Characterization of immobilized tyrosinase-an enzyme that is stable in organic solvent at 100 °C

Article abstract of DOI:10.1039/c8ra07559j

Tyrosinase is a copper-containing enzyme present in plant and animal tissues, which catalyzes the production of melanin and other pigments. In organic solvent, tyrosinase can convert N-acetyl-l-tyrosine ethyl ester (insoluble in aqueous) to a derivative o

Full text of DOI:10.1039/c8ra07559j

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Characterization of immobilized tyrosinase – an
ꢀ
enzyme that is stable in organic solvent at 100 C†
Cite this: RSC Adv., 2018, 8, 39529
ab
bd
b
b
a
Lidong Wu,
Jincheng Li,
*
Brijesh Rathi, Yi Chen, Xiuhong Wu,
Huan Liu,
a
c
a
Anjie Ming and Gang Han
Tyrosinase is a copper-containing enzyme present in plant and animal tissues, which catalyzes the
production of melanin and other pigments. In organic solvent, tyrosinase can convert N-acetyl-L-
tyrosine ethyl ester (insoluble in aqueous) to a derivative of L-dopamine (a drug used for the treatment of
Parkinson's disease). Thus, the performances of tyrosinase in organic solvent have attracted scientific
attention since 1980. In this work, we investigated the stability of immobilized tyrosinase at high
temperature in anhydrous organic solvent. Triethylaminoethyl cellulose (TEAE-Cellulose) performed the
best out of six immobilization platforms. The dry immobilized tyrosinase became extremely thermostable
in organic solvent, and the half-life of the dry immobilized tyrosinase in organic solvent is strongly
related to the polarity of the organic solvent than their log P value. The immobilized tyrosinase loses its
Received 11th September 2018
Accepted 2nd November 2018
ꢀ
activity instantaneously in aqueous solution at 100 C, but it keeps enzymatic activity within 10 min in
DOI: 10.1039/c8ra07559j
hydrophilic methanol and over one month in hydrophobic hexane (log P: 4.66, non-polar) even
ꢀ
rsc.li/rsc-advances
incubating at 100 C. This research provides valuable information for the design of new biocatalysts.
enzymatic catalysis in organic media have been systematically
Introduction
9,10
investigated,
but there is no report about the thermostable
Mimicking tyrosinase has been a longstanding goal of indus- behavior of tyrosinase in different pure organic solvents at high
1
trial and academic chemists. Tyrosinases (polyphenol oxidase, temperature.
11,12
EC 1.14.18.1), copper-containing oxidoreductases, can enable
To study the thermostable behavior of tyrosinase,
the
aerobic oxidation of relatively inert C–H bonds, which remains enzyme needs to be incubated in different organic solvents and
difficult for synthetic catalysts. Most of the synthetic catalysts undergo testing of its activity in the same organic solvent. In
1
3,14
can be stable in most of the organic solvents at high tempera- this way, the enzyme must be easy to reuse.
As previously
ꢀ
ture (mostly higher than 100 C). The behavior of tyrosinase in reported, immobilization techniques are very efficient method
15–17
organic solvents at high temperature is very interesting for to recycle the enzyme.
The enzyme immobilized on a solid
mimicking tyrosinase. Tyrosinase is an enzyme with mono- carrier can make the enzyme reusability easier. It is one of
phenolase and diphenolase activity. Since the 1980s, inno- the key steps to test the thermostability of immobilized
18,19
2–4
20–22
23,24
vative research has shown that tyrosinase can function well in enzymes.
From previous reports,
the adsorption method
2
5–27
anhydrous media with minimal water, pioneered mainly by keeps the original structure of enzyme,
but encounters
5,6
Klibanov et al. This research remained of broad interest in the issues with enzyme leaching. Compared with the covalent
2
8,29
30–32
33,34
last century and mushroom tyrosinase has been utilized as an bonding,
entrapment,
copolymerization
the conditions of the adsorption method
enzymes in organic media. The enzyme regioselectivity and are much milder than the other methods. Furthermore, the
and encap-
35–37
ideal enzyme by many researchers to study the functioning of sulation methods,
7,8
38,39
40,41
42
glass beads,
graphene,
magnetic nanoparticles and
43,44
metal–organic frameworks (MOFs)
were chosen as the
a
Key Laboratory of Control of Quality and Safety for Aquatic Products, Ministry of supports to immobilize the enzyme in previous study. In this
Agriculture, Chinese Academy of Fishery Sciences, Beijing, 100141, China
study, the biocompatible polymers (i.e. cellulose) were utilized
as the ideal matrixes due to their low cost, and renewable and
Besides, we systematically studied
different polymers (i.e. cellulose matrixes, Pluronic F68 and
poly(styrene-co-divinylbenzene)) and traditional glass beads as
b
Department of Chemistry, Massachusetts Institute of Technology, Cambridge,
Massachusetts 02139, USA. E-mail: lidongwu@mit.edu; Tel: +16172533556
45–47
biodegradable nature.
c
Smart Sensing R&D Center, Institute of Microelectronics, Chinese Academy of Science,
Beijing, 100029, China
d
Laboratory for Translational Chemistry and Drug Discovery, Department of
48
Chemistry, Hansraj College University of Delhi, Delhi, 110007, India
enzyme support.
†
Electronic supplementary information (ESI) available: Brief statement in
Aer optimizing the enzyme immobilization material, the
organic solvent effect, the position of substituent group and the
nonsentence format listing the contents of the material supplied as supporting
information. See DOI: 10.1039/c8ra07559j
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Fig. 1 SEM of (A) triethylaminoethyl cellulose (TEAE-Cellulose) and (B) TEAE-Cellulose with tyrosinase; the TEM of (C) triethylaminoethyl
cellulose (TEAE-Cellulose) and (D) TEAE-Cellulose with tyrosinase.
strength of electron-donating substituent group were studied. Preparation of tyrosinase and immobilization onto TEAE-
Furthermore, the activities of immobilized tyrosinase were Cellulose
tested in various hydrophilic and hydrophobic organic solvents
at high temperature. This study provided more reasonable and
systematical explanation of tyrosinase in organic solvents.
ꢁ1
Tyrosinase (6 mg) was dissolved in 0.3 mL of 50 mmol L
phosphate buffer (pH 7.0), and then 60 mg of cellulose was
added. The sticky mixture was spread on a watch glass and le
to dry at room temperature. Tyrosinase immobilization onto
CM-Cellulose or other materials was prepared following
a similar protocol as tyrosinase immobilization onto TEAE-
Cellulose.
Experimental section
Materials
p-Cresol, p-chlorophenol, p-methoxyphenol and other chem-
icals obtained from Sigma Aldrich (USA) were of the highest
purity available. Mushroom Tyrosinase (50 kU, powder) was
purchased from Sigma Aldrich (USA) as a solid with a specic
UV-Vis spectrophotometry detection of the tyrosinase-
catalyzed oxidation product
activity of 2430 unit per mg (1 unit is dened as the enzyme The time course for tyrosinase-catalyzed oxidation of p-cresol in
activity resulting in an increase in absorbance at 280 nm of organic solvents was measured by the following procedure: p-
ꢀ
0
.001 at pH 6.5 at 25 C in a 3 mL reaction volume containing L- cresol was dissolved in a given solvent (its concentration:
ꢁ1
ꢁ1
tyrosine). Poly(styrene-co-divinylbenzene), glass beads, Pluronic 50 mmol L ). 1 mL of p-cresol (50 mmol L ) in organic solvent
F68, cellulose, carboxymethyl cellulose (CM-Cellulose) and was added into a 5 mL round-bottom ask, followed by addition
triethylaminoethyl cellulose (TEAE-Cellulose) were also of 5 mL 50 mmol L phosphate buffer (PBS, pH 7.0). Then, the
ꢁ
1
purchased from Sigma Aldrich (USA).
immobilized tyrosinase was added. Subsequently, the suspen-
sion was stirred by magnetic stirrers at 250 rpm at 25 C.
ꢀ
Aliquots of the liquid were removed and their absorption
spectrum was recorded in the range 400–600 nm. The detection
procedure of other substrates in a given solvent was the same as
p-cresol in a given solvent.
Instrumentation
Scanning electron microscope (SEM) images were obtained
through high-resolution SEM (Zeiss Merlin, Germany) with
a resolution of 0.8 nm at 15 kV and 1.4 nm at 1 kV. Transmission
electron microscope (TEM) images were obtained from FEI
Tecnai Multipurpose TEM (ThermoFisher Scientic, USA). UV-
Stability of tyrosinase in organic solvent at high temperature
Vis spectrum was obtained by a UV-Vis Nanodrop 2000C spec- The immobilized tyrosinase was transferred into a round
troscopy (ThermoFisher Scientic, USA).
bottom ask with anhydrous organic solvent under stirring at
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ꢀ
1
00 C. Aer 1 hour, the anhydrous organic solvent was glass beads. Aer immobilization of tyrosinase onto the surface
removed, and then 1 mL of p-cresol in toluene with 5 mL of materials, cellulose, CM-Cellulose and Pluronic F68
ꢁ
1
5
0 mmol L PBS (pH 7.0) was added. The detection procedure morphologies totally changed, and their surfaces became
was the same as the time course for tyrosinase-catalyzed rougher than before. These alterations showed that the tyrosi-
oxidation of p-cresol in a given solvent.
nase had been immobilized onto these materials.
Immobilized tyrosinase biocatalytic ability
Results and discussion
Six kinds of materials were used for the immobilization of
tyrosinase: the triethylaminoethyl group of TEAE-Cellulose
exhibits positive charge (pH 7.0, PBS solution); cellulose, Plur-
onic F68, poly(styrene-co-divinylbenzene) and glass beads have
neutral charges; and the carboxymethyl group of CM-Cellulose
has negative charge. As shown in Fig. 2, the TEAE-Cellulose
could bind tyrosinase (negative charge at pH 7.0) due to
charge attraction. TEAE-Cellulose is a positively charged resin
used in protein purication or separation. It can lock negatively
charged protein – tyrosinase onto the carrier by charge attrac-
tion and hydrogen-bond interactions. Cellulose can lock part of
tyrosinase only through hydrogen-bond interactions. CM-
Cellulose cannot lock tyrosinase like TEAE-Cellulose through
charge attraction, but a few of tyrosinase can be locked by
hydrogen-bond interactions. The tyrosinase molecules were
physically adsorbed onto the surface of Pluronic F68, poly(-
styrene-co-divinylbenzene) and glass beads. The tyrosinase
molecules immobilization efficiency of different materials
could be arranged as: TEAE-Cellulose > cellulose ¼ Pluronic F68
Physical characterization of TEAE-Cellulose and tyrosinase
immobilization onto TEAE-Cellulose
Fig. 1B displays the SEM image of tyrosinase immobilization
onto TEAE-Cellulose as compared to the original TEAE-
Cellulose (Fig. 1A). There was a signicant change in the
morphology between the original TEAE-Cellulose and tyrosi-
nase immobilization onto TEAE-Cellulose. The original TEAE-
Cellulose had a relative clear edge, and the TEAE-Cellulose
with tyrosinase showed an unclear edge, rough surface and
more porosity than the original one. Fig. 1C and D show the
TEM image of original TEAE-Cellulose and TEAE-Cellulose with
tyrosinase, respectively. The original TEAE-Cellulose surface
was very smooth. Aer tyrosinase immobilization onto TEAE-
Cellulose, its surface was rougher than the original one. Pure
TEAE-Cellulose has a clear edge from the TEM and SEM images.
Aer tyrosinase is immobilized onto TEAE-Cellulose, TEAE-
Cellulose and tyrosinase can cross-link with each other
through electrostatic attraction and hydrogen-bond interac-
tions. In this way, the edge of TEAE-Cellulose becomes unclear
and rough, and there is some porosity on its surface. Fig. S1A-
S1E† display the SEM images of (A) Cellulose, (B) CM-Cellulose,
¼
poly(styrene-co-divinylbenzene)
¼
glass beads > CM-
Cellulose.
p-Cresol was chosen as the test substrate. The tyrosinase can
function in chloroform whereby phenol derivatives can be
converted into corresponding stable o-quinones. The enzymatic
oxidation product of p-cresol is 4-methyl-1,2-benzoquinone,
and its maximum absorbance peak is at 395 nm. Kinetic anal-
(C) Pluronic F68, (D) poly(styrene-co-divinylbenzene) and (E)
o
ysis of tyrosinase is shown in Fig. 3. The V values of immobi-
lized tyrosinase onto TEAE-Cellulose, cellulose, poly(styrene-co-
divinylbenzene), Pluronic F68, glass beads and CM-Cellulose
ꢁ
2
ꢁ2
ꢁ2
ꢁ2
were 6.7 ꢂ 10 , 5.5 ꢂ 10 , 4.4 ꢂ 10 , 3.6 ꢂ 10 , 2.8 ꢂ
Fig. 3
o
V values of tyrosinase molecular immobilization onto CM-
Cellulose, cellulose, TEAE-Cellulose, Pluronic F68, poly(styrene-co-
Fig. 2 Schematic diagram describing the mechanism of tyrosinase divinylbenzene) and glass beads catalyzed oxidations of p-cresol as
immobilization onto (A) CM-Cellulose, (B) cellulose and (C) TEAE- substrate in chloroform. p-Cresol concentration for these reactions is
ꢁ1
Cellulose.
50 mmol L . Error bar means standard deviation.
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Table 1 The activity of tyrosinase immobilization onto TEAE-Cellulose was detected in organic solvent (i.e. CH
Cl
2 2
, CCl
4
and toluene)
Viscosity 10
ꢁ3
ꢁ
1
ꢁ1
p-Cresol
V
o
(mmol L min
)
Std
log P
Pa s
Acetonitrile
Tetrahydrofuran
—
—
—
—
—
2.6 ꢂ 10
2.5 ꢂ 10
5.0 ꢂ 10
4.6 ꢂ 10
3.0 ꢂ 10
1.4 ꢂ 10
ꢁ0.34
0.46
3
0.94
1.25
2.84
1.97
2.83
2.73
0.34
0.46
7.4
0.36
0.42
0.75
0.54
0.9
ꢁ
4
1
-Octanol
tert-Butyl methyl ether
CH Cl
Chlorobenzene
CHCl
CCl
Toluene
<8.3 ꢂ 10
ꢁ4
ꢁ3
ꢁ2
ꢁ2
ꢁ2
ꢁ2
ꢁ4
ꢁ4
ꢁ3
ꢁ3
ꢁ3
ꢁ3
8.3 ꢂ 10
1.5 ꢂ 10
5.7 ꢂ 10
6.1 ꢂ 10
6.3 ꢂ 10
6.7 ꢂ 10
2
2
3
4
0.55
ꢁ
2
ꢁ2
ꢁ1
ꢁ1
10
and 1.5 ꢂ 10 mmol L min , respectively. The V
o
value this rule. 1-Octanol is categorized as a hydrophobic solvent and
of immobilized tyrosinase onto TEAE-Cellulose was the highest the value of log P is relatively high (achieved 3, similarly with
among those onto CM-Cellulose, cellulose, Pluronic F68, toluene and carbon tetrachloride), but the Vo value of the
poly(styrene-co-divinylbenzene) and glass beads, and the Vo immobilized tyrosinase in 1-octanol is relatively low (less than
ꢁ4
value of immobilized tyrosinase onto CM-Cellulose was the 8.3 ꢂ 10 ). In this case, the viscosity of 1-octanol achieves as
ꢁ3
lowest among these materials. The results were consistent with high as 7.4 ꢂ 10 Pa s, and the mass-transfer rate of substrate
the mechanism shown in Fig. 2. The TEAE-Cellulose was in 1-octanol is very slow. This might be one of the most
demonstrated to be the best supporting platform for tyrosinase important parameters in determining the biocatalytic efficiency
and it was chosen as the immobilization material in the of the immobilized tyrosinase. As a result, toluene was chosen
following research work.
as the solvent to test the immobilized tyrosinase activity.
Immobilization enzyme kinetics in organic solvent
The biocatalytic efficiency of the immobilized tyrosinase for
different substituent groups of para-cresol isomers in
hydrophobic organic solvent
Aer optimizing the tyrosinase immobilization platform, the
stability of immobilized tyrosinase was systematically assessed
in different organic solvents. Organic solvents can affect the Aer optimizing the immobilized tyrosinase solvent conditions,
structure of enzymes at the secondary, tertiary and quaternary the relationship between the structure of the substrate and the
7
levels. It is important to note how organic solvents affect the activity of the immobilized tyrosinase was systematically
immobilized tyrosinase activity. From Table 1, the data studied. How the substituent group position (three isomers of p-
demonstrates that the immobilized tyrosinase was completely cresol) inuenced the selectivity of the immobilized tyrosinase
inactive in hydrophilic solvents viz. acetonitrile (log P, ꢁ0.34) in toluene was examined. Table 2 shows that the V
o
value for
and tetrahydrofuran (log P, 0.46). This can be attributed to the tyrosinase in the toluene medium, using p-cresol as substrate,
fact that the hydrophilic solvents could strip the essential water was 8.7-fold higher than those obtained with m-cresol as
from tyrosinase's hydration “shell” (hence deactivate the substrate and more than 30-fold higher than those with o-cresol
enzyme). Moreover, the immobilized tyrosinase had biocatalytic as substrate (the V number was too small to check). The ortho-
o
ability in hydrophobic solvent i.e. tert-butyl methyl ether (log P, substituent group was not signicantly catalyzed by the
0.94), methylene chloride (log P, 1.25), chloroform (log P, 1.97), immobilized tyrosinase because such substrates sterically
carbon tetrachloride (log P, 2.83), chlorobenzene (log P, 2.84) hinder the accessibility of the phenolic hydroxyl moiety to the
and toluene (log P, 2.73). Octanol–water partition coefficient active-site of tyrosinase. The steric inuence of the para
(
log P) is used in QSAR studies and rational chemical design as substituent group of phenol for the biocatalysis of tyrosinase
a measurement of molecular hydrophobicity. Our results sug- was less than that of meta or ortho substituents.
gested that the hydrophobicity of organic solvent played an It was essential to know how the different para substituent
important role in tyrosinase efficiency. As the hydrophobicity of groups inuence the catalytic efficiencies of the immobilized
organic solvents increased, most of the biocatalytic efficiency of tyrosinase. Different para-substituent groups of phenol were
the immobilized tyrosinase increased. There is an exception to tested in toluene. Table 3 shows that the V value of tyrosinase
o
Table 2 The bioactivity of tyrosinase was tested for different substituent sites in different solvent (i.e. CH
2 2 4
Cl , CCl and toluene)
Substrate
p-Cresol (V
o
)
p-Cresol (Std)
m-Cresol (V
o
)
m-Cresol (Std)
o-Cresol (V
o
)
o-Cresol (Std)
ꢁ
ꢁ
ꢁ
ꢁ
2
2
2
2
ꢁ3
ꢁ3
ꢁ4
ꢁ4
ꢁ4
ꢁ4
ꢁ4
Toluene
Chlorobenzene
6.7 ꢂ 10
5.7 ꢂ 10
6.1 ꢂ 10
6.3 ꢂ 10
1.4 ꢂ 10
7.7 ꢂ 10
2.2 ꢂ 10
<8.3 ꢂ 10
<8.3 ꢂ 10
<8.3 ꢂ 10
<8.3 ꢂ 10
—
—
—
—
ꢁ3
ꢁ3
ꢁ5
5.0 ꢂ 10
6.5 ꢂ 10
1.9 ꢂ 10
ꢁ3
ꢁ3
ꢁ4
CHCl
CCl
3
4.6 ꢂ 10
7.0 ꢂ 10
1.2 ꢂ 10
ꢁ3
ꢁ3
ꢁ4
4
3.0 ꢂ 10
7.2 ꢂ 10
1.2 ꢂ 10
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Table 3 The bioactivity of tyrosinase was detected for six kinds of para-substituents of phenol in toluene
Solubility in
water (mol m
ꢁ
1
ꢁ1
ꢁ3
Substrate
V
o
(mmol L min
)
Std
log Kow
)
ꢁ2
ꢁ2
ꢁ2
ꢁ2
ꢁ3
ꢁ3
ꢁ3
ꢁ3
ꢁ3
ꢁ3
ꢁ4
ꢁ5
p-Methyl
p-Ethyl
p-Propyl
p-Chloro
p-Bromo
p-t-Butyl
6.7 ꢂ 10
4.1 ꢂ 10
2.8 ꢂ 10
2.1 ꢂ 10
9.2 ꢂ 10
2.0 ꢂ 10
1.4 ꢂ 10
3.2 ꢂ 10
2.1 ꢂ 10
1.8 ꢂ 10
8.5 ꢂ 10
2.0 ꢂ 10
1.96
2.50
3.20
2.40
2.59
3.65
184.9
65.3
12.7
210.0
85.54
3.861
the immobilized tyrosinase decreased. It indicated that the
tyrosinase activity was related to the electron-donating ability
and steric size of the substitute. In contrast, p-chloro and p-
bromo are electron-withdrawing substituent groups; their cor-
o
responding V value of the immobilized tyrosinase decreased
with their deactivating ability. The conclusion was consistent
with the quantitative assessment of enzymatic catalysis by
49
Dordick et al.
Aer optimizing the immobilized platform, the substrate
and the solvent, the behavior of tyrosinase in anhydrous organic
solvent at high temperature was studied. In this way, the
immobilized tyrosinase was treated as a synthetic catalyst. Nine
kinds of anhydrous organic solvents were used for storing the
ꢀ
immobilized tyrosinase at 100 C. The nine anhydrous organic
solvents are methanol (log P, ꢁ0.77; relative polarity, 0.762),
ethanol (log P, ꢁ0.31; relative polarity, 0.654), acetonitrile
(
log P, ꢁ0.34; relative polarity, 0.46), acetone (log P, ꢁ0.24;
relative polarity, 0.355), methyl acetate (log P, 0.18; relative
polarity, 0.762), tetrahydrofuran (log P, 0.46; relative polarity,
0
.207), methyl t-butyl ether (log P, 0.94; relative polarity, 0.124),
toluene (log P, 2.73; relative polarity, 0.099) and hexane (log P,
.9; relative polarity, 0.009). As shown in Fig. 4, the half-life of
3
the immobilized tyrosinase is strongly related to the relative
polarity value of the storage solvent than their log P values. Aer
the immobilized tyrosinase is immersed in anhydrous hexane at
ꢀ
1
6
00 C for 96 hours, the immobilized tyrosinase still retains over
0% activity. Conversely, the immobilized tyrosinase loses the
ꢀ
activity immediately in water at 100 C. This indicated that the
immobilized tyrosinase could keep its catalytic structure and be
used as a synthetic catalyst in anhydrous organic solvent at high
temperature. This research provides a lot of information for
mimicking enzyme reactions.
Fig. 4 (A) The half-life of the immobilized tyrosinase vs. the log P Conclusion
values of the organic solvent, (B) the half-life of the immobilized
tyrosinase vs. the relative polarity values of the organic solvent. The Triethylaminoethyl cellulose (TEAE-Cellulose) was selected as
organic solvent from right to left is methanol, ethanol, acetonitrile,
acetone, methyl acetate, tetrahydrofuran, methyl t-butyl ether,
Compared with traditional immobilization materials (glass
toluene and hexane.
the best immobilization platform from six kinds of materials.
beads), the activity of tyrosinase immobilization onto TEAE-
Cellulose was two times higher than that of immobilization
onto glass beads. The immobilized tyrosinase showed potential
to be a catalyst in organic solvent without water. The half-life of
the dry immobilized tyrosinase in organic solvent was strongly
related to the polarity of the solvent rather than the log P value
of the organic solvent. This research could be valuable for the
design of new biocatalysts.
biocatalysis for para substituent were: p-methyl > p-ethyl > p-
propyl > p-chloro > p-bromo > p-t-butyl. The methyl, ethyl,
propyl and t-butyl groups are electron-donating substituent
groups. By increasing the electron-donating ability and the
o
steric size of para-substituents, the corresponding V value of
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1
3 R. Plothe, I. Sittko, F. Lanfer, M. Fortmann, M. Roth,
V. Kolbach and J. C. Tiller, Poly(2-ethyloxazoline) as Matrix
for Highly Active Electrospun Enzymes in Organic Solvents,
Biotechnol. Bioeng., 2017, 114(1), 39–45.
Conflicts of interest
There are no conicts to declare.
1
4 F. S. Liao, W. S. Lo, Y. S. Hsu, C. C. Wu, S. C. Wang,
F. K. Shieh, J. V. Morabito, L. Y. Chou, K. C. W. Wu and
C. K. Tsung, Shielding against Unfolding by Embedding
Enzymes in Metal-Organic Frameworks via a de Novo
Approach, J. Am. Chem. Soc., 2017, 139(19), 6530–6533.
Acknowledgements
This study was supported by China Scholarship Council, the
National Natural Science Foundation of China (No. 21307161,
61335008 and 61874137). Authors would like to thank
Alexander M. Klibanov who gave lots of suggestion in experi- 15 U. Guzik, K. Hupert-Kocurek and D. Wojcieszynska,
ment details.
Immobilization as
Properties-Application to Oxidoreductases, Molecules, 2014,
9(7), 8995–9018.
6 K. Min and Y. J. Yoo, Recent progress in nanobiocatalysis for
enzyme immobilization and its application, Biotechnol.
Bioprocess Eng., 2014, 19(4), 553–567.
a Strategy for Improving Enzyme
1
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