In eg a ed Bioca aly ic Pla o m Based on Aqueous Biphasic
Sys ems o he Sus ainable Oligome iza ion o Ru in
Abel Muniz-Mou o, Ana M. Fe ei a, Joao A. P. Cou inho, Ma a G. F ei e, Ana P. M. Ta a es,*
Pa icia Gullón, Sa a González-Ga cía, and Gemma Eibes*
Ci e This: ACS Sus ainable Chem. Eng. 2021, 9, 9941−9950
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sıSuppo ing In o ma ion
ABSTRACT: Ru in is a known an ioxidan compound ha
displays a b oad ange o biological ac i i ies and heal h- ela ed
benefi s bu p esen s a low wa e solubili y ha can be o e come
by i s polyme iza ion. In his wo k, biocompa ible aqueous
biphasic sys ems composed o he ionic liquid cholinium
dihyd ogen phospha e ([CH][DHph]) and he polyme poly-
(e hylene glycol) 600 (PEG 600) we e in es iga ed as an efficien
in eg a ed eac ion−sepa a ion pla o m o he laccase-ca alyzed
oligome iza ion o u in. Two diffe en app oaches we e s udied o
euse laccase in se e al oligo u in p oduc ion cycles, he main
diffe ence be ween hem being he use o monophasic o biphasic egimes du ing he oligome iza ion eac ion. The use o a biphasic
egime in he second app oach (he e ogeneous eac ion medium) allowed he success ul euse o he bioca alys in h ee consecu i e
eac ion−sepa a ion cycles while achie ing no ewo hy u in oligome iza ion yields (95% in he fi s cycle, 91% in he second cycle,
and 89% in he las cycle). These ema kable esul s we e caused by he combina ion o he inc eased solubili y o u in in he PEG-
ich phase oge he wi h he enhanced ca aly ic pe o mance o laccase in he [Ch][DHph]- ich phase, alongside wi h he
op imiza ion o he pH o he eac ion medium s aigh ly linked o enzyme s abili y. Finally, a li e-cycle assessmen was pe o med o
compa e his in eg a ed eac ion−sepa a ion pla o m o h ee al e na i e p ocesses, ein o cing i s sus ainabili y.
KEYWORDS: Ru in oligome iza ion, O ganic- ee, Biocompa ible, Laccase euse, Aqueous biphasic sys em, Li e-cycle assessmen
■INTRODUCTION
Ru in (2-(3,4-dihyd oxyphenyl)-5,7-dihyd oxy-4-oxo-4H-ch o-
men-3-yl 6-O-(6-deoxy-α-L-mannopy anosyl)-β-D-glucopy a-
noside) is a fla onoid compound p esen in ui s, ege ables,
and plan -de i ed be e ages.
1
T adi ionally used in Chinese
medicine,
2
his subs ance exhibi s aluable an ioxidan
ac i i ies and in e es ing pha macological p ope ies ha
allow i s use in he p e en ion o diseases such as cance ,
diabe es, and ca dio ascula and neu odegene a i e condi-
ions.
3
Due o i s an ioxidan ea u es, u in can also be used as
a nu aceu ical compound. Up o da e, he Die a y Supplemen
Label Da abase con ains mo e han 1320 ma ke ed p oduc s
wi h u in as one o hese ing edien s.
4
Howe e , despi e i s
in e es ing biological p ope ies, he la ge-scale use o
fla onoids such as u in aces some challenges due o i s
poo he mal s abili y and low solubili y in aqueous and
non oxic o ganic sol en s.
5
To o e come hese d awbacks, he
enzyma ic modifica ion o na u al fla onoids by laccases (EC
1.10.3.2) and pe oxidases (EC 1.11.1) has gained in e es o e
he pas decades, especially conce ning laccases since hey use
oxygen as a final elec on accep o a he han hyd ogen
pe oxide.
6
These oxida i e enzymes ca alyze he o ma ion o
phenoxyl adicals ha , ollowing a u he oxida i e coupling,
lead o he p oduc ion o highe molecula weigh poly-
phenols,
7
imp o ing he solubili y, s abili y, and, in some cases,
he bioac i e p ope ies o phenolic compounds like u in.
8
Al hough he laccase-ca alyzed polyme iza ion o u in is a
p omising s a egy, since i allows he con ol o he polyme
s uc u e by he na u e o he eac ion medium, while being
ca ied a mild condi ions o empe a u e, pH, and p essu e,
9
he bioca alys cu en ly ep esen s one o he highes cos s o
he p ocess. The e o e, o boos he economic iabili y o hese
bioca aly ic p ocesses, he immobiliza ion o enzymes in solid
suppo s and hei euse has been he a ge o in ensi e
in es iga ion.
10
Howe e , he con en ional enzyme immobili-
za ion echniques p esen some d awbacks, such as mass
ans e limi a ions (in en apmen o encapsula ion), he use
o oxic compounds as a c oss-linking agen ( o bo h
adso p ion on solid suppo s and c oss-linked enzyme
agg ega es), enzyme leaching (in solid suppo s adso p ion),
and enzyme inac i a ion due o modifica ions o he p o ein
Recei ed: May 21, 2021
Re ised: July 2, 2021
Published: July 14, 2021
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s uc u e and changes in i s ac i e si e (co alen binding).
11
Hence, he e is he need o find al e na i e, biocompa ible, and
enzyme- iendly eac ion media which simul aneously allow
he euse o he bioca alys in mul iple p oduc ion s eps. Along
his line, in eg a ed bioca aly ic sys ems can be seen as
p omising app oaches owa d la ge-scale in eg a ion.
12
Liquid−liquid (L−L) biphasic sys ems a e an a ac i e
op ion since hey can allow he in eg a ion o he eac ion and
sepa a ion s eps wi hou implica ing he abo e-men ioned
d awbacks o enzyme immobiliza ion echniques. Indeed,
p e ious wo ks on bioca aly ic p ocesses in biphasic sys ems
composed o wa e −o ganic sol en s (and, in some cases,
su ac an s) ha e epo ed ema kable esul s.
13
Rega ding he
specific pe o mance o laccase in biphasic sys ems, his
enzyme has been used in he deg ada ion o pollu ing a oma ic
compounds (namely es adiol and an h acene), obse ing an
inc ease in subs a e solubili y ha consequen ly p omo ed
mass ans e , enhancing he o e all subs a e con e sion.
13,14
Thus, hese wo ks sugges ha he laccase-ca alyzed oxida ion
o hyd ophobic subs a es can be posi i ely affec ed by he
p esence o a mo e hyd ophilic phase, whe e laccase is
p e e en ially solubilized, and a hyd ophobic phase ha
enhances he subs a e solubili y.
To a oid he use o ola ile o ganic compounds, since some
o hem can be oxic and consequen ly comp omise he final
applica ion o he p oduc s as nu aceu icals, aqueous biphasic
sys ems (ABS) may be in es iga ed as compa ible media o
bioca alyzed p ocesses. ABS a e e na y sys ems comp ised o
wo immiscible wa e - ich phases, o med by mixing wa e and
wo phase- o ming compounds (e.g., polyme −polyme ,
polyme −sal , o sal −sal ). Al hough he po en ial o ABS
o he pu ifica ion o cells, p o eins, enzymes, and o he
biomolecules has been ex ensi ely s udied,
15
hei use in
bioca aly ic p ocesses has no been deeply in es iga ed.
Conce ning laccase, a ious wo ks ha e been epo ed
ega ding he use o ABS o i s pu ifica ion,
16
bu only one
p e ious esea ch s udy ocused on he use o hese sys ems as
an in eg a ed eac ion−sepa a ion pla o m allowing enzyme
euse.
17
This wo k was he fi s s ep o show he iabili y o
ABS o laccase-ca alyzed p ocesses, demons a ing (as a p oo
o concep ) he easibili y o he mo e e sible ABS (composed
o wo diffe en zwi e ions and poly(e hylene glycol) 6000
(PEG 6000)) in pe o ming he oxida ion o ABTS (s anda d
subs a e o laccase used o e alua e i s ac i i y) in a
homogeneous egime ABS, ollowed by laccase and oxidized
subs a e sepa a ion owa d opposi e phases, which occu ed
due o an inc ease/dec ease in empe a u e.
17
When s udying
laccase pu ifica ion, Sil e io e al.
16
analyzed diffe en
polyme −polyme and polyme −sal -based ABS, obse ing
ha polyme −sal ABS led o be e esul s conce ning bo h
laccase ex ac ion and s abili y and showing ha ABS based on
poly(e hylene glycol) (PEG) and phospha e sal combina ions
could lead o p omising esul s and hence should be u he
in es iga ed in he u u e. Mo eo e , Capela e al.
18
analyzed
laccase pu ifica ion and ac i i y in IL-based ABS, iden i ying
cholinium-based ILs as p omising candida es o ABS
o mula ion ega ding bo h enzyme ex ac ion and ac i i y.
Recen ly, PEG-IL-based ABS ha e been in es iga ed wi h
he aim o combining he biocompa ibili y and low oxici y o
PEGs, which a e au ho ized ood addi i es in he Eu opean
Union acco ding o Regula ion (EC) No. 1333/2008,
19
wi h
he inc eased efficiency and selec i i y o he sepa a ion s ep
associa ed wi h IL-based ABS, while also dec easing he
iscosi y o he media when compa ed o polyme -based
ABS.
20
Based on he need o a sus ainable eac ion−sepa a ion
p ocess o he laccase-based oligome iza ion o u in,
especially ocused on he euse o he bioca alys (since i is
he mos expensi e p oduc equi ed in he eac ion), his wo k
aims o in es iga e he use o PEG-IL-based ABS. Laccase om
he ligninoly ic ungus T ame es e sicolo was used. Consid-
e ing ha he final applica ion o oligo u in would be he
o mula ion o ood addi i es, cosme ic p oduc s, o i s use in
pha maceu ical compounds, he oxici y should be aken in o
accoun when selec ing a eac ion medium. Acco dingly,
diffe en cholinium-based ILs (wi h negligible oxici y and
low cos o p oduc ion)
20
we e chosen in his wo k o be
combined wi h PEG in he o mula ion o ABS. Two diffe en
app oaches we e s udied o euse laccase in se e al oligo u in
p oduc ion cycles, i.e., oligome iza ion eac ion ca ied ou in
he monophasic o biphasic egime. Finally, a li e-cycle
assessmen (LCA) was pe o med on he mos p omising
app oach and compa ed o h ee al e na i e p ocesses in o de
o demons a e he iabili y o he ABS o be applied as a
sus ainable in eg a ed eac ion−sepa a ion pla o m o he
p oduc ion o oligo u in, a compound wi h po en ial
comme cial in e es .
■EXPERIMENTAL SECTION
Chemicals. Cholinium dihyd ogen phospha e 99 w % pu e
([Ch][DHph]) and cholinium ace a e 98 w % pu e ([Ch][Ac]) we e
bo h pu chased om IoLiTec (Ionic Liquids Technologies GmbH).
Cholinium glycola e ([Ch][Gly]) was syn hesized as desc ibed by
Quen al e al.
21
Be o e use, all cholinium-based ionic liquids (ILs)
we e pu ified and d ied o a minimum o 24 h a cons an agi a ion,
a mode a e empe a u e (∼50 °C), and unde acuum ( o educe
hei ola ile impu i ies o negligible alues). A e his s ep, he pu i y
o each IL was confi med by 1H and 13C NMR spec a and ound o
be >98%. The polyme poly(e hylene glycol) (PEG) o a e age
molecula weigh 600 g·mol−1(PEG 600) was supplied by Al a Aesa
and used as ecei ed. The chemical s uc u es o such ILs and
polyme a e p esen ed in Figu e S1. Enzyme-ca alyzed eac ions we e
pe o med using comme cial laccase om T. e sicolo , and i s ac i i y
was assessed using 2,2′-azino-bis(3-e hylbenza hiazoline-6-sul onic
acid) diammonium sal (ABTS) as he subs a e, bo h acqui ed
om Sigma. Po assium pe sul a e and T olox (6-hyd oxy-2,5,7,8-
e ame hylch oman-2-ca boxylic acid) o an ioxidan capaci y
measu emen s we e pu chased om Sigma. Ru in ( u in hyd a e 98
w % pu e) was pu chased om TCI (The mo Fische Scien ific).
Laccase-Ca alyzed Ru in Oligome iza ion Using ABS as a
Reac ion Medium. Based on ou p e ious wo k,
22
u in
oligome iza ion was pe o med unde he ca aly ical ac i i y o 1000
U/L o e 24 h and s a ing wi h an ini ial u in concen a ion o 3 g/L
(whe e one uni , U, is defined as he amoun o laccase capable o
oxidizing 1 μmol o ABTS pe minu e). Reac ions we e pe o med in
15 mL alcon ubes filled wi h 4.25 mL o ABS (5 g) in cons an
agi a ion (50 pm) on a Mul i Bio RS-24 agi a o (BioSan) a 25 °C.
Agi a ion o 50 pm was chosen as a p ope o a o y speed
23
since i
p o ided enough con ac be ween he enzyme and he subs a e,
wi hou implying an excess o s i ing. The desi ed pH o he ABS was
achie ed h ough he ca e ul addi ion o 10 M NaOH a e mixing he
polyme , he ionic liquid, and 90% o he dis illed wa e , p o iding he
lacking amoun o wa e o each 5 g o ABS a e he NaOH addi ion.
A e u in addi ion o he ABS, he olume needed o a p e iously
p epa ed and cha ac e ized s ock solu ion o laccase om T. e sicolo
was supplied o s a he syn hesis o oligo u in.
Ru in deple ion du ing he eac ion was ollowed by HPLC
(Shimadzu, PROMINENCE) allowing he calcula ion o he u in
con e sion (also e e ed in his wo k as oligome iza ion yield),
ollowing he p o ocol and calcula ion ully desc ibed in he
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Suppo ing In o ma ion. Laccase ac i i y was also spec opho ome i-
cally assessed (Shimadzu, UV-1800) du ing he eac ion ollowing he
p o ocol desc ibed by Zimme mann e al.
24
Iden ical expe imen s o hose in ol ing 3 g/L u in bu s a ing
wi h an ini ial u in concen a ion o 10 g/L we e addi ionally
pe o med o explo e he easibili y o he de eloped p ocess o allow
he enzyma ic oligome iza ion o u in a highe subs a e
concen a ions.
Reco e y and Laccase Reuse. Th ee consecu i e cycles o
oligo u in syn hesis and phase sepa a ion we e pe o med o assess
he bioca alys eusabili y and oligo u in ex ac ion on ABS eac ion
media. A e 24 h o eac ion, he laccase ac i i y and oligo u in
concen a ion we e measu ed, and he phase o ma ion was quickly
p omo ed by cen i uga ion a 25 °C o 30 min a 5000 pm.
Cen i uga ion was used in his lab-scale wo k bo h o ensu e he
he modynamic equilib ia o he aqueous phases and o s anda dize a
phase- o ma ion s ep o all expe imen s. Howe e , a ema kably apid
phase o ma ion was al eady obse ed in less han 10 min a e
s opping he s i ing o he eac ion media, hus confi ming ha a
cen i uga ion s ep would no be s ic ly needed (which would
posi i ely affec he economic iabili y o la ge -scale eac ions). The
oligo u in concen a ion was measu ed spec opho ome ically
(Shimadzu UV-1800) ollowing he de eloped p o ocol indica ed in
he Suppo ing In o ma ion. Laccase ac i i y (U/L), oligo u in
concen a ion (mg/L), and olume (mL) we e measu ed o bo h
phases o he ABS, allowing he calcula ion o he o al amoun o
oligo u in (mg) and laccase (U) in each phase and, subsequen ly, he
calcula ion o he pe cen age ex ac ion efficiencies (%EE) o bo h
p oduc (%EEoligo u in) and bioca alys (%EElaccase ac i i y), defined as he
a io be ween oligo u in mg o laccase U p esen in ha specific phase
and he o al amoun p esen in he comple e ABS, espec i ely.
The oligo u in-en iched op phase was hen emo ed, and he same
olume o a new op polyme - ich phase wi h he exac same
composi ion (de e mined by he ie-line) and pH, bu lacking
oligo u in, was added o e he emaining laccase-en iched bo om
phase. Ru in was la e added o each a concen a ion o 3 g/L, and
he mix u e was shaken and placed in he Mul i Bio RS-24 agi a o ,
he e o e s a ing a new oligo u in syn hesis s ep. This p ocess,
g aphically ep esen ed in Figu e S2, was epea ed o up o h ee
comple e eac ion−sepa a ion cycles, hus leading o h ee sepa a ed
oligo u in-en iched op phases and a final laccase-en iched bo om
phase.
Reco e y o PEG 600 and [Ch][DHph]. Aiming o explo e he
eco e y o PEG 600 and [Ch][DHph] p esen in he op oligo u in-
en iched phase, an ul afil a ion s ep was conside ed a e phase
sepa a ion. In his p elimina y s udy, a 1 kDa nominal po e size
egene a ed cellulose memb ane was used o e ain he p oduced
oligo u in inside o a 10 mL ul afil a ion Amicon s i ed cell
(Millipo e), while allowing bo h PEG 600 and [Ch][DHph] o pass
h ough he memb ane, he e o e p omo ing he sepa a ion o
oligo u in om he o he chemicals p esen in he op phase. Two
diffe en aqueous dilu ions o op phases (1:2 and 1:4 / ) we e
conside ed as inpu s o his ul afil a ion cell since he o iginal
undilu ed phase would lead o memb ane ouling due o high
iscosi y, and ul afil a ion was p omo ed by an o e p essu e (3 ba )
o igina ed wi h N2(g), while main aining he dilu ed sample unde
cons an s i ing. A final e en a e olume equal o he o iginal
undilu ed op phase olume was ob ained in each case ( he e o e
fil e ing 50% and 75% olume o 1:2 and 1:4 dilu ions, espec i ely).
The composi ion o bo h e en a e and pe mea e s eams was s udied;
he oligo u in concen a ion was spec opho ome ically measu ed
ollowing he p o ocol desc ibed in he Suppo ing In o ma ion, while
he mass pe cen age o PEG 600 + [Ch][DHph] in he e en a e and
pe mea e s eams was calcula ed as he ela ion be ween he mass o a
known olume o each s eam a e and be o e eeze-d ying
(wi hd awing oligo u in mass om his alue). Once hese da a
we e ob ained, he eco e y o PEG 600 + [Ch][DHph] was
calcula ed.
Oligo u in Cha ac e iza ion. Mass spec a o e en a e and
pe mea e p oduc s a e ul afil a ion we e de e mined by MALDI-
TOF (ma ix-assis ed lase deso p ion/ioniza ion− ime-o -fligh )
based on he p o ocol desc ibed by An honi e al.,
25
wi h he sligh
modifica ions indica ed in he Suppo ing In o ma ion.
The an ioxidan ac i i y o he oligo u in p oduced in he h ee
successi e cycles in ol ing laccase euse was measu ed ollowing he
T olox Equi alen An ioxidan Capaci y(TEAC)p o ocolas
desc ibed by Gullon e al.,
26
ully explained in he Suppo ing
In o ma ion. Resul s we e exp essed in T olox equi alen s.
En i onmen al Assessmen . LCA modeling was ca ied ou in
Simap o so wa e 9.0,
27
and he CML 2001 2.05 me hod
28
de eloped by he Cen e o En i onmen al Science o Leiden
Uni e si y was used o he selec ion o he cha ac e iza ion ac o s
equi ed o es ima e he en i onmen al bu dens, conside ing fi e
impac ca ego ies: global wa ming (GW), acidifica ion (AC),
eu ophica ion (EU), ozone laye deple ion (OD), and pho ochemical
oxida ion (PO).
The p ocess de eloped in his wo k, scena io 1 (Sc1), was
compa ed o o he h ee scena ios in ol ing laccase euse o
oligo u in p oduc ion: Sc2, he same biphasic s a egy conside ed as
in ou a ge sys em (Sc1) bu including he euse o PEG 600 and
[Ch][DHph] by implemen ing an addi ional ul afil a ion s ep p io
o eeze-d ying; Sc3, he ope a ion o an enzyma ic memb ane
eac o (EMR) o euse laccase o oligo u in syn hesis;
8
and Sc4, he
use o laccase immobilized on o magne ic nanopa icles (mNPs)
allowing i s euse in mul iple syn hesis s eps.
29
The compa a i e p ofile was pe o med ollowing an a ibu ional
c adle- o-ga e app oach, including all s ages pe o med in he lab om
he p epa a ion o he eac ion medium up o he eeze-d ying s ep o
ob ain a wa e - ee p oduc ich in oligo u in. All his in o ma ion was
di ec ly aken om he lab and co esponds o p ima y da a, as
ecommended. Mo eo e , backg ound p ocesses in ol ed in he
p oduc ion o he inpu s (i.e., wa e , chemicals, enzymes, nano-
pa icles, elec ici y, e c.) and ancilla y ac i i ies (i.e., ea men o
was ewa e ) we e also included wi hin he sys em bounda ies.
The e o e, i has been necessa y o conside seconda y da a o
comple e he in en o y ables o he backg ound p ocesses; he
Ecoin en da abase e sion 3.5
30
has been conside ed as he main
seconda y da a sou ce. Howe e , in he case o laccase and
[Ch][DHph] p oduc ion, en i onmen al p ofiles epo ed by Gilpin
e al.
31
and Zhang e al.
32
ha e been aken in o accoun , espec i ely.
Finally, in Sc4, esea ch om Feijoo e al.
33
has been conside ed o
he iden ifica ion o in en o y da a co esponding o he p oduc ion
o nanopa icles and laccase immobiliza ion.
■RESULTS AND DISCUSSION
Selec ion o he Mos P omising ABS as a Reac ion
Medium. In o de o iden i y an effec i e ABS o he
oligome iza ion o u in, i.e., whe e he enzyme and he
p oduc should p e e en ially concen a e in opposi e phases,
p elimina y s udies o de e mine he pa i ion o laccase and
oligo u in we e ca ied ou wi h ABS o med by combina ions
o PEG wi h a molecula weigh o 600 g·mol−1(PEG 600)
and diffe en cholinium-based ILs, namely cholinium ace a e
([Ch][Ac]), cholinium glycola e ([Ch][Gly]), and cholinium
dihyd ogen phospha e ([Ch][DHph]). De ails on he PEG
and ILs chemical s uc u es a e gi en in Figu e S1. The
sepa a ion pe o mance o each ABS was e alua ed by
de e mining he ex ac ion efficiency o each compound.
Resul s on %EE a e gi en in Table S1. The ABS o med by
PEG 600 and [Ch][Ac], [Ch][DHph], o [Ch][Gly] we e
p e iously cha ac e ized by Pe ei a e al.
20
a 25 °C. Thus,
biphasic mix u e poin s we e selec ed acco ding o ha wo k
(weigh ac ion composi ions being 40 w % PEG + 40 w %
[Ch][Ac], 30 w % PEG + 30 w % [Ch][DHph], and 40 w %
PEG + 35 w % [Ch][Gly], whe e he lacking pe cen age alue
equi ed o comple e 100 w % co esponds o wa e ). A he
condi ions s udied in his wo k, he op phase co esponds o
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he PEG- ich phase, while he bo om phase is mainly
composed o he cholinium-based IL, excep o he [Ch]-
[Ac]-PEG 600 ABS whe e phases display he opposi e
beha io , as p e iously epo ed.
20
As shown in Table S1, he ABS composed o PEG 600 wi h
[Ch][Gly] did no allow he sepa a ion o oligo u in and
laccase since bo h compounds p e e en ially pa i ion o he
IL- ich phase (%EEoligo u in > 90% and %EElaccase ac i i y ∼100,
bo h e e ed o he bo om phase). The ABS cons i u ed by
[Ch][Ac] leads, howe e , o a success ul sepa a ion o bo h
compounds, wi h oligo u in being concen a ed in he PEG-
ich phase (%EEoligo u in > 90) while laccase is en iched in he
IL- ich phase (%EElaccase ac i i y ∼100). Oligo u in p e e en ially
mig a ed o he PEG- ich phase when he ABS was composed
o [Ch][DHph] o [Ch][Ac]. Howe e , he beha io changed
o ABS composed o [Ch][Gly]. The pa i ion o (bio)-
molecules in ABS depends on dispe si e in e ac ions, elec o-
s a ic o ces, hyd ogen bonding, biomolecule size, and
solubili y. Indeed, compa ing he log Kow (oc anol/wa e
pa i ion coefficien ) alues o [Ch][Ac], [Ch][Gly], and
[Ch][DHph], he ollowing he endency was ound: [Ch]-
[Gly] alue o −1.20, [Ch][DHph] alue o −3.70, and
[Ch][Ac] alue o −4.66,
20,34
meaning ha [Ch][Gly] is he
mos hyd ophobic compound, which may be esponsible o
he change in he pa i ion o oligo u in o he IL- ich phase.
The enzyme d ama ically loses i s ac i i y wi hin a ew
minu es in he ABS composed o [Ch][Ac], hus making i
unsui able as an in eg a ed eac ion−sepa a ion pla o m.
Indeed, some ace a e- ype ILs ha e been epo ed o exe a
nega i e effec upon he ac i i y o diffe en enzymes; in
pa icula , o laccase, an in e e ence in bo h he ABTS
binding o he ac i e si e o he enzyme and in ABTS oxida ion
i sel has been p oposed, ep esen ing compe i i e and
uncompe i i e inhibi ion mechanisms, espec i ely.
35
Mo e-
o e , he pH o he mix u e poin o his ABS has a high alue
(pH ∼9.4; measu ed in his wo k), no adequa e o laccase
om T. e sicolo , nei he o i s ela i e ac i i y o s abili y.
36
Rema kably, by using [Ch][DHph] and PEG 600 as ABS
cons i uen s, an app op ia e sepa a ion o oligo u in o he
polyme - ich phase (∼70 o %EEoligo u in) om laccase, which
mainly mig a es o he IL- ich phase (96 o %EElaccase ac i i y),
and wi h no dec ease o he enzyme ac i i y, was success ully
achie ed. Mo eo e , based on p e ious esea ch, [Ch][DHph]
oxici y was defined as negligible, compa able o ha o basic
physiologic sal s,
37,38
hus ein o cing he biocompa ibili y o
he s udied ABS. The e o e, he abili y o his sys em o be
used in he de elopmen o an in eg a ed eac ion−sepa a ion
p ocess o u in oligome iza ion and laccase eco e y and
euse was u he in es iga ed, conside ing wo app oaches as
desc ibed below.
Laccase-Ca alyzed Ru in Oligome iza ion Using ABS
as a Reac ion Medium. In a fi s app oach we s udied he
syn hesis o oligo u in in a homogeneous medium (a 25 °C)
o ake ad an age o he he mo e e sible beha io o he
selec ed ABS, p e iously desc ibed by Pe ei a e al.
20
and he e
confi med o a lowe empe a u e in e al (Figu e S3). In his
app oach, he eac ion can be ca ied ou a in a homogeneous
medium, a e which an inc ease in empe a u e up o 40 °C
leads o he c ea ion o wo phases and he simul aneous
sepa a ion o he enzyme om he p oduc . Howe e , his
app oach did no lead o a high oligome iza ion yield
(calcula ed based on equa ion S1) wi h a good enzyme
ac i i y pe o mance ( u in con e sion o 29% and ela i e
enzyme ac i i y loss o 28%, as epo ed in Table S3).
To o e come his p oblem, a second app oach was
e alua ed, consis ing o he syn hesis o oligo u in by laccase
being pe o med in he biphasic egime (ABS) ha would
allow o simul aneously ca y ou he eac ion, he p oduc
eco e y, and he enzyme euse wi hou any u he ex e nal
s imuli. The biphasic sys em a oom empe a u e was c ea ed
by changes in he pH o he sys em, aking ad an age o he
pH-d i en e e sibili y o his ABS (Figu e S5), achie ed by
he specia ion o he IL anion.
39
P elimina y es s we e
pe o med using ABS consis ing o [Ch][DHph] and PEG 600
a pH 4.5, 5.5, and 6.5 since he biphasic egion is achie ed a
pH alues abo e 4.5, he al eady s udied monophasic egion
being a ibu ed o a pH alue o 4.3. Bo h empe a u e (25
°C) and mix u e composi ion (26.08 w % o PEG + 26.75 w
% o [Ch][DHph]) we e main ained unal e ed as o he
p e ious he mo e e sible app oach. Al hough a mo e alkaline
pH would benefi u in solubili y,
40
highe alues we e no
conside ed since hey could comp omise he laccase s abili y/
ac i i y.
36
I mus be no ed ha such a small change in pH
om 4.3 o 4.5 led o a biphasic egime, which can be
explained by he p oximi y o he mix u e poin o he binodal
cu e o [Ch][DHph]-PEG 600 ABS a pH 4.3 and 25 °C
(Figu e S5). The oligome iza ion o u in was hen pe o med
in hese ABS a 25 °C o 24 h, and he u in con e sion and
ela i e laccase ac i i y (%) we e e alua ed o e ime.
Addi ionally, u in s abili y a such pH alues was also
confi med by eac ion con ols pe o med in he absence o
laccase ( u in concen a ion a e 24 h being highe han 93%
o all pH alues conside ed). The ob ained esul s a e
summa ized in Figu e 1 (Table S4).
A e 2 h o eac ion, a ema kable enhancemen in eac ion
kine ics was obse ed when he pH was inc eased om 4.5 o
6.5, since he oligome iza ion yields change om 22% o 51%,
espec i ely (Figu e 1). The same end was obse ed o all
Figu e 1. (A) Ru in con e sion, %, and (B) ela i e laccase ac i i y, %,
o e ime o he oligome iza ion o u in a pH 4.5 (blue), 5.5
(o ange), and 6.5 (g een), using he [Ch][DHph]-PEG 600 ABS in
he biphasic egime a 25 °C.
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eac ion imes e alua ed, wi h hese esul s being a ibu ed o
a be e con ac be ween he enzyme and i s subs a e due o
highe u in solubili y a mo e alkaline pH alues.
40
Conside ing ha 24 h o eac ion led o he bes
oligome iza ion yield (97%, pH 6.5), his ime was selec ed
o e alua e he effec o pH upon he o e all p ocess. Wi h he
inc ease o pH om 4.5 o 6.5, he u in oligome iza ion yield
eaches alues close o 100% (∼2- old), while laccase ac i i y
dec eases om ∼31 o 17% (Figu e 1). Thus, bo h u in
con e sion and ela i e enzyme ac i i y a e enhanced using
ABS wi h highe pH (pH 6.5). Fu he mo e, he effec o pH
upon oligo u in and laccase ex ac ion efficiencies was also
s udied, as shown in Figu e 2 (de ailed da a in Table S5), wi h
a sligh imp o emen occu ing a pH 6.5 o bo h oligo u in
and laccase (69 o %EEoligo u in and 91 o %EElaccase ac i i y).
In e es ingly, when compa ing he esul s ob ained a pH 4.3
in he monophasic egime (fi s app oach conside ed in his
wo k) o hose eached when using an ABS in he biphasic
egime a pH 4.5, a ema kable imp o emen o he u in
con e sion o ∼1.7- old was obse ed ( u in con e sions o
29% and 49% o pH a 4.3 and 4.5, espec i ely). Such a small
change in pH alone canno explain he inc emen obse ed;
he e o e, o be e unde s and why u in oligome iza ion was
a o ed in a biphasic eac ion medium, he influence o each
phase composi ion on he oligo u in syn hesis was e alua ed.
Fo his pu pose, he eac ion was pe o med in sepa a ed op
and bo om phases a he op imal pH o 6.5. Acco ding o he
esul s (Table S6 and Figu e S6), u in con e sion, laccase
ac i i y, and eac ion kine ics a e imp o ed when he eac ion
akes place in he [Ch][DHph]- ich phase, which can be
explained by he posi i e effec o cholinium ca ions o e bo h
laccase s abili y and ac i i y, as shown in he li e a u e.
18
A e
24 h o eac ion, he final enzyme ac i i y was 10% highe in
his phase han in he PEG- ich phase, eaching he comple e
oligome iza ion o u in, while only 80% yield was achie ed
when he eac ion was pe o med in he PEG- ich phase.
Mo eo e , when he oligome iza ion was pe o med in he
monophasic egion o he ABS, he concen a ion o each ABS
cons i uen was 26.08 and 26.75 w % o PEG 600 and
[Ch][DHph], espec i ely, while in he [Ch][DHph]- ich
phase he composi ion was 6.86 and 41.68 w % o PEG 600
and [Ch][DHph] (Figu e S7). These alues ein o ce he ac
ha high concen a ions o [Ch][DHph] imp o e laccase
enzyma ic pe o mance. Addi ionally, and as men ioned be o e,
u in solubili y benefi s om he p esence o o ganic sol en s,
1
meaning ha he opposi e PEG- ich phase (49.49 w % o
PEG and 8.47 w % o [Ch][DHph]) could inc ease he
amoun o u in a ailable o be ans o med by laccase in o
oligo u in. The s udy o he ex ac ion efficiency o u in o
[Ch][DHph] and PEG- ich phases was also pe o med, and
he esul s (Table S5) show an 88% u in ex ac ion efficiency
owa d he PEG- ich phase, highe han ha obse ed o
oligo u in (∼70%). This end is somehow expec ed gi en
u in hyd ophobici y.
22
Consequen ly, when he eac ion is
ca ied ou in he he e ogeneous egion o he ABS, i.e., in he
p esence o bo h immiscible phases, a highe solubili y o u in
and an enhanced pe o mance o he bioca alys can be
ob ained. Fu he mo e, since he eac ion occu s unde
con inuous s i ing, he highe ac i i y o laccase in he
biphasic medium can also be accoun ed o by he high
a ailabili y o he in e ace, allowing be e in e ac ions
be ween he subs a e and he enzyme.
41
Reco e y and Laccase Reuse. Based on he capaci y o
[Ch][DHph]-PEG 600 ABS o ac as a eac ion medium o
he oligome iza ion o u in by laccase and aking in o
conside a ion i s abili y o sepa a e he enzyme and oligome s,
his ABS was hen s udied as an in eg a ed eac ion−sepa a ion
Figu e 2. Oligo u in (o ange ba ) and laccase (blue ba ) ex ac ion
efficiencies (%EE) in he [Ch][DHph]-PEG 600 ABS a pH 4.5, 5.5,
and 6.5 a e 24 h o enzyma ic eac ion in he biphasic egime a 25
°C.
Figu e 3. (A) Flowcha o he in eg a ed eac ion−sepa a ion p ocess de eloped by applying ABS consis ing o PEG 600 and [Ch][DHph], a pH
6.5 and 25 °C, including he ecycling o he laccase and he IL- ich phase. (B) Rela i e laccase ac i i y and u in con e sion (%) in h ee cycles o
oligome iza ion eac ion, comp ised o bo h he eco e y and he euse o he enzyme and IL- ich phase.
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pla o m o h ee consecu i e eac ion cycles, allowing o
confi m ha laccase main ains i s bioca aly ic ac i i y a e he
sepa a ion s ep. A scheme o he enzyme eco e y/ euse
p ocess is illus a ed in Figu e 3A. A e he fi s cycle o
eac ion, he syn hesized oligo u in was en iched in he PEG-
ich op phase (67 %EEoligo u in), which was hen eco e ed by
sepa a ing bo h phases. The IL- ich bo om phase con aining
laccase (95 %EElaccase ac i i y) was eco e ed and eused in a new
bioca aly ic eac ion cycle. Fo he c ea ion o no el ABS as
eac ion media o s a he nex cycle, a new polyme - ich
phase con aining adequa e amoun s o PEG and IL, namely
49.49 w % o PEG 600 + 8.47 w % o [Ch][DHph], as
ob ained om he binodal cu e (Table S7) and ie-line
(Figu e S7) da a a 25 °C and pH 6.5, was added o he
eco e ed laccase-en iched bo om phase, and new u in was
p o ided o s a he oligome iza ion eac ion. This eac ion−
sepa a ion p ocedu e was epea ed in successi e cycles,
accoun ing o a o al o 3. Images illus a ing he isual aspec
o he end o he eac ion (24 h) and bo h sepa a ed phases a e
p o ided in Figu e S8. The esul s ob ained (Figu e 3B) show
ha i is possible o eco e and euse laccase, o a leas h ee
consecu i e cycles, allowing no ewo hy oligome iza ion yields
o 95% in he fi s cycle, 91% in he second cycle, and 89% in
he las cycle, since only a sligh loss in he enzyme ca aly ic
ac i i y was obse ed du ing he eac ion−sepa a ion cycles
(Table S8). This small loss in enzyma ic ac i i y may be due o
he ime equi ed o u in polyme iza ion (∼25 h) a oom
empe a u e (25 ±2°C) since p e ious s udies demons a ed
ha laccase om T. e sicolo loses ∼20% ela i e ac i i y a e
being incuba ed a oom empe a u e o e 24 h a pH 7.0,
42
hese ope a ional condi ions being simila o hose used in his
wo k (pH 6.5). Mo eo e , laccase ex ac ion efficiencies o
∼94% and oligo u in ex ac ion efficiencies o ∼67% o
opposi e phases we e main ained o e he h ee consecu i e
cycles (Table S9).
When compa ed o ecen ly desc ibed biocompa ible
p ocesses o u in oligome iza ion,
8,22,40
he pla o m he e
designed allows he ob aining o bo h highe o compa able
oligome iza ion yields unde lowe ca aly ic ac i i y a mild
condi ions. Fo ins ance, Pi ec e al.
40
ob ained a 57% u in
oligome iza ion yield using 5000 U/L o laccase ac i i y. In ou
p e ious wo k in ol ing he use o hyd oe hanolic eac ion
media,
22
compa able u in con e sion o ha ob ained in his
wo k (93%) was ob ained only when he enzyme ac i i y was
inc eased up o 10 000 U/L, while 1000 U/L accoun ed o a
70% oligome iza ion yield. Howe e , con a y o hese
p e ious epo s,
22,40
he p ocess de eloped in his wo k also
allows he eu iliza ion o laccase (and he [Ch][DHph]- ich
phase), a leas in h ee successi e eac ion−sepa a ion cycles
as demons a ed, while main aining ou s anding p oduc ion
yields. To he bes o ou knowledge, he e is only ano he
ecen ly published wo k s udying laccase euse in u in
oligome iza ion and biocompa ible eac ion media, based on
ul afil a ion memb ane echnology o sepa a e he enzyme
and he oligome s p oduced.
8
In ha wo k, pH 6.0 phospha e
buffe was used as he eac ion medium, and ∼100% u in
oligome iza ion yields we e ob ained unde 1000 U/L laccase
ac i i y, pe o ming h ee consecu i e eac ion−sepa a ion
cycles, whe e laccase was success ully eused and accoun ing
o a o al ac i i y loss o less han 20% a he end o he hi d
cycle. None heless, while his echnology allowed lowe
enzyme ac i i y losses han hose obse ed in he cu en
wo k, he use o a solid subs a e unde cons an s i ing and in
con ac wi h an ul afil a ion memb ane could comp omise
he in eg i y o he memb ane h ough e osion and ouling
p ocesses. This d awback could be a oided wi h he
echnology de eloped and p oposed he e .
Finally, es s on inc easing he u in ini ial concen a ion up
o 10 g/L (highe han hose es ed in p e ious wo ks no
in ol ing o ganic cosol en s) we e pe o med. The u in
con e sion achie ed a e 24 h o eac ion, using he p oposed
ABS-based echnology, was highe han 93%, while %EE o
bo h oligo u in and laccase ac i i y showed simila alues o
hose obse ed in expe imen s in ol ing 3 g/L o u in
concen a ion (65% and 92%, espec i ely, Table S11).
The e o e, hese esul s suppo he hypo hesis ha his
pla o m would be sui able o highe concen a ions o o he
insoluble subs a es, al hough u he s udies and op imiza ion
o he p ocess would be equi ed. Mo eo e , bo h he esul s
ob ained in consecu i e cycles conside ing laccase euse and
he good oligo u in yield a highe subs a e concen a ions
e ince he obus ness o he de eloped eac ion−sepa a ion
pla o m, which is c ucial o he design o a sus ainable flow
p ocess. I should be u he ema ked ha al hough he
comple e isola ion o oligo u in was no achie ed in a single
s ep, in a cyclic p ocess he comple e eco e y o he p oduc is
no ele an since he final p oduc p esen in he enzyme- ich
phase, i.e., oligo u in, will be ecycled oge he wi h he eused
phase.
Oligo u in Cha ac e iza ion. As al eady epo ed in he
li e a u e, oligo u in shows imp o ed bioac i e and an ioxidan
p ope ies when compa ed o he na u al monome u in,
namely be e xan hine oxidase inhibi o y ac i i y, enhanced
sca enging ac i i y agains biologically impo an adicals such
as NO·,O
2
·,o OH
·, augmen ed Fe2+ chela ing ac i i y,
e c.
8,40,43
These p ope ies, alongside wi h i s ema kably
inc eased aqueous solubili y, boos he po en ial applica ions o
his biosyn hesized p oduc in ei he ood, pha maceu ical, o
cosme ic indus ies. In his wo k, MALDI-TOF analysis o
oligo u in p oduced in he cu en p ocess (Figu e S9)
e ealed a maximum deg ee o u in polyme iza ion o 7,
simila o ha ob ained in hyd oe hanolic medium.
22
On he
o he hand, he an ioxidan ac i i y o oligo u in ob ained in
his wo k, de e mined by he TEAC (T olox Equi alen
An ioxidan Capaci y) assay, shows a dec ease o ∼55% in
adical-sca enging ac i i y when compa ed o u in (Table
S10), also simila o educ ions obse ed in he p e iously
desc ibed syn hesis.
22
Taking in o accoun he s aigh ela ion
be ween he fla onoid s uc u e and hei an ioxidan p ope -
ies,
44,45
bo h he deg ee o polyme iza ion and an ioxidan
capaci y sugges ha he oligo u in p oduced in ABS is
s uc u ally simila o ha ob ained in p e ious wo ks whe e a
mo e ex ensi e analysis o hei p ope ies and s uc u e was
pe o med, p oposing ha he possible bonds be ween
diffe en u in uni s mo e likely happen be ween C2′−C2′,
O4′−C6′, and C6′−C6′a oms p esen in he u in monome
s uc u e.
22,40
Reco e y o PEG 600 and [Ch][DHph]. Ru in oligome s
he e p oduced in an ABS eac ion medium we e concen a ed
in a PEG- ich phase, his polyme being app o ed by he FDA
o be used as a base o ehicle in oods, cosme ics, and
pha maceu ical p oduc s (including injec able, ec al, opical,
and nasal o mula ions).
46
This PEG- ich phase also con ains,
as abo e-men ioned, 8.47 w % [Ch][DHph], he oxici y o
his IL being defined as negligible compa able o ha o basic
physiologic sal s.
37,38
Ne e heless, aiming o inc ease he
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oligo u in pu i y, he implemen a ion o an ul afil a ion s ep
o isola e oligo u in was also s udied, u he allowing he PEG-
ich phase eco e y. As depic ed in Table S12, ul afil a ion o
dilu ed op oligo u in- ich phases caused an inc ease o 1.87
and 3.04- old in oligo u in pu i y in he final eeze-d ied
p oduc , wi h 1:2 and 1:4 aqueous dilu ions, espec i ely.
Indeed, by duplica ing he dilu ion ac o while e aining he
same olume inside o he ul afil a ion cell, an inc ease o
∼20% PEG 600 + [Ch][DHph] eco e y was achie ed (∼70%
eco e y o PEG 600 + [Ch][DHph] om he op phase),
which could be used o pa ially o mula e a no el op phase o
he ABS by simply adding he lacking amoun o bo h hese
compounds o each he co ec composi ion. Mo eo e ,
MALDI-TOF analysis o he e en a e ac ion shows signals
co esponding mainly o oligo u in, while low molecula
weigh u in oligome s appea in he pe mea e s eam in
which signals co esponding o PEG we e mo e ele an
(Figu e S9).
Li e-Cycle Assessmen . Al hough bio echnology p esen s
g ea oppo uni ies o he de elopmen o p oduc ion
pla o ms ha gene a ebioac i ecompounds ha mee
s aigh o wa d en i onmen al p o ec ion goals, hese no el
p oduc ion sys ems a e no always in he pa h o en i on-
men al sus ainabili y.
47
In his ega d, he o e all bio echno-
logical challenge mus be confi med conside ing he co e-
sponding en i onmen al assessmen . Fo his eason, he
en i onmen al benefi s associa ed wi h he ABS-based
eac ion−sepa a ion pla o m de eloped in his wo k (Sc1)
we e app aised by he Li e Cycle Assessmen (LCA)
me hodology and compa ed o h ee al e na i e p ocesses
allowing laccase euse o p oducing oligo u in, b iefly Sc2
in ol ing he same ABS-based s a egy bu including an
addi ional ul afil a ion s ep o euse [Ch][DHph] and PEG
600, Sc3 conside ing an enzyma ic memb ane eac o (EMR),
8
and Sc4 using laccase immobilized on o magne ic nano-
pa icles (mNPs),
29
as abo e-men ioned. No e ha in Sc2,
al hough a u he dilu ion would allow highe PEG 600 and
[Ch][DHph] eco e y (Table S12), da a om 1:2 dilu ion
s udies was conside ed; his choice was based on he need o
an addi ional s ep o educe he wa e con en in he eco e ed
ac ion ob ained in he ul afil a ion p ocess in ol ing 1:4
dilu ion, which would imply an inc ease in elec ici y
equi emen s and wa e consump ion associa ed wi h polyme
and ionic liquid eco e y. The en i onmen al p ofile o hese
p ocesses was s udied in e ms o acidifica ion (AC),
eu ophica ion (EU), global wa ming (GW), ozone laye
deple ion (OD), and pho ochemical oxida ion (PO), co e-
sponding o fi e impac ca ego ies commonly analyzed in
bio echnological s udies.
31,32,48
The main s eps, inpu s, ou -
pu s, and ene gy equi emen s o he diffe en scena ios a e
schema ized in Figu e S10, wi h he in en o y da a sou ces
summa ized in Table S13.
Acco ding o he esul s ob ained pe g am o oligo u in
( unc ional uni ) o each scena io, he e is a ema kable
diffe ence in he p ofiles ega dless o he impac ca ego y
conside ed o analysis, Sc1 being he mos en i onmen ally
iendly s a egy, as shown in Figu e 4. These esul s e ince
ha al hough he addi ion o an ul afil a ion s ep in Sc2
allowed he pa ial eco e y o PEG 600 and [Ch][DHph],
he e o e educing he consump ion o chemicals, i does no
o e come he impac s associa ed wi h he addi ional ul a-
fil a ion s ep equi ed when compa ed o Sc1. Mo eo e ,
impac educ ions highe han 97% a e iden ified in all
ca ego ies o Sc1 compa ed o Sc4, he scena io whe e mNPs
a e in ol ed o allow laccase euse, Sc4 being he p ocess wi h
he wo s en i onmen al p ofile. Specifically, o he pla o m
de eloped in his wo k (Sc1), he ollowing esul s we e
ob ained (Table S14): 5.41 g o SO2equi (AC), 1.30 g o
PO43−equi (EU), 0.80 kg o CO2equi (GW), 0.06 mg o
CFC-11 equi (OD), and 0.39 g o C2H4equi (PO).
The a ionale behind hese impac s is mainly associa ed wi h
elec ici y equi emen s (based on ossil esou ces), as can be
obse ed in Figu e S11, specifically elying on he eeze-d ying
s ep. Indeed, acco ding o he da a, elec ici y equi emen s in
Sc1 a e 61%, 81%, and 99% lowe han in Sc2, Sc3, and Sc4,
espec i ely. I is impo an o ema k ha al hough eeze-
d ying implies high elec ici y consump ion in all scena ios, he
s i ing is he en i onmen al ho spo (i.e., hep ocess
esponsible o he highes con ibu ions o he global p ofile)
in Sc1 and Sc2, obse ing an inc ease o 2.44 kWh/g oligo u in
in Sc2, associa ed wi h he PEG 600 and [Ch][DHph]
eco e y s ep. Howe e , diffe ences ela ed o he main sou ce
o con ibu ing bu dens o he PO ca ego y we e spo ed,
especially in Sc4, whe e he elec ici y equi emen was no he
ho spo bu he uncon olled emission o e hanol (which
ep esen s 30% o he olume o he eac ion medium) in o he
ai in he eeze-d ying s ep. Indeed, his emission ep esen ed
95% o he o al con ibu ing subs ances o he PO ca ego y. I
mus be s a ed ha his impac would be conside ably lowe i
his p ocess was u he op imized by including an e hanol
eco e y s ep, educing bo h e hanol consump ion and
emission. Howe e , a eeze-d ying s ep was assumed o his
LCA s udy ( ime o eeze-d ying based on p ima y
expe imen al da a) o each a ully d y final p oduc , he e o e
making his p ocess mo e easily compa able o he diffe en
scena ios.
I elec ici y- ela ed impac s we e wi hd awn om his
analysis (Figu e S12), he p oduc ion o chemicals, especially
PEG 600 in Sc1 and Sc2, would play a key ole in all he
impac ca ego ies s udied. Ne e heless, in Sc4, he mos
impo an p oduc ion o chemicals esponsible o all he
impac s would be he equi ed e hanol (con ibu ing a ios o
Figu e 4. Compa a i e p ofiles be ween he diffe en oligo u in
p oduc ion scena ios conside ed o analysis in e ms o acidifica ion
(AC), eu ophica ion (EU), global wa ming (GW), ozone laye
deple ion (OD), and pho ochemical oxida ion (PO). Blue ba : Sc1;
ed ba : Sc2; g een ba : Sc3; pu ple ba : Sc4.
ACS Sus ainable Chemis y & Enginee ing pubs.acs.o g/jou nal/ascecg Resea ch A icle
h ps://doi.o g/10.1021/acssuschemeng.1c03399
ACS Sus ainable Chem. Eng. 2021, 9, 9941−9950
9947
mo e han 90% in all he ca ego ies o he impac s associa ed
wi h chemicals p oduc ion, as can be obse ed in Figu e S13).
Finally, he no maliza ion ac o s om he selec ed me hod
o analysis ha e been conside ed o es ablish a final anking o
scena ios. This app oach, al hough con empo a y, is widely
used and well ecognized as i allows he di ec compa ison
be ween equi alen sys ems.
49
The sco es es ima ed o he
scena ios a e 0.502 ×10−13, 1.27 ×10−13, 1.76 ×10−13, and
49.4 ×10−13, o Sc1, Sc2, Sc3, and Sc4, espec i ely.
Acco dingly, Sc1 can be conside ed he bes scena io o
oligo u in p oduc ion allowing laccase euse om an en i on-
men al pe spec i e.
■CONCLUSION
ABS composed o biocompa ible ILs and polyme s we e he e
demons a ed o ac as an en i onmen ally iendly, efficien ,
and in eg a ed eac ion−sepa a ion pla o m o ca y ou he
bioca aly ic con e sion o u in in o oligo u in (a compound
wi h po en ial comme cial in e es ), u he allowing he euse
o he enzyme wi hou comp omising high p oduc ion yields.
O e all, ema kable esul s a e ob ained when he eac ion
akes place in he biphasic egime, benefi ing om bo h an
inc eased subs a e solubili y and an enhanced ca aly ic
pe o mance. These esul s open up a pa hway o he use o
IL-based ABS as sus ainable pla o ms o in eg a ed
bioca aly ic p ocesses.
■ASSOCIATED CONTENT
*
sıSuppo ing In o ma ion
The Suppo ing In o ma ion is a ailable ee o cha ge a
h ps://pubs.acs.o g/doi/10.1021/acssuschemeng.1c03399.
P elimina y s udy on oligo u in and laccase sepa a ion in
biocompa ible ABS; p o ocols used conce ning HPLC,
MALDI-TOF, and an ioxidan ac i i y (TEAC) de e -
mina ion and spec opho ome ic measu emen s; me h-
odology o ABS diag ams and ie-line de e mina ion
and esul s ob ained; de ailed in o ma ion on u in
con e sion, laccase ac i i y, %EElaccase ac i i y, and %
EEoligo u in o he diffe en expe imen s; esul s on he
eco e y o PEG 600 and [Ch][DHph] om he op
phases; da a needed o LCA analysis (PDF)
■AUTHOR INFORMATION
Co esponding Au ho s
Gemma Eibes −CRETUS, Depa men o Chemical
Enginee ing, Uni e sidade de San iago de Compos ela, 15782
San iago de Compos ela, Spain; o cid.o g/0000-0003-
2432-6323; Phone: +34 8818 16016;
Email: [email p o ec ed]
Ana P. M. Ta a es −CICECO−A ei o Ins i u e o Ma e ials,
Chemis y Depa men , Uni e si y o A ei o, 3810-193
A ei o, Po ugal; o cid.o g/0000-0001-9128-6275;
Phone: +351 234 401 520; Email: [email p o ec ed]
Au ho s
Abel Muniz-Mou o −CRETUS, Depa men o Chemical
Enginee ing, Uni e sidade de San iago de Compos ela, 15782
San iago de Compos ela, Spain; o cid.o g/0000-0002-
3199-5958
Ana M. Fe ei a −CICECO−A ei o Ins i u e o Ma e ials,
Chemis y Depa men , Uni e si y o A ei o, 3810-193
A ei o, Po ugal; o cid.o g/0000-0003-3057-5019
Joao A. P. Cou inho −CICECO−A ei o Ins i u e o
Ma e ials, Chemis y Depa men , Uni e si y o A ei o,
3810-193 A ei o, Po ugal; o cid.o g/0000-0002-3841-
743X
Ma a G. F ei e −CICECO−A ei o Ins i u e o Ma e ials,
Chemis y Depa men , Uni e si y o A ei o, 3810-193
A ei o, Po ugal; o cid.o g/0000-0001-8895-0614
Pa icia Gullón −Labo a o io C.A.C.T.I.−PQ Tecnolóxico de
Galicia, Tecnopole, 32901 San Cib ao das Vinas, Ou ense,
Spain
Sa a González-Ga cía −CRETUS, Depa men o Chemical
Enginee ing, Uni e sidade de San iago de Compos ela, 15782
San iago de Compos ela, Spain
Comple e con ac in o ma ion is a ailable a :
h ps://pubs.acs.o g/10.1021/acssuschemeng.1c03399
Au ho Con ibu ions
The manusc ip was w i en h ough con ibu ions o all
au ho s. G.E., A.M.F., A.P.M.T, M.G.F., S.G-G., and A.M-M.
concei ed and designed he expe imen s. A.M-M. pe o med
all he expe imen s. A.M-M., G.E., A.M.F., A.P.M.T, M.G.F.,
S.G-G., P.G., and J.A.P.C. analyzed he da a. All au ho s
con ibu ed o he w i ing o his pape . All au ho s ha e gi en
app o al o he final e sion o he manusc ip .
No es
The au ho s decla e no compe ing financial in e es .
■ACKNOWLEDGMENTS
This esea ch was suppo ed by he Spanish Go e nmen
(AEI) h ough he RTI2018-094482-J-I00 p ojec . This wo k
was de eloped wi hin he scope o he p ojec CICECO-A ei o
Ins i u e o Ma e ials, UIDB/50011/2020 & UIDP/50011/
2020, financed by na ional unds h ough he Po uguese
Founda ion o Science and Technology/MCTES. The
au ho s A.M.-M. and G.E. belong o he Galician Compe i i e
Resea ch G oup (GRC). The p og amme is co unded by
FEDER (UE). The au ho s acknowledge he use o RIAIDT-
USC analy ical acili ies. A.P.M.T. hanks he FCT o he
esea ch con ac CEECIND/2020/01867. G.E. hanks he
Spanish MICIU o he Ramón y Cajal con ac (RYC2018-
024846-I). A.M.-M. hanks he P og ama de axudas á e apa
p edou o al da Xun a de Galicia (ED481A-2018/023).
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