G een Chemis y
PAPER
Ci e his: G een Chem., 2018, 20,
2829
Recei ed 12 h Feb ua y 2018,
Accep ed 8 h May 2018
DOI: 10.1039/c8gc00490k
sc.li/g eenchem
Molecula ools o selec i e eco e y and
de ec ion o lignin-de i ed molecules†
Milla Salmela, *
a
Hanna Sanma k,
b
Elena Efimo a,
a
Alexande Efimo ,
a
Vesa P. Hy önen,
c,d
U po Lamminmäki,
b
Su i San ala
a
and Ville San ala
a
The pulp and pape indus y oge he wi h lignocellulosic bio uel p oduc ion p o ides plen i ul s eams o
lignin and lignin-de i ed molecules (LDMs) ha cu en ly emain unde u ilized. The he e ogenei y and
complexi y o lignin along wi h he lack o con enien ools significan ly hampe i s u iliza ion. Selec i e
sepa a ion o hese LDMs om s eams using specific ools would allow he eco e y o a oma ic com-
pounds, as well as acili a e biological p ocesses aiming a lignin alo iza ion. To his end, he e we epo
he isola ion and cha ac e iza ion o single-chain a iable agmen (scF ) an ibodies agains e ula e, cou-
ma a e, and caffea e, which a e he molecula ep esen a i es o LDMs. Binde s o he a ge LDMs we e
en iched by in e oga ing a syn he ic scF lib a y wi h he phage display echnique. As a esul , scF
binde s specific agains each o he a ge molecules we e ob ained wi h affini ies in he mic omola
ange. The selec i i y o scF s owa ds specific LDMs was p o ed by eco e ing caffea e om simula ed
LDM solu ion, K a lignin, and ice s aw hyd olysa e samples. Fu he p oo o concep s udies wi h
model compounds demons a ed he applicabili y o an ibody-based binde s as a de ec ion ool o
moni o ing mic obial LDM con e sion. O e all, his s udy demons a es he po en ial o scF binde s as a
specific oolse o lignin compound eco e y and analysis.
In oduc ion
Cu en ly, he chemical indus y depends s ongly on c ude oil
e inemen p ocesses designed o anspo a ion uels and
ene gy p oduc ion.
1,2
The e a e, howe e , conce ns o e he
economic and en i onmen al sus ainabili y o hese
indus ies
3–5
as well as on he global sufficiency o oil depos-
i s.
6
These conce ns mo i a e us o dec ease ou eliance on
ossil uels, and he e o e mode n e ine ies equi e new aw
ma e ials independen o he oil indus y.
7
Fo hese pu poses,
lignin is conside ed a p omising eeds ock o a ple ho a o
chemicals and ma e ials. De i ed om sou ces such as he
pulp and pape indus y, as well as om he eme ging indus y
o lignocellulosic bio uel p oduc ion, lignin can p o ide an
en i onmen ally sus ainable and abundan al e na i e o he
oil-based chemical indus y.
8
Fu he mo e, lignin alo iza ion
enables he p oduc ion o bo h ine and bulk chemicals in a
bio e ine y concep .
9–12
Howe e , comme cial applica ions and
indus ial scale p oduc ion ha e been hinde ed due o he
ecalci an and he e ogeneous na u e o lignin
13
and he
p esen indus ial p ocesses disca d he lignin s eams mainly
as a was e o combus hem o hea .
Lignin is an a oma ic he e opolyme ound embedded wi h
cellulose and hemicellulose in lignocellulosic biomass. Plan s
syn hesize lignin p ima ily om 4-hyd oxyphenylp opanoids
ia oxida i e coupling, which esul s in diffe en phenyl-
p opane s uc u es in he polyme .
14
Consequen ly, complex
mix u es o high and low molecula weigh a oma ic s uc-
u es, among o he compounds, can be ound in he lignin
s eams ob ained om biomass p e- ea men p ocesses. In
addi ion, he go e ning a oma ic s uc u es o hese echnical
lignins depend no ably on he o igin o he biomass and on
he chosen ea men me hod.
15
Fo ins ance, e ula e and
couma a e a e ela i ely dominan a oma ic monome s in
lignin ac ions acqui ed om pa icula ag icul u al and
g assy o igins
15–19
and in some cases ace amoun s o caffea e
ha e also been de ec ed.
20
These a oma ic compounds a e examples o indus ially
in e es ing molecules. They possess an ioxidan p ope ies,
which make hem aluable molecules o example o he ood
indus y.
21
In addi ion, in he chemical indus y, he use o
a oma ic monome s om sus ainable sou ces is desi able,
†Elec onic supplemen a y in o ma ion (ESI) a ailable. See DOI: 10.1039/
c8gc00490k
a
Labo a o y o Chemis y and Bioenginee ing, Tampe e Uni e si y o Technology,
Ko keakoulunka u 8, 33720 Tampe e, Finland. E-mail: [email p o ec ed]
b
Depa men o Biochemis y/Bio echnology, Uni e si y o Tu ku, Tykis öka u 6 A,
6 h l ., 20520 Tu ku, Finland
c
Facul y o Medicine and Li e Sciences and BioMediTech, Uni e si y o Tampe e,
Lääkä inka u 1, 33520 Tampe e, Finland
d
Fimlab Labo a o ies, Bioka u 4, 33520 Tampe e, Finland
This jou nal is © The Royal Socie y o Chemis y 2018 G een Chem.,2018,20,2829–2839 | 2829
Open Access A icle. Published on 08 May 2018. Downloaded on 5/4/2020 6:55:20 AM.
This a icle is licensed unde a
C ea i e Commons A ibu ion 3.0 Unpo ed Licence.
View A icle Online
View Jou nal
| View Issue
hough selec i e ca aly ic modi ica ion o hese compounds is
hinde ed due o he difficul lignin ma ix hese compounds
eside in.
22
Biological sys ems, howe e , a e known o be
highly speci ic and wo k in dilu e solu ions. Selec i e binding
o hese molecules, o example, would enable he isola ion o
speci ic a oma ic compounds om a mix u e o diffe en
LDMs, which migh p o e use ul o he eco e y o small
molecules om complex liquo s,
23
dilu e s eams o om
biocon e sion p ocesses accumula ing speci ic a oma ic
compounds.
24
In na u e, ce ain bac e ia ha e he abili y o pe o m selec-
i e con e sion o a oma ic compounds om he e ogeneous
ma e ials such as lignin.
25,26
Bac e ia, such as Acine obac e
baylyi ADP1,
27
Rhodococcus jos ii,
28
and Pseudomonas
pu ida
19,20,29
can, o example, u ilize e ula e, couma a e, and
caffea e as he sole ca bon and ene gy sou ces. Mo eo e ,
me abolic enginee ing and syn he ic biology app oaches
p o ide ools o con e hese subs a es in o aluable p oduc s
such as anillin o polyhyd oxy ca bona es (PHA).
26,27,30
An
ex ended applica ion o LDM selec i e binde s could be he
speci ic and apid de ec ion o LDMs om cul u e b o hs o
he op imiza ion o biocon e sion p ocesses in ol ing lignin.
An ibodies a e p o eins ha can bind speci ically o a ge
analy es e en in complex ma ices. They a e commonly used
in diagnos ic and he apeu ic applica ions and o some
deg ee in en i onmen al analyses.
31–33
Single-chain a iable
agmen (scF ) molecules a e mode n an ibodies enginee ed
o con ain only he smalles immunoglobulin uni s equi ed
o an igen ecogni ion.
34
The scF s can be p oduced in
simple bac e ial exp ession sys ems such as Esche ichia coli,
and hey can easily be u he gene ically modi ied, o
example, o endow hem wi h common labelling p o eins
such as g een luo escen p o ein (GFP) o alkaline phospha-
ase (AP). The ools o gene ic enginee ing can be used o
gene a e highly di e se syn he ic epe oi es o scF s, om
whe e binde s agains a ge s o in e es can be isola ed by
echniques such as he phage display echnique. An igen-
binding si e a chi ec u es o he an ibodies in hese lib a ies
can be designed o a o he ecogni ion o ce ain ypes o
an igens diffe ing o ins ance in e ms o size.
35
Wi h a sui -
able an ibody lib a y, speci ic binde s can be ob ained e en
o e y small hap ens, such as ska ole (MW = 131.2 Da),
36
which ypically a e e y difficul a ge s o an ibody de elop-
men . Thus, scF binde s o small molecules ha a e ele-
an o he lignin indus y, such as hyd oxycinnama es,
migh also be ound om syn he ic an ibody lib a ies, e en
hough hey diffe only in he deg ee o me hoxyla ion and
he numbe o hyd oxyl g oups.
In he p esen s udy, an ibodies capable o speci ic ecog-
ni ion o lignin compounds e ula e, couma a e, and caffea e
we e isola ed and cha ac e ized. As a p oo o concep expe i-
men , caffea e was eco e ed om ea ed lignin con aining
liquo s by a as and simple column pu i ica ion me hod u iliz-
ing a caffea e speci ic scF . The addi ional applica ion o he
scF binde s was demons a ed wi h a highly speci ic caffea e-
binding an ibody by moni o ing he caffea e u iliza ion o
A. baylyi ADP1 by a simple compe i i e enzyme-linked
immunoso ben assay (ELISA).
Ma e ials and me hods
De ailed desc ip ions o he me hods o he chemical syn-
hesis o bio inyla ed a ge molecules, biopanning, cloning
and ecombinan p o ein exp ession a e desc ibed in he ESI.†
Phages, s ains, plasmids, enzymes and bio inyla ed hap ens
The binde s o e ula e, couma a e and caffea e we e isola ed
om a syn he ic an ibody phage lib a y (ScF M) desc ibed by
Huo inen e al.
35
E. coli XL1-Blue (S a agene, USA) cells we e
used o phage in ec ion and p oduc ion, cloning, and p o ein
exp ession. The VCS-M13 helpe phage (S a agene) was used
o escue he phagemid ca ying phage in biopanning. S iI
es ic ion si es we e used o clone he scF genes om he
pEB32x phagemid o exp ession ec o s pLK06H
35
and
pLK04.
37
The pLK06H ec o con ains an ampicillin esis ance
ma ke , as well as an addi ional his idine ag and wo S iI si es
ups eam o an AP gene, whe eas he pLK04H ec o lacks he
AP gene. All o he enzymes and buffe s o cloning we e pu -
chased om The mo Scien i ic, USA. Caffea e consump ion
s udies we e conduc ed wi h A. baylyi ADP1 (DSM 24194,
DSMZ, Ge many).
The bio inyla ed a ge molecules ( a ge hap ens) we e con-
s uc ed by chemical syn hesis. Th ee diffe en conjuga es
we e manu ac u ed ca ying e ula e, couma a e, o caffea e.
Each o he molecules con ained a linke wi h 8 PEG uni s and
a bio in moie y. Cons uc ion o he bio inyla ed a ge mole-
cules (bio in-PEG-[caffea e/ e ula e/couma a e]) is desc ibed in
de ail in he ESI.†
Media componen s
E. coli XL-1 Blue cells we e g own in Supe B o h (SB) medium
( yp one 30 g L
−1
, yeas ex ac 20 g L
−1
, MOPS 10 g L
−1
, and
pH 7) and supplemen ed wi h 1%
w/
glucose and an ibio ics
( e acycline 10 µg mL
−1
, ampicillin 100 µg ml
−1
, chlo am-
phenicol 25 µg mL
−1
o kanamycin 30 µg mL
−1
)whenapp op i-
a e. A. baylyi ADP1 cells we e g own in minimal sal medium
MA/9 (Na
2
HPO·2H
2
O5.518gL
−1
,KH
2
OPO
4
3.402 g L
−1
,NH
4
Cl
0.963 g L
−1
, ni ilo iace ic acid 0.008 g L
−1
, NaCl 1 g L
−1
,
FeCl
3
0.001 mg L
−1
, MgSO
4
240 mg L
−1
, and CaCl
2
11 mg L
−1
)
supplemen ed wi h 10 mM ace a e and 10 mM caffea e. Fo
media p epa a ion, caffeic acid (Sigma) was i s dissol ed in
MA/9 media wi hou supplemen a ion (pH is adjus ed o 8.5
wi h NaOH) and il e ed h ough 0.2 µm il e s. The pH o he
s ock solu ion dec eased a e he caffeic acid addi ion close o
neu al (pH 6.5–7).
Phage en ichmen immunoassay
The scF -phage lib a y scF M was independen ly en iched
agains he diffe en bio inyla ed a ge molecules (bio in-PEG-
[caffea e/ e ula e/couma a e]) in h ee consecu i e ounds o
affini y-based biopanning using supe pa amagne ic beads.
Pape G een Chemis y
2830 |G een Chem.,2018,20,2829–2839 This jou nal is © The Royal Socie y o Chemis y 2018
Open Access A icle. Published on 08 May 2018. Downloaded on 5/4/2020 6:55:20 AM.
This a icle is licensed unde a
C ea i e Commons A ibu ion 3.0 Unpo ed Licence.
View A icle Online
A mo e de ailed desc ip ion o he biopanning p ocess is offe ed
in he ESI.†Subsequen ly, a ime- esol ed luo ome y-based
phage immunoassay was used o e i y he en ichmen o he
a ge speci ic scF -phage. All o he assay s eps we e con-
duc ed in a wo king olume o 200 µL o assay buffe (50 mM
T is-HCl, 150 mM NaCl, 0.1% BSA, 0.0.1% Tween20, pH 8.0) a
oom empe a u e wi h slow shaking on a p ewashed 96-well
s ep a idin (SA) pla e (Kai ogen, Finland). All washing s eps
we e un ou imes wi h a Del ia pla e washe (Pe kinElme ,
Finland) using wash buffe (5 mM T is-HCl, 150 mM NaCl,
0.01% Tween20, pH 8.0). Fo a ge molecule immobiliza ion,
a sa u a ing concen a ion o bio inyla ed a ge molecules
(bio in-PEG-[caffea e/ e ula e/couma a e]) was added in he
s ep a idin-well ollowed by 30 minu es o incuba ion and
washing. Aliquo s o 1 × 10
9
c u mL
−1
phage om all he
panning eac ions we e added independen ly on he pla e,
which was incuba ed o 1 hou and hen washed. Then, 25 ng
o eu opium-labelled an i-M13 phage an ibody 9E7
(Depa men o Biochemis y/Bio echnology, Uni e si y o
Tu ku, Finland) was added and he eac ions we e incuba ed
o 30 min be o e washing. The ea e , DELFIA enhancemen
solu ion (Pe kinElme ) was added, incuba ed o 10 minu es
and hen he luo escence signals we e measu ed wi h ime-
esol ed luo escence measu emen (1420 Vic o Mul ilabel
Coun e (Pe kinElme ) p og am Eu opium). All samples we e
un as duplica e. The wells wi hou he a ge molecules we e
used as con ols.
P ima y, seconda y and compe i i e AP-ELISA sc eening assays
The en iched scF s genes we e cloned om he phagemid
ec o as a pool in o he ec o pLK06H. Depending on he
a ge , 100–200 andomly chosen colonies we e hen picked
om he ans o ma ion pla e o exp ess he chosen clones as
scF -AP- usions. La e on, ce ain scF s we e also exp essed in
he single chain o m using he ec o pLK04H. De ailed
desc ip ions o cloning, AP-scF and scF lysa e p epa a ion as
well as p o ein exp ession and pu i ica ion a e offe ed in he
ESI.†Assessmen s o he ini ial binding o he exp essed
100–200 scF s owa ds hei a ge molecules we e conduc ed
by AP-ELISA. The assay and wash condi ions we e simila o
he phage immunoassay. AP-scF lysa es we e added on he
immobilized molecules in a inal dilu ion o 1 : 10, incuba ed
o 1 hou and washed. Then, 100 µL o 1 mg ml
−1
o 4-ni o-
phenyl phospha e disodium sal hexahyd a e (pNPP) (Sigma-
Ald ich) in pNNP buffe (500 mM T is, 200 mM NaCl, 10 mM
MgCl
2
, pH 9.0) was added in o he wells and incuba ed o
40 min. The abso bance was measu ed wi h a Mul iskan
Ascen pla e eade (The moLabsys ems, Finland) a a wa e-
leng h o 405 nm. The binde s en iched agains couma a e
and e ula e we e sc eened agains bo h couma a e and e u-
la e, whe eas caffea e binde s we e sc eened agains caffea e
and e ula e.
Based on he ini ial sc eening, 18 o 21 clones we e chosen
o a seconda y sc eening. The assay was he same as he
ini ial sc eening, excep ha he eshly p epa ed lysa es we e
dilu ed o a inal concen a ion o 1 : 2, un as duplica es and
pNPP solu ion (Sigma-Ald ich, #P7998) was applied as he
eac an . Using he highes signal- o-backg ound a io and
minimal c oss- eac i i y as he c i e ia, se en scF -APs pe
a ge molecule we e chosen o sequencing.
A compe i i e AP-ELISA was conduc ed o scF -APs wi h
unique sequences in o de o con i m he ecogni ion o he
non-bio inyla ed o ms o he a ge molecules. In his assay,
he lysa es we e incuba ed oge he wi h he bio inyla ed a ge
molecule and inc easing concen a ions o he co esponding
ee acid (0.125–64 µM) o 1–2 h. Then, he mix u e was ans-
e ed o SA-coa ed pla es and incuba ed o 10 minu es. A e
washing he wells, pNPP solu ion was added and he pla es
we e incuba ed o 1 h. The abso bance was measu ed a
405 nm. The samples we e un as iplica e. La e on, simila
assays we e conduc ed wi h pu i ied scF -APs. Fo a caffeic
acid binde a compe i i e assay was also pe o med agains
ca echol and 4-e hylca echol.
E alua ion o binding speci ici y and kine ics by biolaye
in e e ome y
A Fo ebio Oc e RED384 ins umen equipped wi h 16 pa allel
SA-coa ed op ical biosenso s (Fo ebio, Pall Li e Sciences,
Menlo Pa k, USA) was used o u he cha ac e ize he binding
p ope ies o he selec ed binde s. The da a collec ed we e p o-
cessed and analyzed using he Oc e Da a Analysis So wa e
( e sion 7.1). Fo he pu i ied binde s, buffe exchange o
Oc e kine ic buffe (1 mg ml
−1
bo ine se um albumin (BSA),
0.02% Tween 20, 0.05% NaN
3
in PBS) was ca ied ou using
Illus a NAP-10 columns (GE Heal hca e Li e Sciences, USA)
acco ding o he manu ac u e ’s ins uc ions. Fo scF
samples, kine ic buffe was supplemen ed wi h 8% glyce ol.
The SA biosenso s we e soaked p io o un o
15–60 minu es in ITC-buffe (50 mM NaPO
4
, 100 mM NaCl,
pH 7.4). The s i ing speed was se a 500 pm and he em-
pe a u e was a 27 °C. Black, il ed-bo om 384-well pla es
(Fo ebio, Pall Li e Sciences) we e used wi h 80 µL buffe /
sample olumes. The baseline o he senso s was eco ded in
ITC-buffe o 60 seconds. The ea e , he syn hesized ligands
(∼100 µM bio in-PEG-[caffea e/ e ula e/couma a e] in ITC-
buffe con aining 10% e hanol) we e a ached o he senso s
in a 300 s ac i a ion s ep, ollowed by b ie washing and
quenching (sa u a ion o he ee s ep a idins wi h 17 µg ml
−1
bio in in ITC-buffe con aining 10% e hanol) s eps o 60
seconds. The baseline o he senso s in he kine ic buffe was
eco ded o 300 s. The associa ion and dissocia ion o he
scF samples we e measu ed o 600 s. Senso egene a ion
wi h 1 M ace ic acid (pH 2) ollowed by a wash s ep wi h
kine ic buffe was ca ied ou when app op ia e. Senso s
wi hou immobilized ligands and unc ionalized senso s
soaked in an ibody- ee kine ic buffe we e used as con ols.
The effec o ee acids on he dissocia ion o he scF -APs
bound o he unc ionalized biosenso su ace (CAF_3, CAF_5,
FER_9, FER_78, COU_12, and COU_74) was s udied by expos-
ing he scF -AP-soaked senso s o ee caffea e, e ula e, and
couma a e (∼500 µM in kine ic buffe con aining 10%
e hanol). The concen a ions o scF -APs we e 1.2, 1.5, 1.6, 1.6,
G een Chemis y Pape
This jou nal is © The Royal Socie y o Chemis y 2018 G een Chem.,2018,20,2829–2839 | 2831
Open Access A icle. Published on 08 May 2018. Downloaded on 5/4/2020 6:55:20 AM.
This a icle is licensed unde a
C ea i e Commons A ibu ion 3.0 Unpo ed Licence.
View A icle Online
1.5, and 1.6 mg mL
−1
o CAF_3, CAF_5, FER_9, FER_78,
COU_12, and COU_74, espec i ely. The dissocia ion o he
complex in he p esence o each acid o blank kine ic buffe
was hen eco ded. The speci ici y owa ds he acids was de e -
mined by calcula ing he dec ease o he su ace dep h in
nanome e s du ing he dissocia ion s ep. The dec ease was
p opo ioned o ha wi h he blank buffe and no malized.
Fo de e mining he affini ies o he scF s CAF_3s, FER_9s,
and COU_12s owa ds he hap ens, he samples we e dilu ed
o concen a ions o 0.004, 0.016, 0.06, and 0.25 mg mL
−1
. The
biosenso analysis was ca ied ou o each scF sample as
desc ibed abo e, bu in he absence o he compe ing ee
ligand. Local pa ial i (20 s om he beginning o he associ-
a ion and 60 s om he beginning o he dissocia ion phase)
was employed o de e mine he binding affini ies. The
affini ies we e calcula ed as a e ages o he alues de e mined
o each concen a ion.
P epa a ion o scF -based LDM-sepa a ion column
Fo selec i e LDM eco e y, a column coupled wi h he caffea e
binding scF (CAF_3) was assembled. Buffe exchange o pu i-
ied scF CAF_3 was ca ied ou wi h NAP buffe -exchange
columns o coupling buffe (0.2 M NaHCO3, 0.5 M NaCl, pH
8.3) and glyce ol was added o a inal concen a ion o 8%.
The buffe -exchanged scF s we e coupled o a sepha ose
ma ix, in which he coupling occu s ia co alen binding
be ween he lysine esidues o he an ibodies and he NHS-
g oups o he sepha ose ma ix making he scF -coupled bed
eusable. The coupling was conduc ed acco ding o he manu-
ac u e ’s ins uc ions using 0.4 mg o an ibodies pe ml bed
olume, le o e nigh a 4 °C and blocked wi h 0.1 M T is-
HCl, pH 8.5. The eac ion mix u e was washed i e imes wi h
0.1 M T is-HCl (pH8.5) and 0.1 M ace a e buffe , 0.5 M NaCl
(pH 4.5). The coupled ma ix was packed in o a column unde
g a i a ion and used o caffea e eco e y s udies. A o al
olume o 1 mL o sepha ose ma ix was used o he coupling
esul ing in a packed column bed o 1 ml.
Caffea e eco e y wi h scF -based column
Fo he quali a i e selec i i y s udies o he scF CAF_3s, ou
diffe en samples (caffea e, simula ed LDM mix u e, K a
lignin and ice s aw hyd olysa e) spiked wi h caffea e, couma-
a e and e ula e we e pou ed h ough he p epa ed column
and 1 mL o he ac ion samples was analyzed. Each sample
was dilu ed in assay buffe in o a o al olume o 5 mL. The
column was equilib a ed wi h assay buffe (50 mM T is-HCl,
150 mM NaCl, pH 8.0) p io o each expe imen . The samples
we e washed wi h 10 ml o wash buffe (5 mM T is-HCl,
150 mM NaCl, pH 8.0) and elu ed wi h 3 ml o glycine buffe
(pH 2.2). The collec ed ac ions we e analyzed wi h HPLC
(Agilen 1100 se ies, Hewle Packa d, Ge many) equipped wi h
a as acid H + column (Phenomex, USA), a degasse (G1322A)
and an UV-de ec o (G1315A) using 0.005 N H
2
SO
4
as he
eluen . The pump (G1211A) low was adjus ed o 1 ml min
−1
,
he column empe a u e o 80 °C, and peaks we e iden i ied a
a wa eleng h o 310 nm by compa ing he e en ion ime and
spec al p o ile wi h s anda ds p epa ed o couma a e,
caffea e and e ula e. Fo he spiked K a lignin and ice s aw
hyd olysa e, he samples we e analyzed h oughou he spec-
um o 210–900 nm.
To examine he column’s abili y o cap u e caffea e, 50 µM
o caffea e solu ion was passed h ough he column. The
ini ial caffea e eco e y es was e un wi h he same column
a e e-equilib a ion wi h assay buffe . The same column was
used o s udy caffea e eco e y o a simula ed mix u e o
LDMs con aining 1–2mMo caffea e, e ula e and couma a e
(Sigma). To u he es he column’s capabili y o pu i y
LDMs, 0.7 mg o wa e soluble low sul ona e con aining K a
lignin (Sigma) supplemen ed wi h caffea e, couma a e and e -
ula e (1–2 mM each) was used. Due o he high molecula
weigh o he K a lignin (a e age o 10 000 g mol
−1
)–and he
appa en lack o soluble acids ha could be iden i ied wi h he
chosen HPLC equipmen –a es wi h ice s aw hyd olysa e
was also conduc ed. In his expe imen , 50 µM o caffea e, cou-
ma a e and e ula e we e mixed wi h 300 mg o eeze-d ied
ice s aw hyd olysa e (desc ibed by Kannis o e al., 2015),
38
and
passed h ough he column. Addi ionally, quan i a i e caffea e
eco e y was s udied as abo e wi h a educed washing olume
(3 ml) using 2.5 µg o caffea e o 2.6 µg o caffea e dissol ed
wi h 3–5 µg o couma a e and e ula e. Caffea e eco e y pe -
cen ages we e calcula ed om he elu ion ac ions.
Caffea e consump ion s udies
The applicabili y o he scF -AP-based immunoassay as a
moni o ing ool o subs a e con e sion s udies was es ed
wi h he caffea e binde designa ed as CAF_3. Fo hese pu -
poses, A. baylyi ADP1 was p e-g own o e nigh on MA/9
minimal sal media supplemen ed wi h 10 mM Na-ace a e
and 10 mM caffea e. F om he p e-cul u e, 50 ml cul i a ion
(same as p e-cul u e) was inocula ed o he ini ial OD
600
o
0.02, incuba ed a 30 °C and shaken a 300 pm. The samples
we e collec ed e e y 2 h o a du a ion o 12 hou s. The g ow h
was moni o ed wi h op ical densi y measu emen s and 1 ml o
cul u e supe na an was s o ed a −20 °C o subs a e
analysis.
Caffea e deple ion was analyzed wi h compe i i e AP-ELISA.
0.1 µM o bio inyla ed caffea e and 14 µg mL
−1
o he caffea e
binde CAF_3 we e mixed oge he wi h a 1 : 625 dilu ion o
he collec ed samples in a inal olume o 150 µL. F ee caffeic
acid concen a ions anging om 1 µM o 32 µM we e used o
he calib a ion cu e.
To con e he ELISA abso bance alues o mM, a 4 PL
cu e i
39
was applied on he calib a ion cu e. Eqn (1) was
used o sol e he sample concen a ion by eplacing λwi h he
abso bance alue and sol ing x om he equa ion. The alues
c(in lec ion poin ) and b(coefficien o slope s eepness) we e
app oxima ed wi h he Ma lab 2016 so wa e da a i ing ool.
Fo da a i ing, he leas squa es me hod was used.
λ¼λmin þðλmax λminÞ
1þx
c

b
 ð1Þ
Pape G een Chemis y
2832 |G een Chem.,2018,20,2829–2839 This jou nal is © The Royal Socie y o Chemis y 2018
Open Access A icle. Published on 08 May 2018. Downloaded on 5/4/2020 6:55:20 AM.
This a icle is licensed unde a
C ea i e Commons A ibu ion 3.0 Unpo ed Licence.
View A icle Online
λ, he abso bance signal (y-axis), 405 nm; λ
min
, he minimum
eco ded abso bance signal; λ
max
, he maximum eco ded
abso bance signal; x, [ ee caffeic acid] (x-axis); c, he in lec ion
poin ; b, he coefficien o slope s eepness.
Addi ional caffea e concen a ion measu emen s we e
conduc ed wi h UV-Vis a 280 nm (NanoD op 2000
Spec opho ome e , The mo Fische Scien i ic). The ace a e
concen a ions we e measu ed wi h a HPLC (Shimadzu, USA)
equipped wi h an RID-10A de ec o , an SIL-20AC HT au o-
sample and a DGU-20A3 degasse . The empe a u e o he
Resex™RHM-Monosaccha ide H + column (Phenomex, USA)
was adjus ed o 25 °C and he low a e o 0.6 mL min
−1
.
0.01 N H
2
SO
4
was used as he mobile phase.
Resul s and discussion
In p inciple, phage displayed scF -lib a ies p o ide a po en ial
sou ce o binde s owa ds any molecule o in e es . LDMs such
as hyd oxycinnama es a e he molecules o in e es due o
hei po en ial use as building blocks o he chemical indus-
y o as subs a es in bio echnological p ocesses. Thei selec-
i e con e sion o aluable end p oduc s is possible by ce ain
mic oo ganisms, e en hough o many o ganisms used in
cu en bio e ine y p ocesses e en low concen a ions o hese
molecules can be oxic owing o he an ioxidan na u e o he
molecules.
23
In addi ion o hei po en ial as subs a es o
biocon e sion p ocesses, a oma ic LDMs a e also alued as
la o s, ag ances and an ioxidan s.
21,40
As hese compounds
a e ound in he e ogeneous mix u es in ea ed lignin ac-
ions, a sys em a ge ing hese highly simila molecules
speci ically and di ec ly om hyd olysa es could p o e use ul
o he eco e y o pa icula LDMs. Such eco e y migh be
use ul also in cases, whe e he he e ogeneous LDMs a e con-
e ed in o speci ied a oma ics h ough mic obial p ocesses.
In his s udy, we used a syn he ic scF M phage lib a y,
specially designed o a o he ecogni ion o low molecula
weigh compounds o selec binde s agains h ee s uc u ally
ela ed bio inyla ed a ge s (LDMs) wi h a e y low molecula
weigh . The bio inyla ed a ge -molecule conjuga es o couma-
a e, e ula e and caffea e we e chemically syn hesized o ou
LDM-binde de elopmen . Success ul an ibody de elopmen
equi es conjuga es wi h unambiguously de ined s uc u es,
oge he wi h an app op ia ely spaced linke be ween he
bio in moie y and he a ge molecule. Wi hou hese conju-
ga es, i would be necessa y o use immobilized ca ie p o-
eins, such as BSA, du ing an ibody selec ion. Typically, he
a ge hap ens a ach a iably a ound he ca ie p o ein esul -
ing in diffe en epi ope s uc u es. Undesi ably, his phenom-
enon p oduces a mo e miscellaneous g oup o binde s,
whe eas bio inyla ed conjuga es p oduce binde s o mo e
uni o m quali y.
En ichmen o LDM speci ic scF pools
Highly speci ic scF s owa ds h ee common LDMs we e iso-
la ed om a o al numbe o 6 × 10
9
independen clones
s o ed in a scF M phage-lib a y, and used in a no el app oach
o eco e , de ec , and quan i y LDMs. These scF s ac as
binde s owa ds phenolic acids, couma a e, e ula e and
caffea e. F om an immunoassay pe spec i e, he phenolic acid
concen a ions o lignin hyd olysa es
19,20
a e a he high.
Consequen ly, scF s wi h a mode a e affini y can be success-
ully used as binde s, howe e high speci ici y is necessa y due
o he a iabili y o compounds p esen in echnical lignins.
The phages wi h he desi ed p ope ies we e en iched wi h
a ge -molecule coa ed magne ic beads. To minimize he
unspeci ic binding o he pa amagne ic bead ma e ial, he
s ep a idine-coa ed beads we e changed o neu a idin (NA)
in he second ound and back o SA in he hi d ound.
Acco ding o he ob ained da a om he phage immunoassay,
e ula e and couma a e binde s we e en iched in he 2
nd
and
3
d
panning ounds wi hou signi ican unspeci ic binding o
SA (Fig. 1). Fo he caffea e binding phage also unspeci ic
binding o SA occu ed. Wi hin all o he samples, no u he
en ichmen was obse ed be ween he 2
nd
and 3
d
ounds,
hough he o e all yield o he phage inc eased owa ds he 3
d
ound.
Fig. 1 The esul s o phage immunoassay. The ba s ep esen he ela i e fluo escence signals (RFU) ob ained om he assay. Whi e ba s = phages
incuba ed wi h he a ge molecule immobilized on an SA-coa ed mic o i e well; g ey ba s = backg ound signal om he SA-well wi hou he a ge
molecule.
G een Chemis y Pape
This jou nal is © The Royal Socie y o Chemis y 2018 G een Chem.,2018,20,2829–2839 | 2833
Open Access A icle. Published on 08 May 2018. Downloaded on 5/4/2020 6:55:20 AM.
This a icle is licensed unde a
C ea i e Commons A ibu ion 3.0 Unpo ed Licence.
View A icle Online

LDM posi i e clones
A e phage en ichmen , as he phage pool con ains scF s
wi h a ying binding cha ac e is ics, he binde s wi h he
desi ed cha ac e is ics we e sc eened. An immunoassay
(AP-ELISA) was used o sc een 96–192 andomly chosen scF -
AP clones pe a ge molecule o ecogni ion and diffe en-
ia ion be ween simila molecules. The ini ial sc eening
e ealed 86 posi i e clones ou o 96 clones o e ula e and
84posi i eclonesou o 96clones o couma a e(ESI
Fig. 7–9†). In e es ingly, ac i e binde s agains caffea e we e
also ound wi h a ela i ely high equency (56 posi i e clones
ou o 192 clones), despi e he appa en en ichmen o he
s ep a idin in his panning. A clone was de e mined posi i e
when he abso bance signal o he well con aining he a ge
molecule was highe han ha o he bio in sa u a ed back-
g ound con ol wells (= S/B > 1.5). F om hese posi i e clones,
o each a ge molecule, app oxima ely 20 clones we e
chosen o u he alida ion based on he S/B and low c oss-
binding signal (ESI Fig. 7–9†).
Acco ding o he seconda y sc eening, o e ula e a ge ed
binde s some deg ee o c oss-binding occu ed wi h he
majo i y o he clones (16/20 clones), hough e ula e speci ic
clones we e also ob ained (4/20 clones). The speci ic ecog-
ni ion abili y o mos o he binde s a ge ed agains caffea e
and couma a e s uc u es was e i ied by he AP-ELISA. Nea ly
all o he clones selec ed agains couma a e showed a low
backg ound as well as low c oss-binding (19/21 clones),
hough 1/21 clones wi h c oss-binding o e ula e and 1/21
clones wi h no signal a all we e obse ed. Mos o he clones
selec ed agains caffea e showed selec i e binding wi hou
c oss-binding (12/18 clones), hough 3/18 clones indica ed a
high backg ound and c oss-binding, whe eas 3/18 clones
showed no signal a all (ESI Fig. 10–12†).
Cha ac e iza ion o he LDM speci ic ScF -APs and scF s
Fu he scF cha ac e iza ion included sequencing o unique
clones as well as binding speci ici y and affini y s udies. Se en
o eigh clones pe a ge molecule wi h he highes speci ic
signal –compa ed o he backg ound and c oss-signals acco d-
ing o he seconda y sc eening –we e chosen o be sequenced.
The sequencing da a (ESI Table 1†) e ealed 2/8 unique
sequences among binde s a ge ed agains caffea e and 3/7 o
binde s a ge ed agains e ula e, whe eas o binde s a ge ed
agains couma a e 6/7 sequences we e unique.
Speci ici y. Six unique clones we e chosen o u he s udies
based on hei pe o mance in he compe i i e assay, i.e. wo
clones pe a ge molecule ha showed he mos signi ican
esponse (dec ease in signal) upon he addi ion o he co es-
ponding ee acid (ESI Fig. 13†). These clones included wo
binde s agains each hap en, designa ed as CAF_3 and CAF_5
o caffea e, FER_9 and FER_78 o e ula e, and COU_12 and
COU_74 o couma a e. These six AP- usion an ibodies we e
exp essed and pu i ied. The pu i y and co ec size (∼75 kDa)
o he usion an ibodies we e con i med by SDS-PAGE. Speci ic
binding cha ac e is ics o he a ge hyd oxycinnama es we e
u he s udied by biolaye in e e ome y. The binding and
dissocia ion kine ics o he an ibodies we e de e mined using
SA senso s unc ionalized wi h bio inyla ed a ge LDMs. Fo
each binde , he p esence o he cogna e ee acid in he dis-
socia ion phase was ound o accele a e he dissocia ion o he
an ibodies om he senso -bound bio inyla ed ligands com-
pa ed o he ligand- ee buffe (Table 1). The p esence o he
o he ligands had a less p onounced effec on he dissocia ion
kine ics.
All he binde s showed p e e ence owa ds hei cogna e
a ge hap ens, al hough clea diffe ences in speci ici ies we e
obse ed. The caffea e binde s CAF_3 and CAF_5 showed he
highes speci ici y, whe eas o he e ula e and couma a e
binde s also sligh c oss-binding occu ed. In he AP-ELISA,
he couma a e binde s showed e y li le indica ion o c oss-
binding o he bio inyla ed e ula e, whe eas he binding p o-
iles o caffea e and e ula e binde s indica ed simila beha io
agains he bio inyla ed and ee o m o he a ge ed mole-
cule. In s udies whe e single model compounds a e used, he
c oss- eac i i y wi h o he simila compounds is no an issue
and his ype o de ec ion is applicable e en o quan i a i e
con e sion s udies. Howe e , he sligh c oss-binding migh
p o e p oblema ic when mo e complex lignin s uc u es a e
used as a subs a e o i mul iple hyd oxycinnama es a e moni-
o ed a he same ime. To o e come his obs acle, he ools o
gene ic enginee ing can be used o u he op imize he
binding cha ac e is ics o he ob ained an ibodies. In addi ion,
a gene ic binde , which could iden i y all o he hyd oxycinna-
Table 1 Specifici y o he binde s agains he a ge hyd oxycinnama es, caffea e, e ula e, and couma a e. The specifici y was de e mined by com-
pa ing he an ibody dissocia ion om he bio inyla ed hap en in he p esence o he compe i i e ee acid obse ed a 600 seconds o he dis-
socia ion obse ed in he absence o he compe ing ligand
Binde Ta ge an igen
Rela i e dissocia ion
No compe i o F ee caffea e F ee e ula e F ee couma a e
CAF_3 Caffea e 1.0 2.0 1.0 1.0
CAF_5 1.0 1.2 0.8 0.9
FER_9 Fe ula e 1.0 1.2 1.5 1.1
FER_78 1.0 1.3 1.3 1.1
COU_12 Couma a e 1.0 1.6 1.4 1.7
COU_74 1.0 1.6 1.5 1.7
Pape G een Chemis y
2834 |G een Chem.,2018,20,2829–2839 This jou nal is © The Royal Socie y o Chemis y 2018
Open Access A icle. Published on 08 May 2018. Downloaded on 5/4/2020 6:55:20 AM.
This a icle is licensed unde a
C ea i e Commons A ibu ion 3.0 Unpo ed Licence.
View A icle Online
ma es equally and simul aneously, migh also be use ul. Such
gene ic binde s ha e p e iously been de eloped o molecules
such as sulphonamides and luo oquinolones.
41,42
None heless, he speci ic beha io o he caffea e binde
CAF_3 was clea ly p o ed in hese expe imen s indica ing he
applicabili y o scF s as speci ic ools in lignin esea ch.
Based on he speci ici y p o ile, one AP-scF binde agains
each a ge LDM (CAF_3, FER_9, and COU_12) was selec ed o
addi ional compe i i e AP ELISA expe imen s. All o he
selec ed binde s ecognized also he non-bio inyla ed ( ee
acid) o m o he molecule (Fig. 2), simila o he biolaye
in e e ome y expe imen s, e en hough bio inyla ed a ge
molecules we e used in he panning and sc eening s eps. This
implies ha he pa a ope–epi ope pai occu s a he a oma ic
ing s uc u e o he molecule. This claim is u he suppo ed
by he ac ha he diffe en hyd oxycinnama es –diffe ing
only in he deg ee o me hoxyla ion o in he numbe o
hyd oxyl g oups ound in he a oma ic ing s uc u es –we e
ecognized o a lesse deg ee when compa ed o he a ge
molecule in he c oss-binding expe imen s. Addi ionally, he
CAF_3 binde is speci ic o he caffeic acid s uc u e, includ-
ing he alkyl chain o he molecule. Ca echol and 4-e hylca e-
col ha e simila a oma ic subs i u ion o caffeic acid diffe ing
only in he alkyl subs i uen s. In he p esence o ca echol o
4-e hylca echol, he scF CAF_3 showed no compe i i e
binding o hese compounds, in con as o he ee caffeic
acid (ESI 14†). Acco ding o he da a collec ed om he com-
pe i i e immunoassay, he IC50 alues we e app oxima ely
3 µM o he caffea e binde , 7 µM o he couma a e binde
and 17 µM o he e ula e binde . The eliable ecogni ion o
hese scF -AP binde s unde he chosen condi ions spans in
he egion o 1–20 µM.
No ably, wi h mul iple sc eening and selec ion me hods,
binde s wi h speci ic binding p o iles owa ds he a ge mole-
cules we e success ully ob ained. Rega dless o he high back-
g ound in he phage immunoassay, i was also possible o
isola e a highly speci ic an ibody o caffea e, which could dis-
inguish be ween he hyd oxyl and me hoxy g oups o e ula e
and couma a e.
Affini y. In o de o de e mine he affini ies o he binde s,
hey we e p oduced as scF s wi hou he AP usion; due o he
dime ic na u e o AP, he affini ies canno be eliably de e -
mined o an ibody-AP usions. The binde s CAF_3, FER_9,
and COU_12 we e cloned o he ec o pLK04H, exp essed, and
pu i ied. The pu i y and size (∼27 kDa) we e con i med by
SDS-PAGE. The affini ies o he esul ing scF s, designa ed as
CAF_3s, FER_9s, and COU_12s, espec i ely, we e de e mined
by biolaye in e e ome y s udies. Ra he na ow concen-
a ion anges had o be used in he de e mina ion due o he
spon aneous and apid agglome a ion o he single chains a
highe concen a ions. The affini ies o all binde s we e ound
o be in he mic omola ange, CAF_3s possessing he highes
affini y o 2.0 × 10
−6
M (Fig. 3). The mode a e affini y ange is
also a o ed as i enables he elu ion o he a ge ed molecules
o eco e y pu poses.
The mic omola affini y ange is con enien o hei use in
bo h a ge compound moni o ing and eco e y, conside ing
he ele an concen a ions o hyd oxycinnama es in he
lignin-con aining liquo s as well as o allow efficien elu ion o
he a ge compound in hyd oxycinnama e eco e y.
LDM sepa a ion om he e ogeneous mix u es
One po en ial app oach o he alo iza ion o lignin-based
s eams is he selec i e and high eco e y o monome ic com-
Fig. 2 The binding o scF -APs FER_9, COU_12 and CAF_3 owa ds a ge molecules. In his compe i i e assay, he selec ed binde s we e mixed
oge he wi h he bio inyla ed a ge molecule and inc easing concen a ions o he co esponding ee acid (0.125–64 µM). The binde s bound o
he co esponding ee acid we e washed away and he abso bance was measu ed. The abso bance signals o he compe i i e binding esul s a e
scaled o ela i e abso bance B/B0 (B = measu ed signal, B0 = max. signal). Whi e = e ula e binde FER_9, blue = couma a e binde COU_12, and
ed = caffea e binde CAF_3. The s anda d de ia ion o iplica es is also shown in he figu e as e o ba s.
G een Chemis y Pape
This jou nal is © The Royal Socie y o Chemis y 2018 G een Chem.,2018,20,2829–2839 | 2835
Open Access A icle. Published on 08 May 2018. Downloaded on 5/4/2020 6:55:20 AM.
This a icle is licensed unde a
C ea i e Commons A ibu ion 3.0 Unpo ed Licence.
View A icle Online
pounds such as phenolic acids. The commonly used me hods
o phenolic compound eco e y include poly inyl esins o
ac i a ed ca bon ma ices.
43–45
The phenolic compounds bind
o he ma ix ei he ia hyd ophobic in e ac ions o by hyd o-
gen bonding om he a oma ic o ca boxyl g oup o he phe-
nolic compound allowing non-selec i e adso p ion on o he
ma ix. Fo example, da Cos a Lopes e al. (2016) de eloped a
me hod o pu i ying phenols ex ac ed om biomass wi h
ionic liquids using poly inyl esins.
46
The eco e ed ac ion
was u he pu i ied wi h supe c i ical CO
2
. Howe e , he e
we e no a emp s o selec i ely eco e a gi en a ge molecule
om he ac ion esul ing in a mix u e o phenolic acids.
On he o he hand, he adso p ion and deso p ion con-
di ions in a ma ix can be op imized o a o he eco e y o
ce ain molecules. Fo example, he selec i e eco e y o a
e ulic acid om enzyma ically ea ed suga -bee pulp was
s udied by Cou eau & Ma haly (1998) by op imizing a ixed-bed
sys em u ilizing ac i a ed ca bon.
47
In hei s udy, 50% e ulic
acid pu i y was achie ed. As ano he example, e ulic acid
eco e y s udies conduc ed by Tilay e al. (2008) e ealed
50.89% pu i y and 57.97% eco e y efficiency o polyme ic
adso ben s.
48
Howe e , by using mo e complex me hods, such
as subsequen p epa a i e HPTLC, he pu i y o he eco e ed
e ulic acid ac ion was inc eased o 95.35%.
48
Addi ionally,
he molecula imp in ing echnique can be used o inc ease
he selec i eness o he esin. As an example, Michailo e al.
(2008) used molecula ly imp in ed polyme s o eco e caffeic
acid, 4-HBA and o he phenolic compounds wi h eco e y
efficiencies be ween 9–51%. Fo he caffeic acid imp in ed
polyme s ong compe i ion o binding si es occu ed also
wi h p-couma ic acid.
49
To examine he po en ial o he an ibody use in speci ic
LDM eco e y in he con ex o lignin alo iza ion, he selec i-
i y o he chosen scF s was examined in a p oo o p inciple
expe imen by sepa a ing caffea e om a he e ogeneous
mix u e o soluble compounds. Caffea e eco e y om
diffe en mix u es spiked wi h caffea e was demons a ed by
using an affini y column p epa ed wi h immobilized CAF_3
scF . Elu ion o he caffea e molecules was achie ed wi h pH
change, ollowed by immedia e equilib a ion o he column o
e old he co alen ly bound an ibodies, hus making he
column ma ix eusable.
A e e i ying ha he p epa ed column cap u es caffea e
(ESI 14†), he column was used o eco e caffea e om simu-
la ed LDM solu ion, k a lignin and ice s aw hyd olysa e
spiked wi h caffea e, couma a e and e ula e. Caffea e was
eco e ed wi h high pu i y om all o he samples wi hou
aces o e ula e, couma a e o o he de ec able compounds
p oposing he sys em o be highly selec i e owa ds caffea e
(ESI Fig. 15–17†).
In o de o quan i a i ely de e mine he caffea e eco e y
a e o he column, he maximum heo e ical binding capaci y
o he scF -coupled bed was de e mined. Assuming ha all o
he scF s we e coupled unc ionally o he ma ix and each
Fig. 3 De e mina ion o he binding affini y and dissocia ion kine ics o he caffea e (CAF_3s), e ula e (FER_9s), and couma a e (COU_12s)
binde s. Local pa ial fi s we e employed o es ima e he affini ies. The K
D
and K
diss
alues a e p esen ed as an a e age o he affini ies es ima ed o
ou diffe en binde concen a ions ( h ee o FER9s). The de e mina ion was based on he associa ion and dissocia ion o he binde s o/ om he
bio inyla ed LDMs ( he selec ed hyd oxycinnama es) a ached o s ep a idin-coa ed op ical biosenso s.
Pape G een Chemis y
2836 |G een Chem.,2018,20,2829–2839 This jou nal is © The Royal Socie y o Chemis y 2018
Open Access A icle. Published on 08 May 2018. Downloaded on 5/4/2020 6:55:20 AM.
This a icle is licensed unde a
C ea i e Commons A ibu ion 3.0 Unpo ed Licence.
View A icle Online
scF binds he a ge ed molecule in a mola a io o 1 : 1, he
binding capaci y o he column was es ima ed o be 2.6 µg o
caffea e pe 1 ml o so ben in he column. Thus, samples con-
aining 2.5 µg pe 1 ml column o caffea e we e used o quan-
i a i e eco e y expe imen s. Ve y high eco e y a es we e
ob ained o he simula ed LDM mix u e (94%) (Table 2) and
also o he caffea e solu ion (77%). Couma a e and e ula e
we e ound om he low and wash ac ions o he LDM
mix u e e i ying ha hey a e no e ained in he column.
Caffea e binde as a moni o ing ool
Hyd oxycinnama es, such as p-couma a e, e ula e, and
caffea e, a e ound in plan cells and echnical lignin s uc-
u es. These molecules a e po en ial subs a es o bac e ial
lignin upg ading schemes and simple acking me hods o
hese subs a es a e o g ea in e es . Fo example, he speci ic
de ec ion o hese molecules di ec ly om he cul u e medium
and o he complex ma ices is an appealing app oach in bac-
e ial con e sion s udies. To his end, expe imen s wi h he
highly speci ic caffea e binde CAF_3 we e conduc ed o e i y
i s applicabili y as a moni o ing ool o subs a e con e sion.
A. baylyi ADP1 was cul i a ed wi h ace a e and caffea e as
he ca bon sou ces. Acco ding o he cul u e sample analysis
pe o med by he immunoassay, he samples collec ed om
A. baylyi ADP1 cul u es showed he subsequen u iliza ion o
ace a e and caffea e (Fig. 4). This is well in co ela ion wi h he
known subs a e p e e ences o A. baylyi; i s a oma ic ca abo-
lism is ep essed in he p esence o ace a e which is conse-
quen ly u ilized be o e he a oma ics.
50
The de eloped
immunoassay quan i ied he concen a ion o caffea e in he
cul i a ion samples up o he ime poin o 10 hou s.
The ea e , he concen a ions we e below he de ec ion limi .
The calib a ion cu e o he immunoassay ga e a linea
esponse be ween 4–10 µM o he caffea e binde CAF_3 and
he caffea e concen a ions om he samples we e u he con-
i med wi h UV-Vis 208 nm, suppo ing he immunoassay
esul s (Fig. 4).
Conclusions
The s uc u al complexi y and he e ogenei y o lignin make i s
u he u iliza ion and analysis e y challenging. Due o he
high binding speci ici y, an ibody-based ools could p o ide a
unique means o he eco e y, de ec ion and moni o ing o a
a ie y o lignin compounds e en om complex ma ices. In
his s udy, binde s agains h ee lignin-de i ed hyd oxycinna-
ma es e ula e, couma a e and caffea e we e success ully iso-
la ed om a syn he ic an ibody phage lib a y. These scF s can
be used o isola e and collec indi idual chemically pu e sub-
s ances om a complex mix u e o lignin-de i ed compounds
as was demons a ed wi h he speci ic caffea e-binding an i-
body. Fu he mo e, he binde agains caffea e was success ully
employed o ack caffea e consump ion in a bac e ial biop o-
cess. This p oo o concep s udy shows he po en ial o an i-
bodies o he eco e y o small molecules om dilu e and
challenging s eams.
Table 2 Selec i i y and eco e y o caffea e om he CAF_3 scF -
column. LDM model compound solu ions we e un h ough he affini y
column and he yields o he diffe en ac ions we e calcula ed om
he analyzed samples. Reco e y % om he column is calcula ed om
he elu ed samples, whe eas eco e y % o al includes he calcula ed
yields di ided by he ini ial amoun
LDM model compound solu ion
Caffea e
(μg)
Couma a e
(μg)
Fe ula e
(μg)
Sample 2.45 3.84 5.62
Flow h ough 0.00 2.24 3.16
Wash h ough 0.00 1.54 2.14
Elu ion 1 0.24 0.00 0.00
Elu ion 2 1.57 0.00 0.00
Elu ion 3 0.50 0.00 0.00
Reco e y % om
column
94 0.00 0.00
Reco e y % o al 94 98 94
Fig. 4 Uppe figu e: Subs a e consump ion and g ow h o A. baylyi
ADP1 ba ch cul u e o e a ime cou se o 12 hou s. Closed diamonds
ep esen ace a e concen a ion in mM and blue ci cles caffea e con-
cen a ion in mM (immunoassay). The black as e isks ep esen he bac-
e ial g ow h measu ed as he op ical densi y a 600 nm. The open
ci cles indica e he caffea e concen a ion as mM when measu ed wi h
UV-Vis a 280 nm. Bo om figu e: he calib a ion cu e used o calcula -
ing he caffea e concen a ion. Open ci cles ep esen he abso bance
measu ed a 405 nm in diffe en caffea e concen a ions and he do ed
line he coefficien o a ia ion. The black dashed line ep esen s he
da a fi ing o he ob ained abso bance alues o e a ying ee acid
concen a ions.
G een Chemis y Pape
This jou nal is © The Royal Socie y o Chemis y 2018 G een Chem.,2018,20,2829–2839 | 2837
Open Access A icle. Published on 08 May 2018. Downloaded on 5/4/2020 6:55:20 AM.
This a icle is licensed unde a
C ea i e Commons A ibu ion 3.0 Unpo ed Licence.
View A icle Online