A i icial Manganese Me alloenzymes wi h Laccase-like Ac i i y:
Design, Syn hesis, and Cha ac e iza ion
Ca la Ga cia-Sanz, Alicia And eu, Mi osława Pawly a, Ana Vukoicic, Ana Mili oje ic, Blanca de las Ri as,
Dejan Bezb adica, and Jose M. Palomo*
Ci e This: ACS Appl. Bio Ma e . 2024, 7, 4760−4771
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sı Suppo ing In o ma ion
ABSTRACT: Laccase is an oxidase o g ea indus ial in e es due
o i s abili y o ca alyze oxida ion p ocesses o phenols and pe sis en
o ganic pollu an s. Howe e , i is suscep ible o dena u a ion a high
empe a u es, sensi i e o pH, and uns able in he p esence o high
concen a ions o sol en s, which is a issue o indus ial use. To
sol e his p oblem, his wo k de elops he syn hesis in an aqueous
medium o a new Mn me alloenzyme wi h laccase oxidase mime ic
ca aly ic ac i i y. Geobacillus he moca enula us lipase (GTL) was
used as a sca old enzyme, mixed wi h a manganese sal a 50 °C in
an aqueous medium. This leads o he in si u o ma ion o
manganese(IV) oxide nanowi es ha in e ac wi h he enzyme,
yielding a GTL−Mn bionanohyb id. On he o he hand, i s oxida i e
ac i i y was e alua ed using he ABTS assay, ob aining a ca aly ic
e iciency 300 imes highe han ha o T ame es e sicolo laccase. This new Mn me alloenzyme was 2 imes mo e s able a 40 °C, 3
imes mo e s able in he p esence o 10% ace oni ile, and 10 imes mo e s able in 20% ace oni ile han No ozym 51003 laccase.
Fu he mo e, he si e-selec i e immobilized GTL−Mn showed a much highe s abili y han he soluble o m. The oxidase-like
ac i i y o his Mn me alloenzyme was success ully demons a ed agains o he subs a es, such as L-DOPA o phlo idzin, in
oligome iza ion eac ions.
KEYWORDS: me alloenzymes, manganese, nanowi es, a i icial enzymes, laccase-like ac i i y
■INTRODUCTION
Laccase (EC 1.10.3.2, p-diphenol:dioxygen oxido educ ase) is
an enzyme belonging o he g oup o blue coppe oxidases.
1,2
I
con ains oxido educ ases ha couple he educ ion o oxygen
o wa e by ou -elec on educ ion wi h he oxida ion o a
wide ange o o ganic and ino ganic subs a es including
phenols as well as some o ganic subs ances conside ed o be
pe sis en o ganic pollu an s (POPs), anilines, and a oma ic
hiols.
3−5
Due o i s b oad subs a e ange and mul iple oles,
laccase has become a p ominen esea ch a ge in ecen yea s.
As a esul , laccase has wide applica ions in biosensing and
en i onmen al emedia ion, as well as in he ood, pape , and
cosme ics indus ies.
6−9
Indeed, in he las ew yea s, one pa
o he esea ch ocused on enzyma ic applica ions o laccases,
whe eas he o he pa ocused on he p oduc ion o no el
polyphenolic compounds, which ha e been desc ibed as
p omising p ebio ics�chemicals ha a e speci ically used by
hei hos mic obes and ha e a a o able e ec on he
composi ion o he human mic obio a.
10,11
Howe e , na u al laccases ha e se e al d awbacks. The
p ac ical applica ion o na u al laccase is limi ed by i s high
cos , poo s abili y (pH, empe a u e, and s o age ime),
di icul ies in ha sh en i onmen s, sepa a ion p oblems, and
poo eusabili y.
12
Immobiliza ion o he enzyme usually
inc eases i s s abili y and eusabili y.
13
This may be due o
he in e ac ion be ween he ma ix and enzyme, which
acili a es he s abiliza ion o he pep ide wi hin he enzyme.
14
Howe e , enzyme ac i i y on he suppo may be los i he
immobiliza ion me hod al e s he s uc u e o he enzyme, and
he sea ch o a low-cos ca ie ha does no in e e e wi h he
ac ion o he enzyme is s ill a p oblem.
15
While he e is a
necessi y o enhance he s abili y and ecyclabili y o
immobilized laccases, he ongoing explo a ion o new
enzymes wi h heigh ened speci ici y o di e se applica ions
is also unde way. To o e come hese sho comings, e o s
ha e been made o de elop enzyme mimics. A pa icula ly
in e es ing a ea o esea ch in ecen yea s has been he
syn hesis o no el a i icial me alloenzymes by combining
me al o complex o ganome allic sys ems wi h enzymes. This
Recei ed: Ap il 28, 2024
Re ised: June 13, 2024
Accep ed: June 17, 2024
Published: June 25, 2024
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a ea o s udy is g owing bo h in e ms o design and
applica ions.
16
A i icial me alloenzymes (A Ms) esul om he inco po-
a ion o an abio ic me al co ac o wi hin a p o ein sca old.
17
Fou s a egies ha e been epo ed o he syn hesis o A Ms.
18
The i s app oach ocuses on Lewis base amino acids a anged
in a ca i y ha can in e ac wi h a coo dina i e unsa u a ed
me al (co ac o ) h ough da i e bonding. The second s a egy
is whe e he na i e me al o a me alloenzyme can be eplaced
by ano he me al, gi ing he p o ein new ca aly ic ac i i y. The
me al can be a ached only o amino acids, as in he case o
ca boxypep idase A, o i can be a membe o a p os he ic
g oup such as heme. The hi d is based on sup amolecula
in e ac ions be ween a high-a ini y inhibi o and a hos
p o ein, which can be used o bind a me al co ac o . The la e
ocuses on co alen immobiliza ion, which can be achie ed by
i e e sible in e ac ions be ween complemen a y unc ional
g oups on he hos p o ein o ligand.
19−21
Signi ican p og ess
has been made in he design and op imiza ion o a i icial
me alloenzymes using hese ou ancho ing echniques. As a
esul , i has been possible o gene a e me alloenzymes ha a e
mo e selec i e and mo e ac i e han hose ound in na u e, as
well as me alloenzymes wi h unique ca aly ic p ope ies.
22,23
Resea ch in ansi ion-me al manganese-based ca alys s has
ecen ly inc eased due o hei excep ional ca aly ic capabili y
and mul i alen na u e.
24
Mn-based nanoma e ials a e widely
used in ma e ials science, elec onics, en i onmen al p o ec-
ion, and biomedicine because o hei ease o syn hesis, low
cos , en i onmen al iendliness, and excellen physicochemical
p ope ies.
25−27
Wi hin his ield, manganese oxide nanoma-
e ials ha e been epo ed o ha e laccase-like ac i i y.
28,29
Manganese oxides (MnOx) a e a majo componen o soils
and sedimen s and occu na u ally in o e 30 di e en c ys al
o ms. These o ms a e in ol ed in se e al na u al chemical
p ocesses. Ce ain MnOx a e capable o oxidizing subs a es by
he ans e o a single elec on, while he esul ing educed
manganese oxides MnOx ed can be eoxidized o MnOx by
dissol ed oxygen ha is educed o wa e unde ce ain
condi ions leading o a ne esul o elec on shu ling om
subs a es o oxygen, like laccase.
30
In addi ion, manganese
oxides and laccase ha e compa able eac i e capaci ies due o
hei common subs a es, including ABTS (2,2′-azino-bis [3-
e hylbenzo hiazoline-6-sul onic acid]-diammonium sal ). Re-
cen ly, a ew examples ha e been desc ibed in he li e a u e
ega ding manganese oxide ca alys s wi h laccase-like ac i -
i y.
31,32
Howe e , in mos cases, hey ha e la ge nanopa icle
sizes and low s abili y. They also equi e complex syn hesis
condi ions.
The e o e, in his wo k, we desc ibe a new s a egy o he
syn hesis and design o a i icial manganese me alloenzymes
based on he in si u gene a ion o manganese nanopa icles
coo dina ed o he enzyme s uc u e om manganese sal s o
c ea e an enzyme−MnNPs bioconjuga e wi h mime ic laccase-
like ac i i y (Figu e 1).
This equi es he selec ion o an enzyme ype wi h a obus
s uc u e and cha ac e is ics ha imp o e he s abili y and
p ope ies o he laccase. Geobacillus he moca enula us (GTL)
is a he mo-alkalophilic lipase wi h high s abili y o e a wide
ange o pH (9−11), empe a u e (50 °C), and o ganic
sol en s (2-p opanol, ace one, me hanol).
33
GTL has wo
di e en lids (L1 and L2) in i s s uc u e, making i much mo e
complex as i in ol es he mo emen and ea anging o 80
amino acids in he ca aly ic mechanism. In addi ion, his lipase
has been epo ed o ha e high speci ici y o a wide ange o
subs a es and high selec i i y o esol ing key in e media es
in d ug syn hesis.
34
These p ope ies make his enzyme ideal o being used as a
sca old o he syn hesis o he enzyme−MnNPs biohyb ids.
Thus, he hypo hesis consis s in he gene a ion o in si u
manganese nanopa icles induced by he enzyme, whe e hey
will be o med only on he p o ein, by a p e ious coo dina ion
s ep o manganese ions o he enzyme esidues and hen
coalescence and inal nanopa icle o ma ion.
35−37
The e ec s
o he enzyme en i onmen on he syn hesis, mo phology, and
size o MnNPs we e s udied. Finally, he di e en a i icial
me alloenzymes we e es ed as ca alys s in he oxida i e
p ocesses.
■EXPERIMENTAL SECTION
Chemicals. P ime S a HS Taka a DNA polyme ase was ob ained
om Taka a Bio echnology (Jusa su, Japan). PCR eac i es we e
pu chased om Applied Biosys ems (MA). P ime syn hesis was
conduc ed by Fishe Scien i ic. Recombinan plasmid was sequenced
by Secugen S.L. (Mad id, Spain). Res ic ion enzyme DpnI was
p o ided by Roche (Basel, Swi ze land). Bu yl-Sepha ose 4 Fas Flow
was om GE Heal hca e (Uppsala, Sweden). p-Ni ophenylp opio-
na e (pNPP) was ob ained om Al a Aesa (MA). 2,2′-Azino-bis [3-
e hylbenzo hiazoline-6-sul onic acid]-diammonium sal (ABTS) was
pu chased om The mo Scien i ic (MA). T i on X-100, dialysis
ubing cellulose (a g. la 33 mm), sodium ci a e, sodium phospha e,
L-DOPA, phlo idzin, po assium pe mangana e (KMnO4), hyd ogen
pe oxide (33% / ), and laccase om T ame es e sicolo we e
p o ided by Sigma-Ald ich (MA). Laccase om Mycelioph ho a
he mophila exp essed in Aspe gillus o yzae (No ozym 51003) was
om No ozymes (Bags ae d, Denma k). HPLC-g ade ace oni ile
was ob ained om Scha lab (Ba celona, Spain).
Gene al P ocedu e o he Syn hesis, Pu i ica ion, and
Cha ac e iza ion o Manganese Me alloenzymes in he
Colloidal S a e. 400 μL (70 μg p o ein) o he GTL enzyme
solu ion (see he Suppo ing In o ma ion) was added o 3.6 mL o a
solu ion con aining 0.5 mg/mL (2000 equi ), 0.12 mg/mL (500
equi ), o 0.05 mg/mL (200 equi ) o he po assium pe mangana e
sal (KMnO4) in dis illed wa e . This solu ion was kep o 20 h unde
cons an s i ing a 130 pm a 50 °C.
The manganese nanopa icle o ma ion was ollowed by
spec opho ome ic measu emen o he disappea ance o he
cha ac e is ic band o MnO4
−a 520−550 nm and he appea ance
o a peak a 367 nm co esponding o he o ma ion o MnO2. A e
20 h, he manganese me alloenzymes we e ob ained in he colloidal
s a e. Thei colo a ies om he ini ial pu ple o da k o ange-b own
depending on he added concen a ion o he KMnO4sal .
Finally, a memb ane wi h a molecula weigh cu o o 14 kDa was
used o he dialysis pu i ica ion o he manganese me alloenzymes.
This echnique makes i possible o pu i y he me alloenzyme by
emo ing he pe mangana e sal emaining in he colloidal solu ion.
They we e kep unde agi a ion o 4 h a oom empe a u e in a
beake con aining 1 L o dis illed wa e , changing he wa e e e y 30
Figu e 1. Concep ual model o he manganese me alloenzymes as
laccase mimics p oposed in his wo k.
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4761
min (3 imes). A e his ime, 1 mL o he pu i ied manganese
me alloenzymes (0.07 mg/mL) we e ob ained in he colloidal s a e.
The syn hesized manganese me alloenzymes we e called GTL@
Mn2000eq,GTL@Mn500eq, and GTL@Mn200eq.
P epa a ion o Immobilized GTL−Mn Me alloenzymes. 0.1 g
o he immobilized enzyme (Bu−GTL)
34
was added o 9 mL o a
solu ion con aining 0.5 mg/mL (2000 equi ), 0.12 mg/mL (500
equi ), o 0.05 mg/mL (200 equi ) o po assium pe mangana e sal
(KMnO4) in dis illed wa e . This solu ion was kep o 20 h unde
cons an shaking a 130 pm a 50 °C o 20 h. A e his ime, he
solid o each sample was eco e ed by il e ing his solu ion unde
acuum, and hen i was washed wi h dis illed wa e (3 imes, 20 mL)
o ob ain 0.1 g o he immobilized de i a i e. The syn hesized
manganese me alloenzymes we e called BuGTL@Mn2000eq,
BuGTL@Mn500eq, and BuGTL@Mn200eq.
Fluo escence Spec oscopy Measu emen s. A oom empe -
a u e, 3 mL o he co esponding colloidal Mn me alloenzyme was
placed in a qua z cu e e wi h a pa h leng h o 1 cm, and he
exci a ion−emission spec a o he ee GTL and he syn hesized
manganese me alloenzymes we e measu ed. The exci a ion wa e-
leng h was 280 nm, and he emission and exci a ion bandwid hs we e
5 nm. The luo escence emission spec a we e ob ained be ween 200
and 500 nm.
Gel Fil a ion o GTL−Mn Me alloenzymes. Gel il a ion
analyses we e pe o med using a plas ic column packed wi h beaded
aga ose-4BCL (column size 15 nm ×160 nm; column bed olume 8
mL). The elu ing bu e was 10 mM sodium phospha e, pH 7.0; all
sepa a ions we e ca ied ou a 25 °C wi h a low a e o 1.2 mL/min,
whe e 1 mL o GTL@Mn2000eq was added o he column and
elu ed wi h 15 mL o he indica ed bu e . The elua e was collec ed in
0.5 mL aliquo s and he laccase-like ac i i y was de e mined by he
ABTS assay a 420 nm. The molecula weigh o GTL@Mn2000eq
was es ima ed using s anda d p o eins, laccase solu ion om M.
he mophila exp essed in A. o yzae (No ozym 51003) (85 kDa),
Lipase B Candida an a c ica (CALB) (33 KDa), and GTL con aining
0.5% ( / ) T i on X-100 (43 kDa).
The ac i i y o laccase was de e mined using he ABTS assay, while
o CALB and GTL he enzyma ic ac i i y was de e mined using he
pNPP assay a 348 nm.
E alua ion o he Laccase-like Ac i i y o he Manganese
Me alloenzymes (ABTS Assay). The ABTS assay was used o
e alua e he laccase-like ac i i y o he manganese me alloenzymes.
To s a he eac ion, 5 μL o a 0.07 mg/mL dialyzed laccase solu ion
om M. he mophila exp essed in A. o yzae (No ozym 51003), 50 μL
o a 0.07 mg/mL laccase solu ion om T. e sicolo , o di e en
amoun s o he manganese me alloenzymes in he colloidal s a e, i.e.,
5μL (GTL@Mn2000eq), 15 μL (GTL@Mn500eq) o 50 μL
(GTL@Mn200eq), o 50 μL o an emulsion o suppo ed
me alloenzymes (9 mg o he me alloenzyme suppo ed and 500 μL
o dis illed wa e ), we e added unde cons an s i ing o 2 mL o a
s anda d 0.5 mM ABTS solu ion p epa ed in a 1:1 ( / ) a io o 0.1
M sodium ci a e bu e a pH 5 and 0.1 M sodium phospha e bu e
a pH 5. A e he addi ion o he Mn me alloenzyme, he solu ion
changed om anspa en o u quoise blue due o he o ma ion o
he adical species (ABTS+·). This colo change was moni o ed by
measu ing he abso bance (λ= 420 nm) in he kine ic p og am a
oom empe a u e in a 1 cm pa h plas ic cu e e.
To de e mine he laccase-like ac i i y o each me alloenzyme, he
ΔAbs/min alue was calcula ed using he linea po ion o he cu e
(ΔAbs). The speci ic ac i i y (U/mg) was calcula ed using he
ollowing equa ion
U V( moL min mg ) Abs/min 1000
mg
1 1
enzyme
· · = · ·
·
whe e he mola ex inc ion coe icien (ε) o ABTS used was 36,000
M−1cm−1and mg enzyme e e s o mg o p o ein in he me alloenzyme.
S abili y o he Mn Me alloenzymes. The s abili y o di e en
Mn me alloenzymes was e alua ed by incuba ing hem om 2 o 24 h
a di e en empe a u es (40 °C), di e en pHs (25 mM sodium
phospha e a pH 4 and pH 8), o in he p esence o ace oni ile as a
cosol en (10%, 20% ( / )). Then, he laccase-like ac i i y was used
o moni o ing he s abili y, conside ing he ac i i y o a i icial
me alloenzymes a 25 °C in each case as he 100% alue. The ac i i y
was de e mined by using he ABTS assay desc ibed abo e.
L-DOPA Oxida ion. In a 1 cm op ical leng h plas ic cu e e, 50 μL
o GTL@Mn2000eq o ee laccase (No ozym 51003) we e added o
2 mL o a s anda d 1 mM solu ion o L-DOPA (3,4-dihyd oxy-L-
phenylalanine) in 0.1 M sodium phospha e bu e a pH 5 con aining
O2(84 ppm). The solu ion changed om anspa en o a eddish
colo due o he oxida ion o L-DOPA o dopach ome. This colo
change was moni o ed by measu ing i s ca aly ic ac i i y in a
ul a iole − isible (UV− is) abso p ion spec um a 475 nm in he
kine ic p og am. An enzyme ac i i y uni (U) was de ined as he
amoun o enzyme causing an inc ease o abso bance by 0.001/min a
25 °C.
37
Syn hesis o Phlo idzin Oligome s. Oligome iza ion eac ions
we e pe o med in 50 mL E lenmeye lasks on an o bi al shake a
150 pm, in wa e , a a empe a u e o 40 °C. The o al eac ion
mix u e olume was 3 mL. Phlo idzin concen a ion was 2 mg/mL,
and he eac ion was s a ed using 60 mg o me alloenzyme. A e 24
h, he eac ion was s opped by emo ing he bioca alys , GTL@
Mn500eq. Con ol samples wi hou enzymes we e also p epa ed, and
no p oduc s we e de ec ed. The samples we e hen analyzed on
HPLC-UV.
HPLC-UV and HPLC-MS Analysis o Oligome iza ion
P oduc s. Fo he analysis o he eac ion mix u e, e e se-phase
high-pe o mance liquid ch oma og aphy (HPLC) wi h UV− is
de ec ion (Dionex Ul iMa e3000 HPLC sys em, The mo Scien i ic)
was used wi h Ch omeleon 7.2 o da a analysis. The analysis was
pe o med wi h a ZORBAX Ex end-C18 column (4.6 mm ×100 mm,
pa icle diame e 3.5 μm, Agilen Technologies, San a Cla a) wi h a
se empe a u e o 30 °C. As mobile phases, 0.1% ( / ) o mic acid
solu ion in deionized wa e (phase A) and ace oni ile (phase B) we e
used. A g adien elu ion was used as ollows: 0−5 min 0−15% B, 5−
35 min 15−40% B. The low a e was 0.5 mL/min, and he de ec ion
wa eleng h was 280 nm. High-pe o mance liquid ch oma og aphy−
mass spec ome y (HPLC-MS) analysis o he s a ing monome and
he ob ained eac ion mix u e was pe o med using a DionexUl iMa e
3000 HPLC sys em (The mo Scien i ic) coupled o a linea ion ap
LTQ XL (The mo Scien i ic). The p e iously desc ibed me hod o
ch oma og aphic sepa a ion on a ZORBAX Ex end-C18 column was
applied. Analy es we e ionized using elec osp ay ioniza ion (ESI)
echnique in he nega i e mode, o ming dep o ona ed molecula
ions. The op imal ion sou ce pa ame e s we e as ollows: sou ce
ol age (5 kV), shea h gas (18 au, i.e., eigh een a bi a y uni s), and
capilla y empe a u e (270 °C). To al ion ch oma og ams (TIC) we e
ob ained by eco ding mass spec a in he ange m/z50−2000.
■RESULTS AND DISCUSSION
Syn hesis and Cha ac e iza ion o Colloidal GTL@
MnNPs Me alloenzymes. The i s s ep was o p oduce and
pu i y he GTL enzyme.
38−40
Fo ha pu pose, he enzyme
was ini ially adso bed on a bu yl-sepha ose suppo (a
echnique ha allows selec i ely e e sible adso p ion o
lipases in he p esence o o he p o eins).
38
Then, once all o
he lipase a ian s we e abso bed ( es ed by enzyme ac i i y),
he immobilized de i a i e was ea ed in he p esence o a
bu e ed solu ion con aining T i on X-100 (pH 7) o
selec i ely deso b he a ian and eco e he enzyme in
solu ion. This allows he GTL p o ein molecules o be
ob ained in an open o m, s abilized by he de e gen
molecules (Figu e S1).
33
To p epa e he a i icial manganese me alloenzymes, a ious
solu ions o manganese sal s (0.5 mg/mL) such as manganese-
(II) chlo ide e ahyd a e (MnCl2·4H2O), manganese(II)
ace a e e ahyd a e ((CH3COO)2Mn·4H2O), manganese(II)
sul a e monohyd a e (MnSO4·H2O), and po assium pe man-
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4762
gana e (KMnO4) we e p epa ed in dis illed wa e and added o
he p e iously ob ained enzyme (Figu e 2).
In he i s a emp , he syn hesis was pe o med a oom
empe a u e. Howe e , no o ma ion o MnNPs was obse ed
a his empe a u e (da a no shown). This can be explained by
he he mophilic na u e o he GTL enzyme ( he mos able up
o 45 °C). I he e o e equi es empe a u es abo e 45 °C o
ac i a ion. Thus, in he second app oach, he solu ion was
incuba ed wi h cons an s i ing a 50 °C. A his empe a u e,
syn hesis was only ob ained when po assium pe mangana e
was used in he p esence o he enzyme, as indica ed by a
isible colo change o he solu ion ( om pu ple o o ange),
indica ing he o ma ion o MnNPs (Figu e S2). No syn hesis
was obse ed o he o he sal s, as he solu ion emained clea
(Figu e S3). The pe mangana e sal was, he e o e, he choice
o he syn hesis o he me alloenzymes.
Di e en concen a ions o po assium pe mangana e
(KMnO4) we e p epa ed in dis illed wa e and added o he
deso bed GTL, gi ing h ee samples wi h di e en Mn
equi alen s: 2000 equi (0.5 mg/mL), 500 equi (0.12 mg/
mL), and 200 equi (0.05 mg/mL). The o ma ion o
me alloenzymes was ollowed by UV− is spec oscopy by
obse ing a s eady dec ease o all ou abso p ion maxima
co esponding o KMnO4(506, 525, 545, and 566 nm) and
he o ma ion o a b oad dis inc i e peak a ound 360−370 nm
co esponding o he MnO2nanopa icles.
41
I is impo an o
no e ha his colo change is exclusi ely due o he educing
capaci y o he enzyme, as i was no obse ed when he
enzyme was no added (da a no shown).
To unde s and he o ma ion o me alloenzymes, he
syn hesis o me alloenzymes was s udied du ing he i s 20
min o incuba ion. Enzyme ac i i y was measu ed using he
pNPP assay, whe e mos o he enzyme ac i i y was los . This
may be ela ed o he o ma ion o MnNPs in he ac i e si e o
he p o ein. To unde s and his, he luo escence o he
me alloenzyme was measu ed. A sligh dec ease in luo escence
was obse ed, due o he shielding o he yp ophan esidues,
as well as a shi owa d he blue side o he spec a, indica ing
coo dina ion o he enzyme wi h he me al (Mn). This was
con i med by powde X- ay di ac ion (XRD), as he spec um
showed he ini ial o ma ion o MnNPs, al hough he solu ion
Figu e 2. Schema ic illus a ion o he syn hesis o Mn me al-
loenzymes.
Figu e 3. Cha ac e iza ion o he di e en manganese me alloenzymes. (a) UV-abso bance spec opho ome e spec a. (b) Fluo escence spec a
(exci a ion wa eleng h 280 nm). (c) XRD pa e n o GTL@Mn2000eq.
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was s ill pu ple (Figu e S4). The solu ion u ned o ange-
eddish a e 2 h, indica ing he p og ess o he educ ion
eac ion, and inally da k o ange-b own (20 h), indica ing he
onse o he o ma ion o MnO2NPs
42
(Figu e S5). The
maximum o ma ion was ob ained a e 20 h incuba ion
(Figu e S6).
The highes Abs peak alue was 0.7 o GTL@Mn2000eq a
367 nm when using 2000 equi (Figu e 3a). Lowe
concen a ions o Mn (500 and 200 equi ) esul ed in he
o ma ion o MnNPs wi h lowe abso bance (0.29 and 0.13
Abs o GTL@ Mn500eq and GTL@Mn200eq, espec i ely)
(Figu e 3a). These esul s sugges he highe he abso bance,
he highe he concen a ion o manganese in he bioconjuga e.
Fu he mo e, he coo dina ion be ween he enzyme and he
MnNPs is simila in all cases, as he peak wa eleng h does no
shi be ween he samples. In addi ion, luo escence analysis
e ealed ha he h ee-dimensional s uc u e o he p o ein
changes as a esul o he manganese coo dina ion (Figu e 3b)
since he maximum o he na i e enzyme peak (303 nm) is
sligh ly shi ed owa d he blue side o he spec um by λ= 2
nm (301 nm) (Table S1). Nea ci cula dich oism analysis also
con i med he e ec on he p o ein s uc u e by me al
coo dina ion (Figu e S7). This has p e iously been epo ed
wi h o he modi ied p o eins.
43
In addi ion, highe T p-
quenching was obse ed o GTL@Mn2000eq, whe eas
GTL@Mn200eq p esen ed a lowe one. This shows ha
while he ee con o ma ion o lipase is mo e open, he
conjuga ion wi h manganese modi ies he h ee-dimensional
s uc u e, esul ing in a dec ease in luo escence in ensi y and a
shi in he con o ma ion o he lipase�p obably o a mo e
closed o m han he na i e one.
34
Thus, highe concen a ions
o manganese (500 o 2000 equi ) lead o g ea e coo dina ion
o he me al wi h he p o ein and consequen ly o g ea e
shielding o he yp ophan esidues, which in u n educes he
signal. Fu he , inc easing he equi alen o Mn (5000 equi )
esul ed in sa u a ion and p ecipi a ion o he enzyme (da a
no shown).
Figu e 4. Cha ac e iza ion o manganese me alloenzymes (a) GTL@Mn2000eq, (b) GTL@Mn500eq, (c) GTL@Mn200eq: (I) ansmission
elec on mic oscopy (TEM), (II) high- esolu ion TEM (HR-TEM), and (III) HAADF spec a.
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Nex , powde X- ay di ac ion (XRD) analysis was used o
cha ac e ize he manganese s uc u es gene a ed in he
conjuga ion wi h he p o ein (Figu es 3c and S8). The XRD
pa e n shows he peaks o 2θa 37°(210), 38.5°(400), and
65.4°(020) co esponding o he γ-MnO2polymo ph species
in he sample (JCPDS 14−0644).
44
High- esolu ion ansmission elec on mic oscopy (HR-
TEM) e ealed he o ma ion o c ys alline Mn nanowi e
s uc u es ( ilamen s o sho leng h) in all o he colloidal
GTL−Mn bioconjuga es (Figu es S9−S11). This mo phology
has been desc ibed as cha ac e is ic o he γ-MnO2
polymo ph.
45
GTL@Mn2000eq showed he o ma ion o
nanowi es (NWs) wi h a size o abou 60 nm ×3 nm (Figu e
4a), whe eas smalle leng hs we e ob ained o o he
manganese concen a ions wi h an a e age size o 40 ×3
and 20 ×3 nm2 o GTL@Mn500eq (Figu e 4b) and GTL@
Mn200eq (Figu e 4c), espec i ely. The e o e, hese esul s
sugges ha he highe he concen a ion o manganese in he
bioconjuga e, he longe he leng h o he nanowi es.
Fu he mo e, hese nanowi es appea o be o med by he
agg ega ion o sphe ical nanopa icles wi h an a e age diame e
o 3.6 nm, as can be obse ed in GTL@Mn2000eq (Figu e
4c,II).
The chemical composi ion o he nanopa icles was
de e mined by high-annula da k- ield imaging (HAADF)
(Figu e 4(III)). This echnique con i ms he p esence o Mn
and O in he sample, wi h he oxygen signal being wice as high
as he manganese signal, indica ing he p esence o MnO2. The
Cu peaks co espond o he signal de ec ed by he TEM g id,
while he P signal belongs o he bu e .
I is well known ha me al sal s wi h lipases in homogeneous
aqueous media ha e a s ong endency o o m oligome ic
s uc u es.
46
To see i his pe o mance was conse ed in he
Mn me alloenzymes, GTL@Mn2000eq was e alua ed by gel
il a ion ch oma og aphy (Figu e 5). The eluen used in bo h
cases was 10 mM phospha e bu e , pH 7. Unde hese
condi ions, only 43 kDa monome s we e obse ed in GTL in
he p esence o 0.5% ( / ) T i on X-100. In e es ingly,
chemical modi ica ion wi h manganese is able o change he
o m o he na i e enzyme as a di e en elu ion p o ile was
ob ained.
The molecula weigh o GTL@Mn2000eq was es ima ed
om a calib a ion cu e plo ed using s anda d p o eins
(Figu es S12−S14). The esul s showed he o ma ion o a
ime wi h a molecula weigh o 129 kDa.
Finally, he Mn con en in he me alloenzymes was
de e mined by induc i ely coupled plasma op ical emission
spec oscopy (ICP-OES) analysis. Resul s e ealed a a io o
molecules o manganese pe enzyme molecule o 345 in
GTL@Mn2000eq, 112 in GTL@Mn500eq, and 29 in GTL@
Mn200eq (Table S2).
To unde s and he esul s ob ained in he syn hesis o Mn
me alloenzymes, bioin o ma ics analysis o he enzyme
s uc u e was pe o med. In he coo dina ion o he me al
wi h he enzyme, Mn ions p e e o bind “ha d” ligands such as
oxygen o Asp and Glu.
47
Some imes, he N a oms o his idine
can also bind Mn ligands in me alloenzymes.
47
Syn hesis ook
place a pH 5.5, meaning ha in his case he imidazole ing o
his idine is p o ona ed (pKa6) and he e o e MnNPs could
bind o enzymes mainly h ough he ca boxyl g oups o
glu amic and aspa ic esidues (Figu e 6).
The manganese nanopa icles will bind o he p o ein’s mos
exposed ca boxyl g oups, conse ing he nega i e cha ge ee,
no coo dina ed wi h neighbo ing NH3+ g oups (e.g., a ginine
esidues). The analysis o he c ys allog aphic h ee-dimen-
sional p o ein s uc u e shows ha Asp 366 (in he ou e pa
o he oxyanion hole) seems o be he mos p obably accessible
ca boxylic g oup on he p o ein su ace o be able o o m he
ime ic s uc u e obse ed in Figu e S15, wi h he di e en
p o ein molecules being connec ed by he MnNPs coo dina-
ion. This could also be linked o he loss o enzyma ic ac i i y
as he nanopa icles block subs a e access o he ac i e si e.
Ano he aspa ic acid is in ol ed in he coo dina ion si es o
he es o he MnNPs gene a ed.
E alua ion o he Laccase-like Ac i i y o he
Manganese Me alloenzymes. Fi s , he laccase-like ac i i y
o he manganese me alloenzymes was e alua ed in he
selec i e oxida ion o ABTS o ABTS+·. The eac ion was
ca ied ou in an aqueous medium and a oom empe a u e
and compa ed wi h he ee solu ion o he na u al laccase om
T. e sicolo and laccase om M. he mophila exp essed in A.
o yzae (No ozym 51003).
Figu e 7 shows ha he oxida i e ac i i y o he me al-
loenzymes inc eased wi h he Mn equi alen s, wi h he highes
speci ic ac i i y being ob ained o GTL@Mn2000eq (73 U/
Figu e 5. Elu ion p o ile in gel il a ion ch oma og aphy o GTL@
Mn2000eq (blue line) and GTL con aining 0.5%( / ) T i on X-100
(o ange line).
Figu e 6. (a) Su ace c ys al s uc u e o he ac i e con o ma ion o
GTL, wi h ma ked aspa ic acid (o ange) and glu amic acid (g een)
esidues. (b) Ca oon o he ac i e si e o GTL, wi h ma ked aspa ic
acid (pink) and en i onmen esidues (o ange). The p o ein s uc u e
was ob ained om he P o ein Da a Bank (PDB code: 2W22), and
he pic u e was c ea ed using Pymol.
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mg). This alue was h ee imes highe han he one ob ained
o GTL@Mn-500 eq (20 U/mg) and 12- old highe han ha
o GTL@Mn-200 eq (6 U/mg). This high ca aly ic pe o m-
ance could be a ibu ed o he p esence o γ-MnO2NPs in
hei s uc u e, as i has been desc ibed in he li e a u e ha
his manganese polymo ph is he mos po en in oxidizing
ABTS.
31
When he ABTS ac i i y alues o he Mn me alloenzymes
a e compa ed wi h hose ob ained o he na i e enzymes, i
can be obse ed ha GTL@Mn2000eq has hal he speci ic
ac i i y o laccase om M. he mophila (116 U/mg) and mo e
han 300 imes g ea e ac i i y han laccase om T. e sicolo
(0.21 U/mg). The high speci ici y o he 2000 equi
bioconjuga e can be co ela ed wi h he p esence o a highe
longe numbe o γ-MnO2NWs in he me alloenzyme
s uc u e, as has been epo ed in he li e a u e o exhibi
highe ca aly ic pe o mance han he sho e ones.
48
Thus, all o he Mn me alloenzymes showed laccase-like
ac i i y highe han ha o he equen ly used laccase om T.
e sicolo .
S abili y o he Colloidal Manganese Me alloen-
zymes. Ano he impo an p ope y o enzymes is hei
s abili y. In pa icula , laccase has been epo ed as no so
s able unde biological condi ions.
49
In his ega d, he s abili y
o he enzyme−Mn bioconjuga es compa ed o laccase was
e alua ed a di e en empe a u es and pH and in he p esence
o a cosol en (Figu e 8).
Figu e 7. Laccase-like ac i i y o he di e en Mn me alloenzymes a
oom empe a u e ( ) and pH 5.5 exp essed in alues o speci ic
ac i i y (U/mg o p o ein).
Figu e 8. S abili y o he di e en colloidal Mn me alloenzymes (GTL@Mn). (a) 40 °C, (b) sodium phospha e, pH 4 25 mM, (c) 10% ( / )
ace oni ile, and (d) 20% / ace oni ile. The pu ple column e e s o he ini ial ac i i y, and he g een column e e s o he ac i i y a e 2 h
incuba ion.
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The di e en Mn−enzyme bioconjuga es syn hesized
conse ed up o 95% o hei laccase-like ac i i y a 40 °C,
being nea ly as s able as laccase om T. e sicolo (Figu e 8a).
In e es ingly, GTL@Mn2000eq was able o e ain 80% o i s
ac i i y when incuba ed a pH 4, being 20 imes mo e s able
han laccase om M. he mophila and laccase om T. e sicolo
(Figu e 8b). Howe e , i was no possible o measu e he
ac i i y o he enzymes a pH 8 because hey p ecipi a ed.
Fu he mo e, GTL@Mn2000eq u ned ou o be as s able
as laccase om T. e sicolo in he p esence o 10% ( / )
ace oni ile and 2 imes mo e s able han laccase om M.
he mophila. When incuba ed wi h 20% ( / ) ace oni ile, his
alue inc eases up o 6 imes. A hese condi ions, GTL@
Mn500eq and GTL@Mn200eq e ained app oxima ely 60
and 40% o hei ac i i y alues, espec i ely (Figu e 8c−d).
The e o e, hese da a show ha he use o GTL as a sca old
allows me alloenzymes o be mo e s able han na u al laccases.
This could be impo an in e ms o main aining he h ee-
dimensional s uc u e, which was also obse ed in he
luo escence expe imen s.
Immobilized Mn Me alloenzymes. Lipases p esen ed an
ex eme inc ease in s abili y when hey a e immobilized on
hyd ophobic suppo s, in pa icula , GTL in C4- unc ionalized
mac opo ous suppo ma e ials. This esul is ela ed o he
ixing exclusi ely o he open con o ma ion o he lipase on he
suppo , which con e s high s abili y o he enzyme agains
high empe a u e o he p esence o cosol en .
34
Thus, in o de o e alua e a po en ial indus ial applica ion
o hese a i icial me alloenzymes and o inc ease hei s abili y
agains di e en condi ions, he new Mn me alloenzymes we e
p epa ed on he solid phase unde simila condi ions as soluble
ones (Figu es S16−S17). These immobilized me alloenzymes,
named BuGTL@Mn2000eq,BuGTL@Mn500eq, and
BuGTL@Mn200eq, we e hen cha ac e ized in e ms o he
Mn nanos uc u es o med (Figu es S16−S17). TEM analysis
showed he o ma ion o nanowi es as in he soluble
me alloenzymes and also he o ma ion o nanopa icles
exclusi ely in he 2000 equi one. The he mal and sol en
s abili y o he immobilized Mn me alloenzymes was
in es iga ed (Figu e 10). These da a indica e ha he
suppo ed me alloenzymes a e a leas 2 imes mo e s able
han he colloidal ones and as s able as laccase om T.
e sicolo , e aining 100% o hei ini ial ac i i y a high
empe a u es, much mo e s able o e a wide pH ange (4−8)
and in he p esence o cosol en (20% / ) a e 2 h
incuba ion (Figu e 9). The e o e, he use o immobiliza ion
s a egies imp o es he s abili y o he Mn−enzyme bio-
conjuga es.
E alua ion o he Oxidase-like Ac i i y o he
Manganese Me alloenzymes agains L-DOPA. In o de
o e alua e he oxidase ac i i y o he Mn me alloenzymes, L-
Figu e 9. S abili y o he di e en suppo ed Mn me alloenzymes (BuGTL@Mn). (a) 40 °C, (b) sodium phospha e pH 4 25 mM, (c) sodium
phospha e, pH 8 25 mM, and (d) 20% / ace oni ile. The o ange column e e s o he ini ial ac i i y, and he g een column e e s o he ac i i y
a e 2 h incuba ion.
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3,4-dihyd oxyphenylalanine (L-DOPA) eac ion o dopa-
ch ome was pe o med in aqueous media and in he p esence
o O2using na u al laccase M. he mophila and GTL@
Mn2000eq, which was ound o be he mos s able o he
colloidal Mn−enzyme me alloenzymes (Figu e 10a).
Laccase om M. he mophila p esen ed a speci ic ac i i y o
145 U. Howe e , GTL@Mn2000eq exhibi ed a speci ic
ac i i y o 200 U, a ound 50% mo e ac i e han he na u al
laccase. The e o e, his esul again con i ms he oxidase-like
ac i i y o he syn hesized Mn me alloenzymes and indica es
e en highe ac i i y in compa ison wi h na u al bioca alys s.
This ac i i y may be ela ed o i s h ee-dimensional
s uc u e. GTL (Figu e 10b) has a pe ec ihis idine pocke
nea one o he wo lids in ol ed in he enzyme’s ca aly ic
mechanism.
34
This pocke is simila o ha ound in na u al
enzymes (e.g., mush oom y osinase). The wo His pocke s a e
su ounded by di e en amino acid esidues (T p, Phe, Ty ),
which a e impo an o subs a e s abiliza ion o he ca echol
g oup close o he Mn-binding posi ion on he p o ein o allow
he ca aly ic con e sion, as occu s in he na u al enzyme
(Figu e 10c).
50
E alua ion o he Oxidase-like Ac i i y o he
Manganese Me alloenzymes agains Phlo idzin Oligo-
me iza ion. Finally, Mn−enzyme bioconjuga e was es ed in
he s uc u al modi ica ion o phlo idzin in aqueous media a
40 °C (Figu e S18). Suppo ed Mn me alloenzyme (BuGTL@
Mn500eq) was used o his pu pose, as he immobilized
me alloenzyme p o ed o be mo e s able han he colloidal
bioconjuga es. HPLC analysis o he eac ion mix u e e ealed
ha , besides he peak o he s a ing monome , wo addi ional
peaks ha co espond o he p oduc s o he eac ion o
oligome iza ion appea ed on he ch oma og am. A e 24 h o
eac ion, 20% o he s a ing monome was con e ed in o
oligome s. The e o e, he eac ion mix u e was u he
analyzed by HPLC-MS. Figu e S19 shows he ch oma og am
image whe e besides phlo idzin (m/z435, e en ion ime 14
min), wo dime (m/z869) molecules a e en ion imes 21
and 21.5 min, espec i ely, appea ed in he mix u e. Ex inc ion
coe icien s o dime s a e signi ican ly lowe in compa ison
wi h ex inc ion coe icien s o phlo idzin; hence, hey appea
e en smalle in ch oma og ams. A e analyzing he molecula
weigh s o he iden i ied p oduc s, i could be seen ha du ing
he o ma ion o he connec ion be ween he phlo idzin uni s,
he loss o wo hyd ogen a oms occu s.
Figu e 10. (a) L-DOPA oxida ion eac ion o laccase om M. he mophila and GTL@Mn2000eq in he p esence o O2(84 ppm) a and pH 5.5.
(b) Ca oon o he c ys allized GTL wi h ma ked yp ophan in g een, phenylalanine in o ange, and y osine esidues in pink. (c) Ca oon o
c ys allized mush oom y osinase (TYR) wi h ma ked yp ophan in cyan, phenylalanine in pink, y osine esidues in pink, and Cu a oms in blue.
The p o ein s uc u es we e ob ained om he P o ein Da a Bank (PDB code: 2W22 (GTL) and 2Y9W TYR), and he pic u e was c ea ed using
Pymol.
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