Co -immobilization of laccase and mediator through a self -initiated [601669]

Journal of Hazardous Materials
Manuscript Draft

Manuscript Number: HAZMAT -D-15-04570

Title: Co -immobilization of laccase and mediator through a self -initiated
one-pot process for enhanced biocatalysis of malachite green

Article Type: Research Paper

Keywords: Acetylacetone; Biocatalysis; Immobilization; Laccase; Mediator

Abstract: Laccase is essentially a green biocatalyst, which works with
molecular oxygen and produc es water as the only by -product. However, its
practical application is far less than satisfactory due to the low
stability/poor reusability of free laccase and the potential secondary
pollution caused by dissolved mediators. To address those bottlenecks in
laccase-based catalysis, a novel biocatalyst (Immo -LMS) was fabricated by
simultaneously immobilizing both laccase and a mediator (acetylacetone,
abbreviated as AA) into a hydrogel through the laccase -AA initiated
polymerization. This self -initiated immob ilization process avoided the
forced conformation change of laccase in the passive embedding to pre –
existing carriers. Resulting from the effective cooperation of laccase
and AA, the Immo -LMS had the highest substrate conversion quantity to
malachite green , followed by the sole immobilized laccase and the
immobilized laccase with an external mediator. Besides the improved
activity, the Immo -LMS showed enhanced stability. The good performance of
the Immo -LMS suggests that the co -immobilization of laccase and mediator
through the self -initiated one -pot process was a promising strategy for
the immobilization of laccase, which is expected to be helpful to cut
down the running cost as well as the potential toxicity that come from
mediators in the practical applic ation of laccase.

Novelty statement

This is the first work to use acetylacetone (AA) as a mediator for laccase in both free
and immobilized laccase system s. AA played a “Kill Two Birds with One Stone” role in
this novel self -initiated one -pot process : (1) initiate the immobilization process for
laccase and itself , and (2) enhance the biocatalytic transformation through acting as a
redox mediator for laccase. The results proved that AA, even after bounding to polymer
chain s, still possesses redox abili ty. To the best of our knowledge, no other laccase
mediator, such as ABTS and HBT, could work in such a bi -functional way. *Novelty Statement

mediator (AA) monomer Self-immobilizing
Lac
Passive embedding
external mediator
e- e- e- e- e-
e-
e-
e- Kill Two Birds with One Stone Polymerization Transformation AA Graphical Abstract

Highlights
 Laccase and a mediator were co -immobilized through a self -initiated process .
 This self -initiated process was favorable to retain the enzymatic activity.
 The mediator played important roles in both immobilization and transformation.
 The biocatal ysis was enhanced by the co -immobilization of laccase and mediator .
Highlights (for review)

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Co-immobilization of laccase and mediator through a self-initiated one-pot process
for enhanced biocatalysis of malachite green
Hongfei Sun, Wenguang Huang, Hua Yang, Shujuan Zhang *

State Key Laboratory of Pollution Control and Resource Reuse, School of t he Environment,
Nanjing University, Nanjing 210023, China

Submitted to Journal of Hazardous Materials for possible publication
(Original Article )

* Corresponding authors ( S. Zhang )
Tel.: +86-25-89680389
E-mail addresses: sjzhang @nju.edu.cn

*Manuscript
Click here to view linked References

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Abstra ct 1
Laccase is essentially a green biocatalyst, which works with molecular oxygen and 2
produces water as the only by -product. However, its practical application is far less than 3
satisfactory due to the low stability /poor reusability of free laccase and the potential 4
secondary pollution caused by dissolved mediators . To address th ose bottlenecks in 5
laccase -based catalysis, a novel biocatalyst (Immo -LMS) was fabricated by 6
simultaneously immobilizing both laccase and a mediator (acetylacetone, abbreviated as 7
AA) into a hydrogel through the laccase -AA initiated polymerization. This self -initiated 8
immobilization process avoided the forced conformation change of laccase in the passive 9
embedding to pre -existing carriers. Resulting from the effective cooperation of la ccase and 10
AA, the Immo -LMS had the highes t substrate conversion quantity to malachite green, 11
followed by the sole immobilized laccase and the immobilized laccase with an external 12
mediator. Besides the improved activity, the Immo -LMS showed enhanced stabili ty. The 13
good performance of the Immo -LMS suggests that the co -immobilization of laccase and 14
mediator through the self -initiated one -pot process was a promising strategy for the 15
immobilization of laccase , which is expected to be helpful to cut down the runn ing cost as 16
well as the potential toxicity that come from mediators in the practical application of 17
laccase. 18
19
Keywords : Acetylacetone; Biocatalysis; Immobilization; Laccase; Mediator 20

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1． Introduction 21
Laccase (benzenediol oxygen oxidoreductase, EC 1.10.3.2) is an efficient and 22
environmentally frien dly catalyst for bioremediation [1]. It is able to oxidize a wide range 23
of substrates with no additional co -factor other than molecular oxygen [2-4]. However, the 24
poor reusability, low operational stability, and high p roduction costs restrict its practical 25
applicability in water treatment. 26
Immobilization is a useful strategy to optimize the operational performance of lacc ase 27
in large -scale applications [5-7]. There are plenty of strategies for enzyme immobilization, 28
including non -covalent adsorption, cov alent attachment and entrapment [8-10]. Most of 29
the existing methods are limited to the technical route that loading or embedding laccase 30
passively to the surface and/or interior of variant carriers. The conformation and even 31
chemical structure of the bound laccase might undergo forced changes during the passive 32
adaptation process to the pre -existing carri er space [11-14]. As a result, the catalytic 33
activity of the immobilized laccase is far less than satisfied. Therefore , new immobilization 34
approaches are needed to let the laccase to establish the supporting space in its own way. 35
Laccase cannot directly catalyze the oxidization of many nonphenolic substrates due 36
to the relatively low redox potential [15-17]. Some soluble redox mediators, such as 37
diammonium 2,2’-azino-bis(3 -ethyl benz othiazoline -6-sulfon ate) (ABTS), 38
1-hydroxybenzotriazole (HBT), and syringaldehyde have to be employed to enhance the 39
oxidation capabilities of laccase towards aromatic compo unds with high redox potentials 40

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[15,17 ]. Even for phenolic compounds, the laccase -mediator system (LMS) is often 41
recommended because the use of a mediator could maximize the efficacy of laccas e by 42
enhanced electron transfer [16,18]. However, the current available synthetic med iators, 43
such as ABTS and HBT, are expensive and toxic, whereas the efficacy of the natural 44
mediators, e.g., syr ingaldehyde, is not high enough [16,19,20 ]. Given the high cost and 45
potential toxici ty of these dissolved mediators [21], how to recover and reus e them is 46
another attractive but challenging task. Immobilization provides a solu tion for the recovery 47
of enzyme [22]. However, the recovery and reuse of redox mediators were almost 48
neglected in the current immobilization of enzymes. 49
β-Diketones are ubiqui tous in nature as metabolic intermediates in the microbial 50
metab olism of aromatics and terpenes [23]. Our recent work has proved that acetylacetone 51
(2,4-pentanedione, denoted as AA) was a potential redox mediator for laccase in the 52
catalytic transformatio n of a toxic triarylmethane dye [24]. The addition of AA in a free 53
laccase system significantly increased the substrate conversion quantity [24]. On the other 54
hand, several articles have reported that laccase could induce free radical polymerization of 55
vinyl monomers with the aid of AA [4,25-27]. However, few reports paid attention to how 56
much laccase activity was retained after this polymerization process, and whether such 57
polymers could be used as immobilized enzyme for wastewater treatment. 58
In our recent work [28], a sponge -like hydrogel was prepared through grafting 59
copolymerization of chitosan (CTS) and polyacrylamide (PAM) with potassium persulfate 60

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(K2S2O8) as a free -radical initiator. This hydrogel was proved as a goo d supporting matrix 61
for laccase [28]. Therefore, in the present work, we attempted to immobilize both laccase 62
and AA into the CTS -g-PAM hydrogel through a one -pot process with the laccase -AA, 63
instead of K 2S2O8, as the self -initiating system. The obtained material was expected to 64
have good laccase activity and useful internal mediators. 65
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2． Experimental 67
2.1 Materials 68
Trametes versicolor laccase (Lac) with a nominal activity of ≥10.0 U/mg (lot result: 69
12.9 U/mg) was provided by Sigma -Aldrich Corporation. Malachite green (MG), AA, 70
sodium acetate (NaAc), K 2S2O8, CTS (80 %-95% deacetylation), disodium hydrogen 71
phosphate dodecahydrate (Na 2HPO 4 ·12H 2O), and citric acid monohydrate (C 6H8O7 ·H2O) 72
were all of analytical grade and were purchased from Shanghai Reagent Station, China. 73
Deuterated water (D 2O) with isotopic purity of 99 atom% D was purchased from Beijing 74
SeaSkyBio Technology Corporation. Other chemicals including ABTS, acetic a cid (HAc), 75
HBT, syringaldehyde, acrylamide (AM), and N, N’ -methylene -bis-acylamide (MBA) were 76
of analytical grade and were purchased from Sigma -Aldrich Corporation. All the 77
chemicals were used as received without any treatment. 78
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2.2 Fabrication of immobilized laccase -AA system 80

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The synthesis procedure was optimized through an L 9 (3)4 orthogonal array design 81
matrix with the following four factors at three levels: CTS (5, 10, 15 g L-1), AM (0.53, 1.06, 82
1.60 g), MBA (0.1, 0.2, 0.3 g), AA (150, 300, 600 μL). Based o n the mechanical structure 83
and the recovered enzyme activity of the obtained materials, the following conditions were 84
selected for further investigation: First, 16 mL CTS solution (10 g L-1 in a degassed HAc 85
solution (1 wt%)) together with 1.60 g AM and 0. 1 g MBA were added to a three -necked 86
flask. Then, 35 mL Lac solution (0.8 mg mL-1 in a HAc -NaAc buffer (0.2 M, pH 5.0)) was 87
firstly mixed with 300 μL AA for 10 min and was then added to the flask. The mixed 88
solution in the flask was purged with nitrogen fo r 5 min. Thereafter, the solution was gently 89
stirred (120 rpm) in a water bath at 45oC for 2 h under nitrogen purging till the reaction 90
mixtures were completely gelled. The fresh hydrogel was immediately immersed in 91
ultrapure water for 48 h to remove the u n-reacted components and oligomers. The washed 92
hydrogel was dried with a freeze drier (Labconco, America) and then was crushed into 93
small particles with swelling particle sizes ranging from 0.2 mm to 1.8 mm. The resultant 94
immobilized Lac -AA system (denoted as Immo -LMS) was stored at -22oC for subsequent 95
use. A similar procedure was conducted for the preparation of immobilized Lac (Immo -Lac) 96
with K 2S2O8, instead of AA , as the initiator [28]. A portion of the Immo -LMS was 97
immersed into methanol for 24 h to ob tain the deactivated counterpart (Immo -deLMS). 98
In order to verify whether the other redox mediators could work with Lac to initiate a 99
similar free radical polymerization, the same L 9 (3)4 orthogonal tests were conducted 100

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except that AA was replaced by ABTS, HBT, and syringaldehyde, respectively. 101
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2.3 Characterization of the Immo -LMS 103
Scanning electron microscope (SEM) (S -3400N II, Hitachi Corporation, Japan) and 104
field emission scanning electron microscope (FESEM) (S -4800, Hitachi Corporation, 105
Japan) were used to study the morphology of the Immo -LMS. Nitrogen 106
adsorption -desorption analysis of the Immo -LMS was carried out at 77 K by using a 107
surface analyzer (ASAP 2020, Micromeritics Instrument Corporation, USA). 13C 108
solid -state nuclear magnetic resonance (NMR) spect ra were conducted with a Bruker 109
A V ANCE III 400WB spectrometer. More details are a vailable in a previous report [28]. 110
Cyclic voltammetry (CV) experiments were conducted with an electrochemical 111
workstation (Shanghai Chenhua Instruments Co. Ltd.). A glassy ca rbon electrode was 112
served as the working electrode. A saturated Ag/AgCl electrode was served as the 113
reference electrode and a P t wire as the counter electrode. The electrocatalytic process was 114
studied in a 0.1 M citric acid -phosphate buffer (pH 4.0). The c oncentrations of AA and Lac 115
were 5 mM and 2 mg ml-1, respectively. A drop of swollen Immo -LMS or Immo -Lac was 116
spread on the surface of the glassy carbon electrode. The coating was dried at room 117
temperature. Then the electrocatalytic process was conducted i n the aqueous electrolyte. 118
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2.4 Determination of enzyme activity 120

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The enzyme activity was determined by monitoring the product formation rate of 121
ABTS (2 mM) with a UV -vis spectrophotometer (Beijing Purkinje General Instrument Co., 122
Ltd.) at 420 nm [29]. The reco vered activity (%) of the Immo -LMS was defined as the ratio 123
between the determined activity of the Immo -LMS and the total enzyme activit y that was 124
dosed in fabrication . 125
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2.5 Stability test of the Immo -LMS 127
The residual activities at different pHs were determine d to evaluate the pH resistance. 128
The free Lac (0.8 mg protein per mL) and the Immo -LMS were incubated in 129
citrate -phosphate buffer solutions (50 mM, pH 3.0 to 8.0) at 25oC for 48 h. The 130
experiments were conducted in triplicate. 131
The thermal stability was eva luated with the simplified deactivation model by 132
incubating the free Lac and the Immo -LMS in a HAc -NaAc buffer solution (200 mM, pH 133
5.0) at 65oC for 10 h [30]. 134
The Immo -LMS was stored at -22oC for 10 months. The storage stability was 135
calculated as the rati o of the residual activity to the initial activity. 136
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2.6 Degradation kinetics of dyes 138
Prior to decoloration experiments, the Immo -LMS and Immo -Lac were soaked in 139
distilled water for 4 h to reach swelling equilibrium. One aliquot of swollen Immo -LMS, 140

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two aliquo ts of swollen Immo -Lac, and a free Lac solution of same enzyme activities were 141
individually added into MG solutions (50 μM, pH 5.0, 50 mL). An HBT solution (5 mM) 142
was added to one of the Immo -Lac systems. The four mixtures were magnetically stirred at 143
25oC. During the decoloration process, the concentrations of the sample solutions were 144
determined at certain time intervals by measuring the absorbance of the supernatants at 617 145
nm with an UV -vis spectrophotometer (UV -2700, Shimadzu). When the MG removal 146
reached equilibrium, a small volume of a fresh MG stock solution was added to the 147
enzymatic degradation systems to start the next cycle run. 148
The supernatants of the treated MG solutions were analyzed with a Dionex Ultimate 149
3000 HPLC system equipped with an UV detector. Prior to analysis, samples were 150
centrifuged for 10 min and then were filtered through a 0.22 μm filter. An Agilent 4.6×150 151
mm, 5 μm ZORBAX Eclipse Plus C18 column was used for the separation of the 152
transformation products. A mixture of 0.1% HCOOH solution and methanol was employed 153
as the mobile phase. The insoluble products formed in the Lac -treated MG solutions were 154
centrifuged and dried in a vacuum freeze dryer (Labconco, America) and then analyzed by 155
a 13C NMR (Bruker AV ANCE III 400WB spectrome ter). 156
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3． Results and discussion 158
3.1 Characteristics of the Immo -LMS 159
Consistent with a previous r eport [4], ABTS, HBT, and syringaldehyde were unable to 160

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initiate free radical polymerization with the Lac. On the contrary, the Lac -AA system 161
worked in a wide range. The fresh Immo -LMS was clear, transparent and homogeneous. 162
After being soaked in distilled water for 48 h, the un -reacted components, including free 163
AA, Lac, and oligomers were thoroughly washed out from the hydrogel. As shown in Fig. 164
S1a, channels with di ameter of 10 -20 μm were regularly distributed throughout the 165
monolithic Immo -LMS. Besides, there were massive mesopores and macropores in the 166
lamellar walls of the channels ( Fig. S1 b and S1c). Due to the resolution limit, the pores of 167
diameter less than 10 nm were unobservable with the FESEM. The nitrogen adsorption 168
analysis demonstrates that there were also abundant micropores in the lamellar walls. As 169
shown in Fig. S1 d, most of the pores concentrated at 2.5 -4.0 nm. The highly opened 170
channels and the abund ant pores facilitated the diffusion of substrate molecules th roughout 171
the Immo -LMS [31]. The recovered activity of the Immo -LMS was 32.0% (± 1.9%) of the 172
dosed activity. Herein, the dosed activity means that all the Lac that was dosed into the 173
synthesis so lution. This recovered activity was comparable to or higher than th e reported 174
data, e.g., 2.0 -6.0% [21], 28% [32], 79% based on a 40% effective binding yield of laccase 175
(The activity recovery was defined as the apparent activity of the immobilized laccase 176
divided by the laccase activity effectivel y immobilized onto the support) [6], and 65% 177
based on a 28.9% effective binding yield [33]. 178
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3.2 Stability of the Immo -LMS 180

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In general, enzymatic biocatalysts are susceptible to environmental change during the 181
incubatio n process, which greatly limits their practical application in was tewater treatment 182
[34]. Fig. S2 a shows that the Immo -LMS suffered less activity loss than the free Lac after 183
incubation for 48 h. At pH 3, the residual activity of the Immo -LMS was 45.1%, wh ile only 184
4.3% was remained for the free Lac. In addition, when the solution was neutral or alkaline, 185
the catalytic activity of the free Lac was almost undetectable, while there was still 186
considerable residual activity in the Immo -LMS. 187
Good storage stabili ty is an essential requirement for a biocatalyst to be used in 188
indus trial applications [34]. A slower deactivation rate was observed for the Immo -LMS 189
than that for the free Lac when incubated at 65oC (Fig. S2b) . The half -life time of the Lac in 190
the Immo -LMS (1.4 h) was around 3.5 -time longer than that of the free Lac (0.4 h), 191
suggesting that the Immo -LMS could be stored for a longer time without significant loss in 192
its catalytic activity. In fact, after storage for 10 months at -22oC, the Immo -LMS well 193
retained its original activity. 194
195
3.3 Catalytic performance of the Immo -LMS 196
According to our previous work [28], there was electrostatic repulsion between the 197
cationic CTS -g-PAM hydrogel and the cationic MG, whereas the hydrogel had a large 198
uptake to an anionic az o dye. To check the adsorption ability of the Immo -LMS to the 199
substrate, the same amount of deactivated Immo -LMS (Immo -deLMS) was added to a MG 200

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solution. As shown in Fig. S3 , a large amount of insoluble products were accumulated 201
within the Immo -LMS after i ncubated in the MG solution for two days. The Immo -LMS 202
hydrogel was significantly darkened after the incubation, whereas the color of the 203
Immo -deLMS hydrogel had almost no change. This difference suggests that the 204
decoloration of the tested MG solution was exclusively from enzymatic transformation 205
rather than adsorption. 206
The catalytic activity of the Immo -LMS was compared with the free Lac, Immo -Lac, 207
and Immo -Lac with external HBT (denoted as Immo -Lac/HBT) side -by-side with MG as 208
the target substrate. As cl early shown in Fig. 1 a, at the end of the fifth run, the free Lac and 209
the Immo -Lac/HBT systems were nearly ineffective for the decoloration of MG. The 210
decoloration rate of the Immo -Lac system was reduced to 33.3% at the end of the sixth run. 211
However, the d ecoloration rate of th e Immo -LMS system was still above 87.3% at the 212
seventh run. As a result, the Immo -LMS system had the highest substrate conversion 213
quantity (6.94 μmol U-1), followed by the Immo -Lac system (4.64 μmol U-1) and the 214
Immo -Lac/HBT system (3.25 μmol U-1). No surp rising, the free Lac system had the lowest 215
substrate conversion quantity (2.54 μmol U-1). However, it is worth noting that there was 216
an interesting phenomenon in the decoloration kinetics. The pseudo -first-order 217
decoloration rate constant ( k1) of MG in the Immo -LMS system was initially much lower 218
than those of the other three systems ( Fig. 1 b), but was significantly accelerated after a 3 -h 219
reaction. This acceleration was sustained to the second run and then the rate constant was 220

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quite stable during the next several runs. In the other three systems, especially in the 221
Immo -Lac/HBT system, the rate constants were apparently reduced as the cycle runs 222
continued and were far lower than that of the Immo -LMS from the second run ( Fig. 1 c). 223
This phenomenon was exactly the same as that observed i n the free Lac/mediator systems 224
[24], from which two conclusions could be drawn: First, in terms of recyclability, AA was a 225
better mediator than HBT [24,35 ]. Second, AA was a useful mediator for Lac even after 226
immobilization. In other words, the covalent bonding of AA to polymer chains did not 227
eliminate its activity. 228
229
3.4 Role of AA in the synthesis of the Immo -LMS 230
The feasibility of the laccase -AA system in free radical polymerization has been 231
reported in several works [4,25-27]. However, these studies focused mainly on the yields 232
of polymers. Little attention was paid to the residual enzyme. The polymerization initiated 233
by the laccase -AA system was generally interpreted as a free radical mechanism. The 234
formation of a carbon -centere d AA radical was proposed as a key step in the 235
polymerization process. Nevertheless, this proposal on the formation of AA radicals was 236
actually deri ved from a peroxidase -AA system [36-38]. In our previous work, strong 237
radical signals were observed as the L ac and AA were mixed [24]. Although the possible 238
mechanisms for the enzymatic formation of AA radicals were still mooted, according to the 239
diketone -cleaving enzyme -catalyzed cleavage of AA [23], the observed radical signals 240

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might originate from either keto -oxygen radicals or carbon -centered radicals, owing to the 241
hydrogen atom transfer or electron transfer between the Lac and AA. 242
1H NMR was used to identify the structure of AA after bonding to the polymers. As 243
shown in Fig. 2 , after adding AM into the mix tures of free Lac and AA, the contents of both 244
the methine (CH) hydrogen of the enolic AA and the methylene (CH 2) hydrogen of the keto 245
AA were reduced, whereas the methyl (CH 3) hydrogen was relatively increased. The peaks 246
at 1.57 and 1.69 ppm corresponded to the CH 2 of the PAM segments [39,40 ] confirming the 247
formation of polymer chains. The change of hydrogen contents in different forms indicates 248
that AA might be covalently bonded to the polymer chains through a carbon -centered 249
radical (Scheme 1). It is rep orted that modifications of the CH 2 group in a β-diketone 250
molecule usually do not impact the activity of the resulting derivative in further reactions 251
[41,42 ], which explained the similar role of AA in both free and immobilized states. 252
253
3.5 Decoloration mecha nism in the Immo -LMS 254
As illustrated in Fig. S4 , the soluble transformation intermediates in the Immo -LMS 255
were different from those in the Immo -Lac and the Immo -Lac/HBT systems, 256
demonstrating that the presence of AA altered the transformation pathways of MG . There 257
were also lots of insoluble products in the Immo -LMS and Immo -Lac. Nevertheless, it was 258
difficult to separate these insoluble products from the hydrogels. Therefore, incubation 259
systems composed of free Lac and mediators were explored to simulate th e corresponding 260

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decoloration processes in the immobilized systems. The precipitates appeared in the MG 261
solutions was analyzed with 13C solid -state NMR ( Fig. S5). It was clear that regardless of 262
which mediator was used, the precipitates came from the coupli ng reactions between Lac 263
and some transformation intermediates of MG, which w as similar to the other reports 264
[43,44 ]. The shift at 40 ppm in the MG/Lac system indicates that the CH 3 groups on the 265
benzene rings still existed in the coupling precipitates. Th e disappearance of the CH 3 shift 266
in the MG/Lac/HBT system was indicative of the N -demethylation react ions caused by the 267
HBT radicals [44]. As for the precipitates in the MG/Lac/AA system, the resonances in the 268
region from 118 to 143 ppm was more complex th an those of the MG/Lac and 269
MG/Lac/HBT systems, indicating the formation of different benzene derivatives. Except 270
the shift at 40 ppm, new alkane shifts at 29.5 ppm and 12.7 ppm and carbonyl shift at 193 271
ppm were detected. It was highly possible that a few radicals from AA became a part of the 272
precipitates. Besides, some AA might be degraded by the Lac to smaller fragments, such as 273
methylglyoxal and a cetate [23]. However, considering the stable concentration of AA in 274
the solution system, the majority of AA ( over 90% based on the UV absorbance at 270 nm) 275
in the Lac system might act as a mediator, which was in agr eement with our previous report 276
[24]. 277
Both the decoloration experiments and the product analyses demonstrate that the 278
bonded AA acted as a redox media tor for the Lac in the biocatalytic transformation of MG. 279
To understand how the bonded AA worked in the LMS, a CV experiment was conducted to 280

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compare the redox behaviors of AA in different systems. As illustrated in Fig. S6a, the 281
reduction current of the L ac solution was higher than that of the control with an apparent 282
anode peak at about 20 mV versus Ag/AgCl electrode (The standard electrode potential is 283
198 mV). The direct electron transfer (DET) process between Lac and electrodes has been 284
report ed by a l arge number of studies [45-47]. A widely accepted mechanism was that the 285
electrons were transferred from the electrode to the T1 site of the laccase and then through 286
an internal electron transfer (IET) to the T2/T3 cluster, where molec ular oxygen is reduce d 287
to water [45,47 -49]. The redox potentials of the T1 site in the Lac was 780 mV versus 288
normal hydrogen electrode (NHE) [48] and it was generally believed that the redox 289
potential of the T2/T3 cluster in copper oxidase was far lower than that of T1 site [45,50]. 290
So, the anode peak might indicate that the redox potential of the T2/T3 cluster was about 291
218 mV versus NHE in the current system. It is worth noting that the weakly catalytic wave 292
of the Lac substrate (molecular oxygen) started at about 700 mV [45,48]. In the presence of 293
AA, the oxidation current was apparently enhanced ( Fig. S6a). 294
The closest distance between the surface of the enzyme and the T1 site was less than 295
10 Å [51,52 ]. If properly oriented on the electrode surface, such a distance would al low 296
efficient DET from the electrode to the T1 site. The electron would be then transferred 297
through an IET mechanism to the T2/T3 cluster [45,46,53 ]. When the Lac was embedded 298
within polymers (Immo -Lac), the DET process might be difficult to continue effec tively 299
(Scheme 2a). On the contrary, the polymer attached on the electrode would occupy the 300

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valid sites and hinder the normal diffusion of the electrolyte ions. As illustrated in Fig. S6b, 301
the voltammetric wave of the Immo -Lac was similar to that of the fr ee Lac but with a 302
weaker hydrogen evolution reaction. 303
As for the Immo -LMS, AA acted like an electronic bridge between the electrode and 304
the immobilized Lac (Scheme 2b). During the electrolytic reduction, AA was firstly 305
reduced and then transferred electron s to the Lac. As a result, the reduction current was 306
correspondingly enhanced. The obvious catalytic wave started from about 700 mV should 307
come from the superimposed effect of both AA oxidation current and O 2 electroreduction. 308
Therefore, the results could confirm that AA after being covalently bonded into polymers 309
still possessed redox ability. Because of the additional electron transfer process mediated 310
by AA, the electrical resistance of the Immo -LMS was higher than that of the Immo -Lac, 311
which explained t he lower k1 in the Immo -LMS at the beginning of reactions. 312
313
4． Conclusions 314
Low stability, poor reusability, and secondary pollution caused by synthetic mediators 315
are the bottlenecks in the application of laccase. The simultaneous immobilization of Lac 316
and AA through the self-initiated polymerization generated a sponge -like hydrogel with 317
competitive enzyme activity and good stability . In this way, both the recycle of laccase and 318
mediator were facilitated, which might be helpful to cut down the running cost as w ell as 319
the potential toxicity that come from synthetic mediators. We believe that this innovative 320

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immobilization pattern is promising for the application of immobilized laccase in 321
wastewater treatment. 322
323
Acknowledgments 324
This work was supported by the Natio nal Natural Science Foundation of China 325
(51378254 , 21522702 ) and the Fundamental Research Funds for the Central Universities . 326
327
328
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parameters of laccase catalyzed direct electrochemical oxygen reduction reaction, 461
ACS Catal . 2 (2012) 38 -44. 462
[50] Christenson, S. Shleev, N. Mano, A. Heller, L. Gorton, Redox potentials of the blue 463
copper sites of bilirubin oxidases, Biochim . Biophys . Acta 1757 (2006) 1634 -1641 . 464
[51] K. Piontek, M . Antorini, T. Choinowski, Crystal structure of a laccase from the fungus 465
Trametes versicolor at 1.90 -Å resolution containing a full complement of coppers, J. 466
Biol. Chem. 277 (2002) 37663 -37669 . 467
[52] E.I. Solomon, U.M. Sundaram , T.E. Machonkin, Multi copper oxidases and 468
oxygenases, Chem. Rev. 96 (1996) 2563 -2605 . 469
[53] H.B. Gray, J.R. Winkler, Electron tunneling through proteins, Quar. Rev. Biophys. 36 470
(2003) 341 -372. 471
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Figure and Scheme Legends 473
Scheme 1. The formation route of the Immo -LMS and its role in substrate con version. 474
Scheme 2. Proposed mechanism for electron transfer from the glass carbon electrode to (a) 475
Immo -Lac and (b) Immo -LMS. 476
Fig. 1. (a) Decoloration percentage of MG solutions (50 μM, pH 5.0, 50 mL) in a 477
continuous seven -cycle run. The initial enzyme act ivity in all the systems was 2.5 478
U. (b) The decoloration kinetic profiles in the first run. (c) The pseudo -first-order 479
decoloration rate constants of MG in the seven cycle runs. The columns in (a) 480
represent the same items as those in (c). 481
Fig. 2. The 1H NMR spectra of the mixture of Lac (2 mg ml-1) and AA (15 mM) with AM 482
at concentrations of 7.5 mM (AM1), 15 mM (AM2), and 30 mM (AM3), 483
respectively. 484

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Scheme 1 486

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Scheme 2 488

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Fig. 1 490

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Fig. 2 492

1
Supplementary Material 1
2
Co-immobilization of laccase and mediator through a self-initiated one-pot process 3
for enhanced biocatalysis of malachite green 4
Hongfei Sun, Wenguang Huang, Hua Yang, Shujuan Zhang * 5
6
State Key Laboratory of Pollution Control and Res ource Reuse, School of the Environment, 7
Nanjing University, Nanjing 210023, China 8
9
Submitted to Journal of Hazardous Materials for possible publication 10
(Original Article ) 11
12
* Corresponding authors ( S. Zhang ) 13
Tel.: +86-25-89680389 14
E-mail addresses: sjzhang @nju.edu.cn 15
16
17
18
19
The S upporting Information contains 8 pages, including Figs. S1-S6. 20 Supplementary Material

2
Figure Caption s 21
Fig. S1. The pore structures of the Immo -LMS: (a) SEM image of the monolithic 22
Immo -LMS; (b) and (c) FESEM images of the lamellar walls around the channels; 23
(d) Pore size distribution of the Immo -LMS. 24
Fig. S2. Thermal and chemical stability of the Immo -LMS and free Lac. (a) Incubated at 25
25oC for 48 h; (b) Incubated at 65oC in a solution of pH 5.0. The relative activity 26
was calculated with the one determined at 25oC and pH 5.0 as the reference. 27
Fig. S3. Picture of the Immo -LMS and the Immo -deLMS after incubated in a MG 28
solution for 2 days. 29
Fig. S4. HPLC profiles of MG solutions after incubated in the Immo -Lac, Immo -LMS and 30
Immo -Lac/HBT (5 mM) systems. 31
Fig. S5 . The 13C NMR spectra of MG, Lac, and their precipitates. The data labeled on the 32
MG molecule was the NMR shifts simulated by the ChemDraw. The arrows 33
indicate the attacking positions by the different species. 34
Fig. S6. Cycle voltammetric profiles of AA and Lac in (a) f ree and (b) immobilized 35
systems. 36
37

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Fig. S1 39

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Fig. S4 45

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Fig. S5 47

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Fig. S 6 49

Response to technical check

1. Cover letter should state the manuscript word count, which includes text, figure
captions, and table legends, but not references. Also make sure manuscript should
be no more than 5000 words including text, figures, and table legends, but not
references.
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supplementary material document is created to provide these figures. The word count
of th e revised manuscript is now 4880 equivalent. A new ve rsion of cover letter
containing the word count information, a new version of the manuscript and an
additional supplementary material document are supplemented in the resubmission. Responses to Technical Check Results