Covalently Immobilized Lipase on a Thermoresponsive Polymer with an Upper Critical Solution Temperature as an Efficient and Recyclable Asymmetric Catalyst in Aqueous Media

Article abstract of DOI:10.1002/cctc.201701512

A thermoresponsive lipase catalyst with an upper critical solution temperature (UCST) of about 26 °C was exploited by covalent immobilization of an enzyme, Pseudomonas cepacia lipase (PSL), onto poly(acrylamide-co-acrylonitrile) by glutaraldehyde coupling. The experimental conditions for the PSL immobilization were optimized. The immobilized PSL was much more stable for wide ranges of temperature and pH than free PSL. The material was also evaluated as an asymmetric catalyst in the kinetic resolution of racemic α-methylbenzyl butyrate at 55 °C in an aqueous medium and exhibited high catalytic performance and stability. Up to 50 % conversion and 99.5 % product enantiomeric excess were achieved, thus providing highly pure enantiomers. This biocatalyst could be easily recovered by simple decantation for reuse based on temperature-induced precipitation. It showed good reusability and retained 80.5 % of its original activity with a well reserved enantioselectivity in the 6th cycle. This work would shed light on the future development of new UCST-type enzyme catalysts.

Full text of DOI:10.1002/cctc.201701512

Accepted Article
Title: Covalently Immobilized Lipase on a Thermoresponsive Polymer
with an Upper Critical Solution Temperature as an Efficient and
Recyclable Asymmetric Catalyst in Aqueous Media
Authors: Wuzong Zhou, Lan-lan Lou, Huaxin Qu, Wenjun Yu, Bei
Wang, Lezi Ouyang, and Shuangxi Liu
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Link to VoR: http://dx.doi.org/10.1002/cctc.201701512
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ChemCatChem
10.1002/cctc.201701512
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Covalently Immobilized Lipase on a Thermoresponsive Polymer
with an Upper Critical Solution Temperature as an Efficient and
Recyclable Asymmetric Catalyst in Aqueous Media
Lan-Lan Lou,[a,b] Huaxin Qu, Wenjun Yu, Bei Wang, Lezi Ouyang, Shuangxi Liu,* and Wuzong
[a]
[a]
[a]
[a]
[a,c]
Zhou**[b]
Abstract: A thermoresponsive lipase catalyst with an upper critical
solution temperature (UCST) of about 26 °C was exploited by covalent
immobilization of an enzyme, Pseudomonas cepacia lipase (PSL),
onto poly(acrylamide-co-acrylonitrile) via glutaraldehyde coupling.
The experimental conditions for the PSL immobilization were
optimized. The immobilized PSL was much more stable for wide
ranges of temperature and pH than the free PSL. The material was
also evaluated as an asymmetric catalyst in the kinetic resolution of
racemic α-methylbenzyl butyrate at 55 °C in an aqueous medium and
exhibited high catalytic performance and stability. Up to 50%
conversion and 99.5% product enantiomeric excess were achieved,
thus providing highly pure enantiomers. More importantly, this
biocatalyst could be easily recovered by simple decantation for reuse
based on temperature-induced precipitation. It showed good
reusability and retained 80.5% of its original activity with a well
However, the free enzymes often present some disadvantages,
such as low stability for long-term operation, high sensitivity
towards environmental conditions, as well as difficulty in
separation and recovery from the reaction system. Their practical
applications are therefore severely limited.[3] Immobilization of
enzymes can usually overcome these shortcomings and has thus
been recognized as an appropriate method to improve the
catalytic properties of enzymes.[3,4] Consequently, many efforts
have been devoted to the immobilization of lipases on different
solid carriers, including various inorganic materials and organic
polymers, through a variety of processes, such as entrapment,
physical adsorption, and covalent attachment.[5]
On the other hand, diffusional limitations of reactants to the
catalytic active sites often have an adverse effect on the catalytic
activity of immobilized lipases. In addition, low reusability in
aqueous media is common issue over the immobilized lipases by
entrapment and physical adsorption due to a large tendency for
enzymes to leach from the carriers, further limiting their
applications, especially for hydrolytic reactions.
th
reserved enantioselectivity in the 6 cycle. This work would shed light
on the future development of new UCST-type enzyme catalysts.
In the past decades, water-soluble thermoresponsive polymers
have attracted considerable interests in both academia and
Introduction
industry owing to their ‘‘smart’’ nature of reversible solubility
Owing to the increasing demands for enantioselective syntheses
of fine chemicals and environmentally benign processes in the
chemical industry, enzymes have recently received extensive
attention as a class of highly specific, efficient and stereoselective
biocatalysts.[1] Lipases are among the most useful enzymes and
are widely utilized in numerous industrial biosynthetic processes
for the production of optically active compounds, pharmaceuticals,
agrochemicals, food, biodiesel, detergent, etc.[2]
changes in response to temperature,[6] which makes it highly
possible to design polymer-based smart heterogeneous
biocatalysts that can realize
a
desirable process of
“
homogeneous catalysis, heterogeneous recycle” by changing
the system temperature. Some thermoresponsive polymers,
especially polymers with a lower critical solution temperature
(
LCST) have been widely used as carriers for enzyme
[7]
immobilization. In general, the LCST-type thermoresponsive
polymers, typically poly(N-isopropylacrylamide) and poly(N-
vinylcaprolactam), exhibit LCSTs of about 30~35 °C, which are
lower than the reaction temperature for optimum activity of most
enzymes. In other words, these polymers are often insoluble and
would separate from the aqueous system at the optimum
temperature for enzymes, leading to unsatisfactory catalytic
activity. Moreover, the catalyst separation process for a multi-
cycle operation performed at elevated temperatures may always
deactivate the enzymes.
[a]
Dr. L.-L. Lou, Ms. H. X. Qu, Dr. W. J. Yu, Ms. B. Wang, Mr. L. Z.
Ouyang, Prof. S. X. Liu
Institute of New Catalytic Materials Science and Key Laboratory of
Advanced Energy Materials Chemistry (Ministry of Education),
National Institute for Advanced Materials, School of Materials
Science and Engineering
Nankai University
Tianjin 300350 (China)
E-mail: sxliu@nankai.edu.cn
[
b]
c]
Dr. L.-L. Lou, Prof. W. Z. Zhou
School of Chemistry
University of St Andrews
St Andrews, Fife KY16 9ST (United Kingdom)
E-mail: wzhou@st-andrews.ac.uk
Prof. S. X. Liu
In contrast, polymers with an upper critical solution temperature
(
UCST), above which polymers are soluble in water, display more
promising potential for enzyme immobilization. As the UCST is
often below the optimum temperature of enzymes, the soluble
polymer molecules would remarkably reduce the mass transfer
limitation for the immobilized enzymes and thus enhance the
catalytic activity in such a homogeneous catalytic system.
[
Collaborative Innovation Center of Chemical Science and
Engineering (Tianjin)
Tianjin 300072 (China)
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However, the reports about the immobilized enzymes with UCST-
type thermoresponsive polymers to date have been still very
limited. As a rare example, Limadinata et al. [8] reported the
synthesis of UCST-type polymer nanoparticles poly(glycidyl
transmittance of these polymer samples with a total concentration
of 0.5 wt% in a phosphate buffer solution (pH 7.0, 0.05 M).
100
methacrylate)/poly(acrylic
acid-co-acrylamide)
and
their
pAA
pAA-PSL
application as carriers to immobilize cellulase and cellobiase, also
popular enzymes, for the hydrolysis of cellulose to glucose. These
biocatalysts showed good catalytic performances above the
UCST of 14 °C and could be easily separated from the liquid
phase by cooling down the solution to a temperature below the
UCST. Till now, however, the UCST-type polymers have not been
investigated for their application in enantioselective biocatalysis.
In the present work, we explore immobilization of Pseudomonas
cepacia lipase (PSL) using a UCST-type thermoresponsive
polymer, poly(acrylamide-co-acrylonitrile) (pAA), as the carrier,
and investigate its enantioselective application in an aqueous
medium. PSL was covalently attached on pAA by using
90
8
7
6
0
0
0
50
4
0
0
3
o
o
20
22 C
30 C
10
20
25
30
35
40
45
o
Temperature ( C)
glutaraldehyde (GA) as
a coupling agent. The produced
immobilized lipase, with a UCST of about 26 °C, was applied in
asymmetric hydrolysis of racemic α-methylbenzyl butyrate. The
immobilization conditions, catalytic activity, enantioselectivity,
stability and reusability of the immobilized enzyme are
investigated in detail. This preliminary research on UCST-type
thermoresponsive lipase catalyst may shed light on the future
development of new enzyme catalysts.
Figure 1. Turbidity curves of 0.5 wt% of pAA and pAA-PSL in a phosphate buffer
solution (pH 7.0, 0.05 M) and photographs of pAA-PSL in the phosphate buffer
solution at 22 °C and 30 °C (inset).
As seen in Figure 1, the polymer pAA shows a UCST of about
26 °C. This phase transition behaviour can be mainly attributed to
the increased inter-/intra-chain hydrogen bonding interaction
C=O···H-N) in the polymer molecules upon cooling.[
10]
The
(
Results and Discussion
immobilized enzyme pAA-PSL also exhibits a thermoresponsive
behaviour with a very similar UCST to the pAA carrier, although
the overall transmittance of the former is about 10% lower than
the latter, indicating that incorporation with the enzyme reduces
the solubility of the polymer. Consequently, the phosphate buffer
solution of pAA-PSL is cloudy at 22 °C and becomes clear at
Synthesis and turbidity measurement of immobilized PSL on
pAA
The polymer pAA was synthesised through the polymerization of
30 °C. This UCST-behaviour is highly desirable since the reaction
acrylamide and acrylonitrile with a molar ratio of 82 : 18 by using
temperature for an optimum activity of most enzymes is higher
than this UCST. Thus efficient catalysis can be expected above
the UCST over the immobilized enzymes that are well dissolved,
and separation of the catalysts can be easily achieved by
precipitation below the UCST.
,2-azobisisobutyronitrile (AIBN) as an initiator.[ Enzyme PSL
9]
2
was then covalently immobilized on pAA by using GA, in which
the aldehyde groups reacted with the amino groups in the polymer
as well as in the PSL molecules (Scheme 1). The resulting
specimen was denoted as pAA-PSL.
Optimization of conditions for PSL immobilization
The experimental conditions for the PSL immobilization were
optimized by investigating the effects of the GA concentration,
PSL concentration, and immobilization time on the enzyme
loading and specific activity measured in tributyrin hydrolysis of
the immobilized PSL.
Scheme 1. Glutaraldehyde-mediated covalent immobilization of PSL on the
polymer pAA.
The effects of the GA concentration on the enzyme loading and
specific activity of pAA-PSL were studied in the range of 0.03 mol
L to 0.19 mol L1 at 45 °C, and the results are depicted in Figure
1
2
a. It is observed that the enzyme loading increases notably with
The thermoresponsive behaviour of pAA is important as the
activity of enzyme immobilized on it can be adjusted by changing
the temperature above and below the UCST. To characterize the
thermoresponsive behaviours of pAA and pAA-PSL, turbidity
observation was carried out by measuring the 500 nm UV
1
the increase of the GA concentration from 0.03 to 0.08 mol L ,
1
and then reaches a steady stage between 0.08 and 0.13 mol L ,
followed by a sharp decrease with further raising the GA
concentration. The specific activity of immobilized PSL exhibits a
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FULL PAPER
1
1
1
1
400
300
200
1
600
400
1
350
(a)
(b)
700
(c)
600
550
500
450
400
620
1
6
00
00
1300
1250
1200
1150
1200
000
600
580
560
5
1
8
6
4
00
00
00
400
300
200
100
0
.00
0.05
0.10
0.15
0.20
1
2
3
4
5
1
2
3
4
5
-1
)
-1
GA concentration (mol L
PSL concentration (mg mL )
Immobilization time (h)
Figure 2. Effects of the GA concentration (a), the PSL concentration (b), and immobilization time (c) on enzyme loading and specific activity of the immobilized PSL.
1
similar trend to that of enzyme loading, and the highest value is
obtained at the GA concentration of 0.11 mol L .
loading is 1.33 mg g pAA-PSL and the specific activity of
immobilized PSL is 610 U g1 pAA-PSL. With prolonging the
immobilization time, the overloaded PSL molecules may undergo
multilayer adsorption, which can not only change the
conformation of enzyme molecules, but also make some of the
enzyme molecules inaccessible by the substrate.[12]
1
This phenomenon is well consistent with the previous reports.[11]
It was reported that the GA concentration has a noticeable effect
on its own structure and the activity of immobilized enzyme. With
an appropriate GA concentration, most GA molecules link the
polymer support and PSL molecules with a dimer form, which is
beneficial to reach a high activity for immobilized enzyme by
exposing sufficiently the active sites on the enzyme. Whereas GA
exists mainly in a monomeric or polymeric form when a lower or
higher GA concentration is adopted. In both cases, the enzyme
PSL is apt to closely attach on the polymer carrier, which is
unfavorable for the access of the substrate to the active sites of
enzymes, thus leading to an inferior activity.
This optimum enzyme loading, however, is at a relatively low
level as compared with those for other immobilized biocatalysts
reported in the literature.[
concentration made an insignificant change in the enzyme
loading. The low loading may be resulted from the inferior reaction
efficiency between the polymer and GA as well as from the low
enzyme content in the PSL reagent.
11b,13-16]
Increase of the added enzyme
The influence of the PSL concentration on the enzyme loading
and specific activity of the immobilized PSL was studied in a range
Activity and stability of immobilized PSL
1
of 1 to 5 mg mL . The results are presented in Figure 2b. A
significant dependence of enzyme loading as well as the activity
of immobilized PSL on the PSL concentration can be found. The
enzyme loading monotonically increases with increasing the PSL
concentration.
The activity of pAA-PSL for tributyrin hydrolysis was studied at
different temperatures and pH values. For comparison, free PSL
was also tested under the same reaction conditions. The influence
of temperature on hydrolytic activity of pAA-PSL and free PSL
was investigated in a range of 40-65 °C with pH of 7.0. The results
are given in Figure 3a. It can be found that both pAA-PSL and free
PSL give the highest activity at 55 °C. At this point, pAA-PSL
As for the specific activity of the immobilized PSL, a remarkable
ascent is observed with increasing the PSL concentration up to 3
1
1
mg mL , where a maximum value of 610 U g is reached,
followed by a gradual decline with further increase of the PSL
exhibits a specific activity of 650 U g1 pAA-PSL, namely 489 U
mg-1 protein, suggesting a high retainment of ~70% of the free
1
concentration from 3 to 5 mg mL . The decreased activity of
immobilized PSL at high PSL concentration may be ascribed to
the increased intermolecular reaction of the immobilized lipase
with a relatively high enzyme loading, when the enzyme
undergoes a multilayer aggregation on the polymer.[7b,12] With the
decrease in the enzyme loading, on the other hand, a monolayer
of the immobilized enzyme may be presented on the polymer
support,[ which is favorable to the maintenance of the active
conformation of the PSL molecules as well as to the accessibility
of the active sites of enzyme by the substrate. In consequence,
the maximum activity of the immobilized PSL can be achieved.
The influence of immobilization time on the enzyme loading and
specific activity was investigated in a range of 1 ~ 5 h. The results,
depicted in Figure 2c, show that the enzyme loading increases
monotonically with the increase of immobilization time, while the
specific activity of immobilized PSL shows the best result when
the immobilization time is about 4 h. At this point, the enzyme
mg1 protein. In addition, at other
PSL activity, 693
U
temperatures below or above 55 °C, remarkably more stable
activities are achieved over immobilized PSL compared with the
free PSL, suggesting the stabilization of PSL activity upon
immobilization on the polymer. The improvement in stability of the
enzyme activity can be mainly attributed to the multipoint
attachment between the polymer pAA and the PSL molecules,
which can effectively prevent the interaction between enzyme
molecules as well as thermal denaturation of PSL.
13]
Figure 3b shows the influence of pH value on hydrolytic activity
of pAA-PSL and free PSL in a range of 6.0-8.0 with a constant
temperature of 55 °C. The optimum pH value is determined to be
7.0 for both pAA-PSL and free PSL. Apart from this, compared
with free PSL, highly stable activities of above 90% were also
achieved over the immobilized PSL at other pH values. For free
PSL, however, notably decreases in activities were observed
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ChemCatChem
10.1002/cctc.201701512
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above or below 7.0, especially, a low relative activity of 48.5% was
obtained at pH 8.0. All the results indicate that the immobilization
of PSL onto pAA effectively enhances the stability of the enzyme
activity towards variations of temperature and pH.
produce (R)-1-phenylethanol in aqueous media at 55 °C (Scheme
2). For comparison, the free PSL with the same protein content
was also examined under the identical reaction conditions. Blank
experiment showed that no reaction occurred in the absence of
PSL. Table 1 gives the conversions and enantioselectivities of the
enzymatic kinetic resolution at different reaction time and
substrate concentration using pAA-PSL and free PSL.
100
100
(a)
(b)
9
8
7
6
0
0
0
0
90
80
70
60
50
Free PSL
pAA-PSL
50
40
Free PSL
pAA-PSL
40
45
50
55
60
65
6.0
6.5
7.0
pH
7.5
8.0
o
Temperature ( C)
Scheme 2. The chemical reaction of kinetic resolution of (R,S)-α-methylbenzyl
butyrate to produce (R)-1-phenylethanol using immobilized enzyme pAA-PSL
as catalyst.
Figure 3. Effects of temperature (a) and pH (b) on hydrolytic activity of free and
immobilized PSL.
In order to get further insight into the thermal stability of the
immobilized enzyme, pAA-PSL as well as free PSL was incubated
at 60 °C for various periods and then the residual activities were
measured. As shown in Figure 4, the immobilized PSL exhibits a
superior thermostability compared with the free PSL, e.g. they
retain 97.7% and 73.3% respectively of the original activity after 1
h of incubation. With increasing the incubation time, the residual
activities of both the enzyme samples decrease, whereas the
immobilized PSL shows a slower descending tendency than free
PSL. For example, after 12 h of incubation, free PSL only presents
a residual activity of 34.4%, while pAA-PSL still maintains 68.8%
of its original activity. These results suggest that the thermal
stability of PSL is significantly enhanced upon immobilization onto
pAA, which is very favorable from the point of view of practical
application.
With 0.05 M of racemic α-methylbenzyl butyrate, pAA-PSL
exhibits 96.3% of the free enzyme activity at 6 h (33.8/35.1, Table
, entry 2) and furnishes higher conversions after 9 h compared
with free PSL. 50.0% conversion is achieved over pAA-PSL at 15
h, whereas the free PSL shows a conversion of 45.9% under the
same conditions (Table 1, entry 5). In addition, the immobilized
PSL shows high enantioselectivity throughout the reaction with
1
p
the product enantiomeric excesses (ee ) above 98.9% and E >
2
00. These results indicate that pAA-PSL possesses high
catalytic performance and that (R)-α-methylbenzyl butyrate is
almost completely hydrolysed at 15 h to produce highly pure
enantiomers. The higher conversion over pAA-PSL compared to
free enzyme can be attributed to the enhancement of enzyme
stability through immobilization.
With the increase of substrate concentration,
a gradual
decrease in the conversion was observed for both the immobilized
and free enzymes. The conversions were reduced to 46.5% and
1
00
Free PSL
pAA-PSL
41.1% for pAA-PSL and free PSL, respectively, at 15 h of reaction
8
6
4
2
0
0
0
0
0
as the substrate concentration was raised to 0.15 M (Table 1,
entry 8). Further increasing the substrate concentration to 0.25 M,
only 22.3% and 17.2% conversions over pAA-PSL and free PSL
respectively were obtained at the same reaction time (Table 1,
entry 11). It is noted that in all cases higher conversions were
achieved over immobilized enzyme at extended reaction time
compared to the free enzyme, confirming the better stability of
immobilized enzyme.
As for the enantioselectivity, a slight declining trend was
detected for the two enzyme catalysts when the substrate
concentration was increased. For instance, the ee and the E
p
0
0.5
1
2
3
4
12
Incubation time (h)
value over pAA-PSL at 3 h of the reaction decreased from 98.9%
and >200 to 97.1% and 74.4, respectively, with the substrate
concentration increased from 0.05 M to 0.25 M (Table 1, entry 1
versus 9).
Figure 4. Thermal stability of immobilized and free PSL at 60 °C for 12 h.
The reusability of UCST-biocatalyst pAA-PSL was studied in the
kinetic resolution of (R,S)-α-methylbenzyl butyrate. Each
experiment was carried out in a phosphate buffer solution (2.5 mL,
pH 7.0, 0.05 M) at 55 °C for 12 h with 0.125 mmol of fresh
substrate. After each reaction, the catalyst was easily precipitated
by cooling the reaction mixture below the UCST (Figure 5a). The
Kinetic resolution of (R,S)-α-methylbenzyl butyrate catalyzed
by immobilized PSL in aqueous media
The immobilized enzyme pAA-PSL was evaluated as a soluble
catalyst in kinetic resolution of (R,S)-α-methylbenzyl butyrate to
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Table 1. Kinetic resolution of (R,S)-α-methylbenzyl butyrate catalyzed by pAA-PSL and free PSL[a]
Conv (%)[c]
ee
(%)[d]
pAA-PSL
E[e]
p
Entry
C
s
(M)[b]
Time (h)
Free PSL
25.9
35.1
39.8
43.3
45.9
16.9
32.2
41.1
6.3
pAA-PSL
21.9
33.8
42.9
48.9
50.0
14.8
33.8
46.5
8.8
Free PSL
Free PSL
pAA-PSL
>200
>200
>200
>200
>200
106.3
>200
>200
74.4
1
2
3
4
5
6
7
8
9
0.05
3
6
98.7
99.0
99.3
99.5
99.5
97.3
99.1
99.3
97.2
98.1
98.5
98.9
99.1
99.3
99.5
99.5
97.8
99.0
99.5
97.1
98.8
99.0
>200
>200
>200
>200
>200
88.8
9
12
15
3
0.15
0.25
9
>200
>200
73.7
15
3
1
0
1
9
13.7
17.2
18.8
22.3
121.8
160.7
>200
>200
1
15
[
a]
[b]
Reaction condition: 10 mg pAA-PSL or 1.6 mg free PSL, 2.5 mL phosphate buffer solution (pH 7.0, 0.05 M), 55 °C.
C
s
represents the substrate
[
c]
concentration in the reaction system. Conv (%) represents the substrate conversion and is determined by GC with a RESTEK RT-BetaDEXse chiral
column. ee
represents the enantiomeric ratio of the reaction and is calculated by the expression ln[1-Conv(1+ee
[
d]
[e]
p
(%) represents the enantiomeric excess of alcohol product and is determined by GC with a RESTEK RT-BetaDEXse chiral column.
)]/ln[1-Conv(1-ee )].
E
p
p
furnishes 96.4% of its initial activity. After six cycles, the catalyst
still remains 80.5% of its original activity. In addition, the
(c)
p
enantioselectivity of the reaction is well reserved (99.5% ee and
E > 200) during the recycling experiments. According to the
previous researches, it is quite common for immobilized enzymes
to drop their catalytic activity after several runs. For example,
three lipases covalently immobilized on polymer-encapsulated
magnetic nanobeads retained about 40~90% of the initial activity
after six cycles in the hydrolysis of rac-α-methylbenzyl butyrate in
aqueous solutions.[14] Lipase from Burkholderia cepacia
immobilized into calcium carbonate microcapsule showed about
(a)
4
7% of its original activity after five cycles in the kinetic resolution
(b)
[
15]
of rac-2-acetoxyhexyl tosylate in an aqueous phase. 65% of the
initial activity was kept after ten cycles in the hydrolysis of olive oil
by Candida rugosa lipase covalently immobilized on core/shell
magnetic composite microspheres.[16] Han et al. reported that the
Figure 5. Photographs of catalytic reaction solutions after cooling to 0 °C (a)
and under a new cycle at 55 °C (b). (c) Reusability of pAA-PSL in the kinetic
resolution of (R,S)-α-methylbenzyl butyrate. Relative activities were calculated
by taking the initial activity for the first time as 100%.
immobilized Pseudomonas fluorescens lipase on porous polyuria
remained 73% of its initial activity after five cycles in the hydrolysis
of p-nitrophenyl palmitate.[11b] The recycling experiments in the
present work demonstrate that the new immobilized biocatalyst
has high stability and durability for the kinetic resolution reaction.
The obtained result is among the best results with regard to the
reusability for the immobilized lipase catalysts towards hydrolytic
reactions.
catalyst recovered via simple decantation and washing was then
well redispersed in the phosphate buffer solution for next cycle
(
Figure 5b). The Bradford assay showed that there was no
detectable protein leaching in the supernatant and washings after
each cycle, indicating that the enzyme PSL was strongly attached
on the polymer.
The recycling results are depicted in Figure 5c. It shows that
pAA-PSL maintains a high catalytic activity in the third cycle and
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Conclusions
(S)- and (R)-1-phenylethanol after reaction, respectively. The enantiomeric
ratio of the reaction (E) was calculated using ln[1-Conv(1+ee
p
)]/ln[1-
Conv(1-ee
p
)] as described by Chen et al.[17]
The thermoresponsive polymer pAA with a UCST of 26 °C was
synthesised, surface-modified with GA and then applied to
covalently immobilize an enzyme of PSL to afford a biocatalyst
with high catalytic performance and stability. The optimal
experimental conditions for PSL immobilization, including GA
concentration, PSL concentration and immobilization time, were
determined, under which the highest activity of 650 U g1 was
achieved over immobilized PSL with an enzyme loading of 1.33
mg g pAA-PSL, suggesting a high specific activity retention of
about 70% compared with the free enzyme. Upon immobilization,
significantly enhanced stability of enzyme activity was obtained
towards different temperature and pH. After 12 h of incubation at
Synthesis of polymer pAA
The UCST-thermoresponsive polymer pAA was prepared according to a
literature method [9] with a slight modification. In a 25 mL round-bottom
flask, acrylonitrile (1.08 mmol) and acrylamide (4.92 mmol) were dissolved
in distilled dimethyl sulfoxide (DMSO, 5 mL), followed by an addition of
AIBN (0.03 mmol) at room temperature. The flask was then placed into an
oil bath for polymerization at 65 °C for 6 h under nitrogen protection. After
that, the reaction mixture was quickly cooled to room temperature in an
ice-water bath, and the polymer was precipitated in excess methanol and
isolated by filtration. The obtained crude polymer was further purified by
washing with methanol thrice, and then dried in vacuum at 70 °C for 24 h.
1
60 °C, the immobilized PSL still maintained 68.8% of its original
activity comparing with only 34.4% for free PSL. The immobilized
biocatalyst pAA-PSL, with a similar UCST to the carrier, was
evaluated in the kinetic resolution of (R,S)-α-methylbenzyl
butyrate in aqueous media. Higher catalytic performance than the
free PSL was achieved over pAA-PSL at 55 °C with conversion
1
H NMR (DMSO-d6, 300 MHz): δ (ppm) 1.2-1.9 (polymer backbone,

2
CH ), 2.0-2.7 (polymer backbone, CHCONH
2
), 2.3-3.0 (polymer
). FT-IR: 3420 (NH), 2930 (CH), 2242
backbone, CHCN), 6.8 (NH
2
(C≡N), 1669 (C=O) cm
1
.
p
and ee values of up to 50% and 99.5%, respectively, giving highly
pure enantiomers. Apart from this, the immobilized biocatalyst
was easily separated and recovered from the reaction system at
Immobilization of PSL on pAA
0
°C and showed good durability retaining 80.5% of its original
Enzyme PSL was immobilized on the polymer pAA via covalent bonding.
To accomplish this, pAA was first modified with aldehyde functional groups
through reacting with GA.[18] In a typical process, pAA (50 mg) and GA (9
activity with a well reserved enantioselectivity after six cycles.
These results indicate that the UCST-type thermoresponsive
polymer pAA is a promising carrier for enzyme immobilization and
application.
~
57 mg) were stirred in deionized water (3 mL) at 45 °C for 6 h. Then the
modified polymer, denoted as pAA-GA, was separated by cooling
precipitation and rinsed thoroughly with deionized water to remove
unreacted GA.
Experimental Section
Thereafter, the immobilization of PSL was performed as follows. PSL (3 ~
15 mg) was dissolved in a phosphate buffer solution (3 mL, pH 7.0, 0.05
General
M), to this solution pAA-GA (50 mg) was added, and the mixture was
stirred at 45 °C for 1 ~ 5 h. After the reaction mixture was cooled in an ice-
water bath, the immobilized enzyme pAA-PSL was separated by
decantation, washed twice with the phosphate buffer solution (1 mL), and
then freeze-dried. The protein concentration in the supernatant and
washings was determined by the Bradford assay using bovine serum
albumin as a standard.[19] The enzyme loading of immobilized PSL, which
was defined as the amount of the protein bound per gram of pAA-PSL,
was thus calculated by subtracting the residual amount of the protein after
immobilization from the initial protein content.
Acrylonitrile (99%, Aladdin Ind. Corp.) was purified by 8% NaOH before
use. Acrylamide (99%, Energy Chemical) was recrystallized from acetone.
AIBN (AR, Wako Pure Chemical Ind., Ltd.) was recrystallized from ethanol
and dried under vacuum. Tributyrin (98%, Energy Chemical), GA (50 wt %
in water, Aladdin Ind. Corp.), (R,S)-α-methylbenzyl butyrate (99%, Damas-
beta), and PSL (“Amamo Enzyme”, Novocata) were used as received. The
protein content of PSL was determined to be 0.83 wt %.
1
The H NMR spectrum was recorded at 300 MHz using a Varian Mercury
Vx-300 spectrometer. The FT-IR spectrum was taken in the transmission
mode by using a Bruker Tensor 27 FT-IR instrument. The turbidity
Determination of the activity and thermal stability of PSL
measurements were carried out on
a
Shimadzu UV-2550
spectrophotometer at 500 nm with deionized water as a reference (100%
transmittance). Temperature was controlled by a Shimadzu TCC-240A
temperature controller. Before each measurement, the temperature point
was held for 2 min to ensure the thermal equilibrium of the sample cell.
The specific activity of enzyme PSL was measured by the hydrolysis of
tributyrin as follows. Typically, immobilized PSL (10 mg) or free PSL (2 mg)
was mixed with a phosphate buffer solution (2 mL, pH 7.0, 0.05 M) at 45 °C
under stirring for 2 min, followed by addition of tributyrin (20 μL). The
mixture was stirred for another 15 min. After that, methanol (10 mL) was
added to terminate the reaction. The yield of butyrate was measured by
titration with a sodium hydroxide solution (0.05 M). The specific activity of
enzyme PSL was described as U per g of catalyst, where U represents the
enzyme unit and one U is defined as the amount of PSL required to release
Conversion and enantioselectivity were determined by gas
chromatography (GC) equipped with a flame ionization detector and a
RESTEK RT-BetaDEXse chiral capillary column (30 m × 0.25 mm × 0.25
μm). The column temperature was set at 120 °C. The substrate conversion
1
μmol of butyrate per minute under the assay conditions.
was expressed as Conv (%) =100 × [1- (S
are the concentrations of (S)- and (R)-α-methylbenzyl butyrate before
reaction, S and R are the concentrations of (S)- and (R)-α-methylbenzyl
butyrate after reaction, respectively. The ee was defined as ee (%) =100
(R – S )/(S + R ), where S and R are the product concentrations of
1 1 0 0 0
+ R )/(S + R )], where S and
R
0
1
1
To study the thermal stability of enzyme PSL, the immobilized PSL or free
PSL in the phosphate buffer solution was incubated in a water bath of
60 °C for 0.5 ~ 12 h. The remaining activity was measured by hydrolysis
p
p
×
2
2
2
2
2
2
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ChemCatChem
10.1002/cctc.201701512
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of tributyrin at 45 °C as described above. The initial activity without the
thermal incubation was preset to be 100%.
[5]
a) Z. Zhou, M. Hartmann, Chem. Soc. Rev. 2013, 42, 38943912; b) W.
Feng, P. Ji, Biotechnol. Adv. 2011, 29, 889895; c) S. A. Ansari, Q.
Husain, Biotechnol. Adv. 2012, 30, 512523; d) J. Liu, Q. Yang, C. Li,
Chem. Commun. 2015, 51, 1373113739; e) X. Wu, M. Hou, J. Ge, Catal.
Sci. Technol. 2015, 5, 50775085.
a) M. A. C. Stuart, W. T. S. Huck, J. Genzer, M. Müller, C. Ober, M.
Stamm, G. B. Sukhorukov, I. Szleifer, V. V. Tsukruk, M. Urban, F. Winnik,
S. Zauscher, I. Luzinov, S. Minko, Nat. Mater. 2010, 9, 101113; b) J.
Zhuang, M. R. Gordon, J. Ventura, L. Li, S. Thayumanavan, Chem. Soc.
Rev. 2013, 42, 74217435; c) J. P. A. Custers, S. F. G. M. van Nispen,
A. Can, V. R. de La Rosa, S. Maji, U. S. Schubert, J. T. F. Keurentjes, R.
Hoogenboom, Angew. Chem. Int. Ed. 2015, 54, 1408514089; Angew.
Chem. 2015, 127, 1429114295.
a) B. Trzebicka, R. Szweda, D. Kosowski, D. Szweda, Ł. Otulakowski, E.
Haladjova, A. Dworak, Prog. Polym. Sci. 2017, 68, 3576; b) H. Chen,
L.-H. Liu, L.-S. Wang, C.-B. Ching, H.-W. Yu, Y.-Y. Yang, Adv. Funct.
Mater. 2008, 18, 95102; c) K. J. Mackenzie, M. B. Francis, J. Am. Chem.
Soc. 2013, 135, 293300; d) Q. Wu, T. Su, Y. Mao, Q. Wang, Chem.
Commun. 2013, 49, 1129911301.
Kinetic resolution of racemic α-methylbenzyl butyrate
[6]
The immobilized lipase pAA-PSL was evaluated as an enantioselective
catalyst in the kinetic resolution reaction of racemic α-methylbenzyl
butyrate. For comparison, the free enzyme PSL was also examined. A
typical procedure for the enzyme-catalyzed kinetic resolution reaction was
as follows. (R,S)-α-methylbenzyl butyrate (0.125 mmol) was dispersed in
a phosphate buffer solution (2.5 mL, pH 7.0, 0.05 M), to which was added
pAA-PSL (10 mg) or 1.6 mg of free PSL. The mixture was stirred at 55 °C
for 3 ~ 15 h. After that, the mixture was cooled in an ice-water bath and
the precipitated catalyst was separated by simple decantation, washed
thoroughly with the phosphate buffer solution, and then submitted to the
next cycle. The resulting supernatant together with the washings was
subjected to GC analysis.
[7]
[
8]
9]
P. A. Limadinata, A. Li, Z. Li, Green Chem. 2015, 17, 11941203.
J. Seuring, S. Agarwal, Macromolecules 2012, 45, 39103918.
[
Acknowledgements
[10] L. Hou, P. Wu, Soft Matter 2015, 11, 70597065.
[
11] a) L. Betancor, F. López-Gallego, A. Hidalgo, N. Alonso-Morales, G. D.-
O. C. Mateo, R. Fernández-Lafuente, J. M. Guisán, Enzyme Microb.
Tech., 2006, 39, 877882; b) H. Han, Y. Zhou, S. Li, Y. Wang, X. Z. Kong,
ACS Appl. Mater. Interfaces 2016, 8, 2571425724.
This work was financially supported by the National Natural
Science Foundation of China (Grant No. 21203102), the Tianjin
Municipal
1
2
Natural
4JCQNJC06000), China Scholarship Council (Grant No.
01606200087), MOE (IRT13R30) and 111 Project (B12015).
Science
Foundation
(Grant
No.
[12] E. Lai; Y. Wang; Y. Wei, G. Li, G. Ma, J. Appl. Polym. Sci. 2016, 133,
4334343351.
[13] G. Peng, C. Zhao, B. Liu, F. Ye, H. Jiang, Appl. Surf. Sci. 2012, 258,
5435552.
5
[
[
[
[
[
[
14] Y. K. Sim, S. Jung, J. Y. Lim, J. Kim, S.-H. Kim, B. K. Song, B. T. Kim, H.
Keywords: Immobilization • Lipase from Pseudomonas cepacia
Thermoresponsive polymer • Upper critical solution
temperature • Kinetic resolution
Lee, S. Park, Tetrahedron Lett. 2011, 52, 10411043.
15] M. Fujiwara, K. Shiokawa, K. Yotsuya, K. Matsumoto, J. Mol. Catal. B:
Enzym. 2014, 109, 94100.
•
16] L. Fan, B. Zhang, H. Zhang, X. Jia, X. Chen, Q. Zhang, RSC Adv. 2016,
6
, 6591165920.
17] C.-S. Chen, Y. Fujimoto, G. Girdaukas, C. J. Sih, J. Am. Chem. Soc.
982, 104, 72947299.
18] A. Hamerska-Dudra, J. Bryjak, A. W. Trochimczuk, Enzyme Microb. Tech.
007, 41, 197204.
19] M. M. Bradford, Anal. Biochem. 1976, 72, 248254.
[
1]
2]
a) S. J. Benkovic, S. Hammes-Schiffer, Science 2003, 301, 11961202;
b) C. M. Clouthier, J. N. Pelletier, Chem. Soc. Rev. 2012, 41, 15851605.
P.-Y. Stergiou, A. Foukis, M. Filippou, M. Koukouritaki, M. Parapouli, L.
G. Theodorou, E. Hatziloukas, A. Afendra, A. Pandey, E. M.
Papamichael, Biotechnol. Adv. 2013, 31, 18461859.
1
[
2
[
3]
4]
R. A. Sheldon, S. van Pelt, Chem. Soc. Rev. 2013, 42, 62236235.
a) D. N. Tran, K. J. Balkus Jr., ACS Catal. 2011, 1, 956968; b) R. C.
Rodrigues, C. Ortiz, Á. Berenguer-Murcia, R. Torres, R. Fernꢀndez-
Lafuente, Chem. Soc. Rev. 2013, 42, 62906307; c) P. Adlercreutz,
Chem. Soc. Rev. 2013, 42, 64066436.
[
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Entry for the Table of Contents (Please choose one layout)
FULL PAPER
Thermoresponsive lipase catalyst:
UCST-type thermoresponsive
polymer-lipase conjugate was
exploited and applied as an
Lan-Lan Lou, Huaxin Qu, Wenjun Yu,
Bei Wang, Lezi Ouyang, Shuangxi
Liu*, and Wuzong Zhou*
asymmetric biocatalyst in the kinetic
resolution of racemic α-methylbenzyl
butyrate, exhibiting high catalytic
performance, easy recovery and good
durability.
Page No. – Page No.
Covalently Immobilized Lipase on a
Thermoresponsive Polymer with an
Upper Critical Solution Temperature
as an Efficient and Recyclable
Asymmetric Catalyst in Aqueous
Media
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