi, P.Q., 2003. A lectin with mycelia differentiation and antiphytovirus activities from the edible mushroom Agrocybe aegerita. J. Biochem. Mol. Biol. 36, 214–222. https://doi.org/10.5483/bmbrep.2003.36.2.214 Suryanarayan, S., 2003. Current industrial practice in solid state fermentations for secondary metabolite production: The Biocon India experience. Biochem. Eng. J. 13, 189–195. https://doi.org/10.1016/S1369703X(02)00131-6 Taboada-Puig, R., Eibes, G., Lloret, L., Lú-Chau, T.A., Feijoo, G., Moreira, M.T., Lema, J.M., 2016. Fostering the action of versatile peroxidase as a highly efficient biocatalyst for the removal of endocrine disrupting compounds. N. Biotechnol. 33, 187–195. https://doi.org/10.1016/j.nbt.2015.05.003 Taboada-Puig, R., Lu-Chau, T.A., Eibes, G., Feijoo, G., Moreira, M.T., Lema, J.M., 2015. Continuous removal of endocrine disruptors by versatile peroxidase using a two-stage system. Biotechnol. Prog. 31, 908–916. https://doi.org/10.1002/btpr.2116 Tan, J., Wang, C., Xiang, B., Han, R., Guo, Z., 2013. Hydrogen peroxide and nitric oxide mediated coldand dehydration-induced myo-inositol phosphate synthase that confers multiple resistances to abiotic stresses. Plant, Cell Environ. 36, 288–299. https://doi.org/10.1111/j.13653040.2012.02573.x Ten Have, R., De Thouars, R.G., Swarts, H.J., Field, J.A., 2001. Veratryl alcoholmediated oxidation of isoeugenyl acetate by lignin peroxidase. Eur. J. Biochem. 265, 1008–1014. https://doi.org/10.1046/j.14321327.1999.00808.x Teodoro, T.S., Oliveira, F. de, Poffo, C., Braga, L.P., Arbigaus, A., Rampinelli, J.R., Wisbeck, E., Bonatti-Chaves, M., Furlan, S.A., 2018. The influence of Tween 80 on laccase production by Pleurotus sajor-caju and the efficiency of crude enzyme broth in the removal of bisphenol-A. Arq. Inst. Biol. (Sao. Paulo). 85. https://doi.org/10.1590/1808-1657001022017 References 281 Terpe, K., 2006. Overview of bacterial expression systems for heterologous protein production: From molecular and biochemical fundamentals to commercial systems. Appl. Microbiol. Biotechnol. 72, 211–222. https://doi.org/10.1007/s00253-006-0465-8 Thoa, L.T.K., Thao, T.T.P., Hung, N.B., Khoo, K.S., Quang, H.T., Lan, T.T., Hoang, V.D., Park, S.-M., Ooi, C.W., Show, P.L., Huy, N.D., 2022. Biodegradation and detoxification of malachite green dye by extracellular laccase expressed from Fusarium oxysporum. Waste and Biomass Valorization 13, 2511–2518. https://doi.org/10.1007/s12649-02201692-2 Tien, M., Kirk, T.K., 1988. Lignin Peroxidase of Phanerochaete chrysosporium. Methods Enzymol. 161, 238–249. Tinoco-valencia, R., Gómez-cruz, C., Galindo, E., Serrano-carreón, L., 2014. Toward an understanding of the effects of agitation and aeration on growth and laccases production by Pleurotus ostreatus. J. Biotechnol. 177, 67–73. https://doi.org/10.1016/j.jbiotec.2014.02.013 Tonin, F., Tieves, F., Willot, S., van Troost, A., van Oosten, R., Breestraat, S., van Pelt, S., Alcalde, M., Hollmann, F., 2021. Pilot-scale production of peroxygenase from Agrocybe aegerita. Org. Process Res. Dev. 25, 1414– 1418. https://doi.org/10.1021/acs.oprd.1c00116 Torres, E., Aburto, J., 2005. Chloroperoxidase-catalyzed oxidation of 4,6dimethyldibenzothiophene as dimer complexes: Evidence for kinetic cooperativity. Arch. Biochem. Biophys. 437, 224–232. https://doi.org/10.1016/j.abb.2005.03.006 Tran, N.H., Reinhard, M., Gin, K.Y.H., 2018. Occurrence and fate of emerging contaminants in municipal wastewater treatment plants from different geographical regions-a review. Water Res. 133, 182–207. https://doi.org/10.1016/j.watres.2017.12.029 Tufail, A., Alharbi, S., Alrifai, J., Ansari, A., Price, W.E., Hai, F.I., 2021. Combining enzymatic membrane bioreactor and ultraviolet photolysis for enhanced removal of trace organic contaminants: Degradation efficiency and by-products formation. Process Saf. Environ. Prot. 145, 110–119. https://doi.org/10.1016/j.psep.2020.08.001 Tummler, K., Lubitz, T., Schelker, M., Klipp, E., 2014. New types of References 282 experimental data shape the use of enzyme kinetics for dynamic network modeling. FEBS J. 281, 549–571. https://doi.org/10.1111/febs.12525 Ullrich, R., Hofrichter, M., 2005. The haloperoxidase of the agaric fungus Agrocybe aegerita hydroxylates toluene and naphthalene. FEBS Lett. 579, 6247–6250. https://doi.org/10.1016/j.febslet.2005.10.014 Ullrich, R., Le, M.H., Nguyen, L.D., Hofrichter, M., 2005. Laccase from the medicinal mushroom Agaricus blazei: Production, purification and characterization. Appl. Microbiol. Biotechnol. 67, 357–363. https://doi.org/10.1007/s00253-004-1861-6 Ullrich, René, Nueske, J., Scheibner, K., Spantzel, J., Hofrichter, M., 2004. Novel haloperoxidase from the agaric basidiomycete Agrocybe aegerita oxidizes aryl alcohols and aldehydes. Appl. Environ. Microbiol. 70, 4575–4581. https://doi.org/10.1128/AEM.70.8.4575 Ullrich, René, Nuske, J., Scheibner, K., Spantzel, J., Hofrichter, M., 2004. Novel haloperoxidase from the agaric basidiomycete Agrocybe aegerita oxidizes aryl alcohols and aldehydes. Appl. Environ. Microbiol. 70, 4575–4581. https://doi.org/10.1128/AEM.70.8.4575-4581.2004 Ullrich, R., Poraj-Kobielska, M., Scholze, S., Halbout, C., Sandvoss, M., Pecyna, M.J., Scheibner, K., Hofrichter, M., 2018. Side chain removal from corticosteroids by unspecific peroxygenase. J. Inorg. Biochem. 183, 84–93. https://doi.org/10.1016/j.jinorgbio.2018.03.011 Umar, A., Ahmed, S., 2022. Optimization, purification and characterization of laccase from Ganoderma leucocontextum along with its phylogenetic relationship. Sci. Rep. 12, 1–14. https://doi.org/10.1038/s41598-02206111-z Umezawa, K., Niikura, M., Kojima, Y., Goodell, B., Yoshida, M., 2020. Transcriptome analysis of the brown rot fungus Gloeophyllum trabeum during lignocellulose degradation. PLoS One 15, 1–19. https://doi.org/10.1371/journal.pone.0243984 Valderrama, B., Ayala, M., Vazquez-Duhalt, R., 2002. Suicide inactivation of References 283 peroxidases and the challenge of engineering more robust enzymes. Chem. Biol. 9, 555–565. https://doi.org/10.1016/S1074-5521(02)00149-7 Valles, M., Kamaruddin, A.F., Wong, L.S., Blanford, C.F., 2020. Inhibition in multicopper oxidases: a critical review. Catal. Sci. Technol. 10, 5386– 5410. https://doi.org/10.1039/D0CY00724B van Schie, M.M.C.H., Spöring, J.D., Bocola, M., Domínguez de María, P., Rother, D., 2021. Applied biocatalysis beyond just buffers - From aqueous to unconventional media. Options and guidelines. Green Chem. 23, 3191–3206. https://doi.org/10.1039/d1gc00561h Vandenberg, L.N., Maffini, M. V., Sonnenschein, C., Rubin, B.S., Soto, A.M., 2009. Bisphenol-a and the great divide: A review of controversies in the field of endocrine disruption. Endocr. Rev. 30, 75–95. https://doi.org/10.1210/er.2008-0021 Vannini, C., Domingo, G., Marsoni, M., De Mattia, F., Labra, M., Castiglioni, S., Bracale, M., 2011. Effects of a complex mixture of therapeutic drugs on unicellular algae Pseudokirchneriella subcapitata. Aquat. Toxicol. 101, 459–465. https://doi.org/10.1016/j.aquatox.2010.10.011 Vázquez, G., Freire, S., Rodríguez-Bona, C., González, J., Antorrena, G., 1999. Structures, and reactivities with formaldehyde, of some acetosolv pine lignins. J. Wood Chem. Technol. 19, 357–378. https://doi.org/10.1080/02773819909349617 Veiter, L., Rajamanickam, V., Herwig, C., 2018. The filamentous fungal pellet—relationship between morphology and productivity. Appl. Microbiol. Biotechnol. 102, 2997–3006. https://doi.org/10.1007/s00253018-8818-7 Venkatesagowda, B., 2019. Enzymatic demethylation of lignin for potential biobased polymer applications. Fungal Biol. Rev. 33, 190–224. https://doi.org/10.1016/j.fbr.2019.06.002 Vipotnik, Z., Michelin, M., Tavares, T., 2022. Rehabilitation of a historically contaminated soil by different laccases and laccase-mediator system. J. Soils Sediments 22, 1546–1554. https://doi.org/10.1007/s11368-021-031254 Vipotnik, Z., Michelin, M., Tavares, T., 2021. Ligninolytic enzymes production References 284 during polycyclic aromatic hydrocarbons degradation: effect of soil pH, soil amendments and fungal co-cultivation. Biodegradation 32, 193–215. https://doi.org/10.1007/s10532-021-09933-2 Vrsanska, M., Buresova, A., Damborsky, P., Adam, V., 2015. Influence of different inducers on ligninolytic enzyme activities. J. Met. Nanotechnologies 3, 64–70. Wan, C., Li, Y., 2012. Fungal pretreatment of lignocellulosic biomass. Biotechnol. Adv. 30, 1447–1457. https://doi.org/10.1016/j.biotechadv.2012.03.003 Wang, D., Zhu, R., Lou, J., Baek, N., Fan, X., 2023. Plasticizer phthalate esters degradation with a laccase from Trametes versicolor: effects of TEMPO used as a mediator and estrogenic activity removal. Biodegradation. https://doi.org/10.1007/s10532-023-10030-9 Wang, F., Ai, M., Yang, G., Chen, J., Chen, X., Huang, F., 2016. Influence of carbon source on the production of extracellular ligninolytic enzymes by Phanerochaete chrysosporium. BioResources 11, 5676–5686. https://doi.org/10.15376/biores.11.3.5676-5686 Wang, F., Kuai, J., Pan, H., Wang, N., Zhu, X., 2018. Study on the demethylation of enzymatic hydrolysis lignin and the properties of lignin–epoxy resin blends. Wood Sci. Technol. 52, 1343–1357. https://doi.org/10.1007/s00226-018-1024-z Wang, M., Zhao, Y., Li, J., 2018. Demethylation and other modifications of industrial softwood kraft lignin by laccase-mediators. Holzforschung 72, 357–365. https://doi.org/10.1515/hf-2017-0096 Wang, S., Meng, X., Zhou, H., Liu, Yang, Secundo, F., Liu, Yun, 2016. Enzyme stability and activity in non-aqueous reaction systems: A mini review. Catalysts 6, 32. https://doi.org/10.3390/catal6020032 Wang, Y., Vazquez-Duhalt, R., Pickard, M.A., 2002. Purification, characterization, and chemical modification of manganese peroxidase from Bjerkandera adusta UAMH 8258. Curr. Microbiol. 45, 77–87. https://doi.org/10.1007/s00284-001-0081-x Wang, Z., Ren, D., Cheng, Y., Zhang, X., Zhang, S., Chen, W., 2022. References 285 Immobilization of laccase on chitosan functionalized halloysite nanotubes for degradation of Bisphenol A in aqueous solution: degradation mechanism and mineralization pathway. Heliyon 8, e09919. https://doi.org/10.1016/j.heliyon.2022.e09919 Wang, Z., Zhu, H., Cheng, Y., Jiang, Y., Li, Y., Huang, B., 2019. The polyubiquitin gene MrUBI4 is required for conidiation, conidial germination, and stress tolerance in the filamentous fungus Metarhizium robertsii. Genes (Basel). 10, 1–15. https://doi.org/10.3390/genes10060412 Wariishi, H., Akileswaran, L., Gold, M.H., 1988. Manganese peroxidase from the basidiomycete Phanerochaete chrysosporium: spectral characterization of the oxidized states and the catalytic cycle. Biochemistry 27, 5365–5370. https://doi.org/10.1021/bi00414a061 Webster, G.K., Chang, J.C., Heflin, J.L., 2021. Stability indicating method for polysorbate 80 in protein formulations. J. Chromatogr. Sci. 59, 706–713. https://doi.org/10.1093/chromsci/bmaa116 Wendiro, D., Wacoo, A.P., Wise, G., 2019. Identifying indigenous practices for cultivation of wild saprophytic mushrooms : responding to the need for sustainable utilization of natural resources 1–15. Wojcik, M., Miłek, J., 2016. A new method to determine optimum temperature and activation energies for enzymatic reactions. Bioprocess Biosyst. Eng. 39, 1319–1323. https://doi.org/10.1007/s00449-016-1596-7 Wong, D.W.S., 2009. Structure and action mechanism of ligninolytic enzymes. Appl. Biochem. Biotechnol. 157, 174–209. https://doi.org/10.1007/s12010008-8279-z Wong, M.K.L., Krycer, J.R., Burchfield, J.G., James, D.E., Kuncic, Z., 2015. A generalised enzyme kinetic model for predicting the behaviour of complex biochemical systems. FEBS Open Bio 5, 226–239. https://doi.org/10.1016/j.fob.2015.03.002 Wong, M.R., 2013. Evaluation of Oxygen Mass Transfer in Fungal Fermentation using Airlift/Bubble Column Bioreactors. University of Hawaiʻi at Mānoa. https://scholarspace.manoa.hawaii.edu/server/api/core/bitstreams/1927 906c-1350-42fb-9190-2dc25293b888/content References 286 Wu, V.W., Thieme, N., Huberman, L.B., Dietschmann, A., Kowbel, D.J., Lee, J., Calhoun, S., Singan, V.R., Lipzen, A., Xiong, Y., Monti, R., Blow, M.J., O’Malley, R.C., Grigoriev, I. V., Benz, J.P., Glass, N.L., 2020. The regulatory and transcriptional landscape associated with carbon utilization in a filamentous fungus. Proc. Natl. Acad. Sci. U. S. A. 117, 6003–6013. https://doi.org/10.1073/pnas.1915611117 Xu, A., Li, W., Zhang, Y., Xu, H., 2016. Eco-friendly polysorbate aqueous solvents for efficient dissolution of lignin. RSC Adv. 6, 8377–8379. https://doi.org/10.1039/c5ra24570b Xu, C., Ma, F., Zhang, X., 2009. Lignocellulose degradation and enzyme production by Irpex lacteus CD2 during solid-state fermentation of corn stover. J. Biosci. Bioeng. 108, 372–375. https://doi.org/10.1016/j.jbiosc.2009.04.023 Xu, F., 1997. Effects of redox potential and hydroxide inhibition on the pH activity profile of fungal laccases. J. Biol. Chem. 272, 924–928. https://doi.org/10.1074/jbc.272.2.924 Yadav, M., Yadav, H.S., 2015. Applications of ligninolytic enzymes to pollutants, wastewater, dyes, soil, coal, paper and polymers. Environ. Chem. Lett. 13, 309–318. https://doi.org/10.1007/s10311-015-0516-4 Yang, N., Li, D.F., Feng, L., Xiang, Y., Liu, W., Sun, H., Wang, D.C., 2009. Structural basis for the tumor cell apoptosis-inducing activity of an antitumor lectin from the edible mushroom Agrocybe aegerita. J. Mol. Biol. 387, 694–705. https://doi.org/10.1016/j.jmb.2009.02.002 Yang, Y., Xia, J., Li, J., Chu, J., Li, L., Wang, Y., Zhuang, Y., Zhang, S., 2012. A novel impeller configuration to improve fungal physiology performance and energy conservation for cephalosporin C production. J. Biotechnol. 161, 250–256. https://doi.org/10.1016/j.jbiotec.2012.07.007 Yao, M., Li, W., Duan, Z., Zhang, Y., Jia, R., 2017. Genome sequence of the white-rot fungus Irpex lacteus F17, a type strain of lignin degrader fungus. Stand. Genomic Sci. 12, 55. https://doi.org/10.1186/s40793-0170267-x References 287 Yao, Y., Zhou, G., Lin, Y., Xu, X., Yang, J., 2020. A highly thermotolerant laccase produced by Cerrena unicolor strain CGMCC 5.1011 for complete and stable malachite green decolorization. AMB Express 10, 178. https://doi.org/10.1186/s13568-020-01118-z Ye, Q., Han, Y., Zhang, J., Zhang, W., Xia, C., Li, J., 2019. Bio-based films with improved water resistance derived from soy protein isolate and stearic acid via bioconjugation. J. Clean. Prod. 214, 125–131. https://doi.org/10.1016/j.jclepro.2018.12.277 Yee, K.L., Jansen, L.E., Lajoie, C.A., Penner, M.H., Morse, L., Kelly, C.J., 2018. Furfural and 5-hydroxymethyl-furfural degradation using recombinant manganese peroxidase. Enzyme Microb. Technol. 108, 59–65. https://doi.org/10.1016/j.enzmictec.2017.08.009 Ying, G.G., Kookana, R.S., Ru, Y.J., 2002. Occurrence and fate of hormone steroids in the environment. Environ. Int. 28, 545–551. https://doi.org/10.1016/S0160-4120(02)00075-2 Yoshida, H., 1883. Chemistry of Lacquer (Urushi). J. Chem. Soc. Trans. 43, 472–486. Yu, X., Wei, Z., Lu, Z., Pei, H., Wang, H., 2019. Activation of lignin by selective oxidation: An emerging strategy for boosting lignin depolymerization to aromatics. Bioresour. Technol. 291, 121885. https://doi.org/10.1016/j.biortech.2019.121885 Yusuf, M., 2017. Agro-industrial waste materials and their recycled valueadded applications: Review, in: Handbook of Ecomaterials. Springer International Publishing, Cham, pp. 1–11. https://doi.org/10.1007/978-3319-48281-1_48-1 Zaier, H., Maktouf, S., Roussos, S., Rhouma, A., 2021. Filamentous fungi isolated from Tunisian olive mill wastes: use of solid-state fermentation for enzyme production. Not. Bot. Horti Agrobot. Cluj-Napoca 49, 1–18. https://doi.org/10.15835/nbha49112125 Zdarta, J., Nguyen, L.N., Jankowska, K., Jesionowski, T., Nghiem, L.D., 2022. A contemporary review of enzymatic applications in the remediation of emerging estrogenic compounds. Crit. Rev. Environ. Sci. Technol. 52, 2661–2690. https://doi.org/10.1080/10643389.2021.1889283 References 288 Zelena, K., Eisele, N., Berger, R.G., 2014. Escherichia coli as a production host for novel enzymes from basidiomycota. Biotechnol. Adv. 32, 1382–1395. https://doi.org/10.1016/j.biotechadv.2014.08.006 Zhang, Y., Geißen, S.U., 2010. In vitro degradation of carbamazepine and diclofenac by crude lignin peroxidase. J. Hazard. Mater. 176, 1089–1092. https://doi.org/10.1016/j.jhazmat.2009.10.133 Zhao, X., Huang, X., Yao, J., Zhou, Y., Jia, R., 2015. Fungal growth and manganese peroxidase production in a deep tray solid-state bioreactor, and in vitro decolorization of poly R-478 by MnP. J. Microbiol. Biotechnol. 25, 803–813. https://doi.org/10.4014/jmb.1410.10054 Zhou, S., Zhang, J., Ma, F., Tang, C., Tang, Q., Zhang, X., 2018. Investigation of lignocellulolytic enzymes during different growth phases of Ganoderma lucidum strain G0119 using genomic, transcriptomic and secretomic analyses. PLoS One 13, 1–20. https://doi.org/10.1371/journal.pone.0198404 Zhou, Z.-W., Liu, C.-X., Cai, C.-X., Wang, N., Yu, X.-Q., 2023. Co-immobilization of laccase and PEG modified COFs into Cu doped gel beads to achieve synergistic effect of photocatalysis and enzymatic catalysis for pollutants removal. Colloids Surfaces A Physicochem. Eng. Asp. 656, 130201. https://doi.org/10.1016/j.colsurfa.2022.130201 Zhu, P., Gu, Z., Hong, S., Lian, H., 2018. Preparation and characterization of microencapsulated LDHs with melamine-formaldehyde resin and its flame retardant application in epoxy resin. Polym. Adv. Technol. 29, 2147–2160. https://doi.org/10.1002/pat.4323 Zhu, Y., Tao, H., Janaswamy, S., Zou, F., Cui, B., Guo, L., 2021. The functionality of laccaseor peroxidase-treated potato flour: Role of interactions between protein and protein/starch. Food Chem. 341, 128082. https://doi.org/10.1016/j.foodchem.2020.128082 Zuo, S., Niu, D., Zheng, M., Jiang, D., Tian, P., Li, R., Xu, C., 2018. Effect of Irpex lacteus, Pleurotus ostreatus and Pleurotus cystidiosus pretreatment of corn stover on its improvement of the in vitro rumen fermentation. J. Sci. Food Agric. 98, 4287– 4295. https://doi.org/10.1002/jsfa.8951 LIST OF PUBLICATIONS Fungal enzymes are considered attractive biocatalysts because of their versatility and ability to catalyse multiple oxidation reactions. Their use in biotechnological ields has been widely explored in recent years, demonstrating that they can offer different advantages compared to conventional technologies. However, in view of their application, it is necessary to advance in the improvement of enzyme production. The present PhD Thesis focuses on the production of ligninolytic enzymes by valorisation of different agro-industrial wastes. The results obtained show the importance of integrating waste streams as main substrates for fungal fermentation media and the great potential of enzyme cocktails in environmental biotechnology processes.