Enzyme entrapped in polymer-modified nanopores: The effects of macromolecular crowding and surface hydrophobicity

Article abstract of DOI:10.1002/chem.201203833

Macromolecular crowding is an ubiquitous phenomenon in living cells that significantly affects the function of enzymes in vivo. However, this effect has not been paid much attention in the research of the immobilization of enzymes onto mesoporous silica. Herein, we report the combined effects of macromolecular crowding and surface hydrophobicity on the performance of an immobilized enzyme by accommodating lipase molecules into a series of mesoporous silicas with different amounts of inert poly(methacrylate) (PMA) covalently anchored inside the nanopores. The incorporation of the PMA polymer into the nanopores of mesoporous silica enables the fabrication of a crowded and hydrophobic microenvironment for the immobilized enzyme and the variation in polymer content facilitates an adjustment of the degree of crowding and surface properties of this environment. Based on this system, the catalytic features of immobilized lipase were investigated as a function of polymer content in nanopores and the results indicated that the catalytic efficiency, thermostability, and reusability of immobilized lipase could all be improved by taking advantage of the macromolecular crowding effect and surface hydrophobicity. These findings provide insight into the possible functions of the macromolecular crowding effect, which should be considered and integrated into the fabrication of suitable mesoporous silicas to improve enzyme immobilization.

Full text of DOI:10.1002/chem.201203833

FULL PAPER
DOI: 10.1002/chem.201203833
Enzyme Entrapped in Polymer-Modified Nanopores: The Effects of
Macromolecular Crowding and Surface Hydrophobicity
Jia Liu, Juan Peng, Shuai Shen, Qianru Jin, Can Li,* and Qihua Yang*[a]
Abstract: Macromolecular crowding is
an ubiquitous phenomenon in living
cells that significantly affects the func-
tion of enzymes in vivo. However, this
effect has not been paid much atten-
tion in the research of the immobiliza-
tion of enzymes onto mesoporous
silica. Herein, we report the combined
effects of macromolecular crowding
and surface hydrophobicity on the per-
formance of an immobilized enzyme by
accommodating lipase molecules into a
series of mesoporous silicas with differ-
ent amounts of inert poly(methacry-
late) (PMA) covalently anchored
inside the nanopores. The incorpora-
tion of the PMA polymer into the
nanopores of mesoporous silica enables
the fabrication of a crowded and hy-
drophobic microenvironment for the
immobilized enzyme and the variation
in polymer content facilitates an adjust-
ment of the degree of crowding and
surface properties of this environment.
Based on this system, the catalytic fea-
tures of immobilized lipase were inves-
tigated as a function of polymer con-
tent in nanopores and the results indi-
cated that the catalytic efficiency, ther-
mostability, and reusability of immobi-
lized lipase could all be improved by
taking advantage of the macromolecu-
lar crowding effect and surface hydro-
phobicity. These findings provide in-
sight into the possible functions of the
macromolecular crowding effect, which
should be considered and integrated
into the fabrication of suitable mesopo-
rous silicas to improve enzyme immo-
bilization.
Keywords: biocatalysis · immobili-
zation · lipase · mesoporous materi-
als · nanopores
Introduction
ed as one of the main reasons for this decreased activity.
Previous studies have shown that the nature of the mesopo-
rous silica, such as its textural properties (pore size, surface
area, and pore volume), surface charge(s), and surface hy-
drophobicity/hydrophilicity play important roles in deter-
mining the catalytic efficiency and stability of the immobi-
lized enzymes.[3,12–14] However, the macromolecular crowd-
ing effect of an enzyme, one of the key characteristics in the
cellular environment and intimately coupled to the enzyme
function in vivo, has not been paid enough attention in
terms of enzyme immobilization.[15–17]
Cells are a crowded environment, in which the macromo-
lecules, such as proteins, nucleic acids, lipids, and other cy-
toskeletons, can occupy up to 40% of the total cellular
volume.[16–18] In such a crowded environment, the enzymes
behave in different ways to their dilute solutions in test
tubes.[19] Experimental and theoretical studies have been
performed to mimic the intracellular environment by adding
crowding agents, such as ficoll, dextran, and PEG, into the
buffer solution of enzymes. The presence of these crowding
agents, which do not participate directly in a particular bio-
logical process, can decrease the amount of free space and
the volume of solvent available to the protein and, thus, can
significantly affect the dynamics and conformation of the
enzymes. It has been demonstrated that macromolecular
crowding could stabilize the folded (native) state of enzyme,
restrain the dissociation of multiple enzyme, increase the ef-
fective concentrations of the enzyme molecules, and modu-
late the affinity between the enzyme and the substrate
whilst inducing alteration in the enzyme structure.[16,20–24]
Enzymes that display high catalytic efficiency and selectivity
under mild conditions are promising catalysts for use in the
chemical and pharmaceutical industries.[1,2] However, the
practical application of native enzyme suffers from severe
limitations owing to its low stability against heat, extreme
pH values, and organic solvents, as well as the difficulty in
its separation and reuse.[3,4] Pleasingly, the immobilization of
the native enzyme onto solid materials could partly solve
these problems and facilitate its use in continuous produc-
tion processes, which are favorable for industrial applica-
tions.[5–8] Mesoporous silicas have emerged as a new class of
promising candidates for enzyme immobilization owing to
their large surface area, ordered porous structure, narrow
pore-size distribution, facile functionalization, and chemical
and mechanical stability.[9–11] However, enzymes that are ac-
commodated inside mesoporous silicas often exhibit lower
activity compared with the native enzymes. The altered mi-
croenvironment around the immobilized enzymes is regard-
[a] J. Liu, J. Peng, S. Shen, Q. Jin, Prof. Dr. C. Li, Prof. Dr. Q. Yang
State Key Laboratory of Catalysis
Dalian Institute of Chemical Physics
Chinese Academy of Sciences
457 Zhongshan Road, Dalian 116023 (China)
Fax : (+86) 411-84694447
E-mail: canli@dicp.ac.cn
yangqh@dicp.ac.cn
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201203833.
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However, most of the information on the macromolecular
crowding effect is based on research conducted in solution;
very little is known about the crowding effect for immobi-
lized enzymes. Eggers and Valentine found a general en-
hancement in the thermal stability of enzymes that were
confined inside the pores of silica glass.[15,25] Zhou and Dill
suggested that confining a protein inside a small inert space
should stabilize it against reversible unfolding.[26] Wei and
co-workers demonstrated by using computer simulation
studies that enzyme stability was promoted after entrapment
inside silica gels.[27,28] Based on these pioneering findings,
any research into the immobili-
Results and Discussion
Synthesis and characterization of PMA-n-MS: PMA-func-
tionalized mesoporous silicas (PMA-n-MS, where n is the
À
molar percentage of PMA organosilane in the initial mix-
ture of the silane precursors) were synthesized by the co-
À
condensation of PMA organosilane (see the Supporting In-
formation, Figure S1) with sodium silicate and tetraethoxysi-
lane (TEOS), with P123 as the structure-directing agent
under mildly acidic conditions (HAc/NaAc buffer solution,
pH 4.4), as outlined in Scheme 1.[36,37] The FTIR spectra of
zation of enzymes onto meso-
porous silicas should also pay
attention to the macromolecu-
lar crowding effect.
Lipases have been found to
be versatile and efficient bioca-
talysts that can be used in a
broad variety of esterification,
interesterification, and ester-hy-
drolysis reactions with high
chemo-, regio-, and stereoselec-
tivity.[29–31]
A
substantial
number of investigations have
been performed to entrap lipas-
es inside mesoporous silicas
that were tailored with various
organic groups. Owing to its
unique catalytic mechanism, li-
pases feature notable sensitivity
towards hydrophilic/hydropho-
bic surfaces. Therefore, most re-
search has focused on the
impact of the surface properties
of mesoporous silicas on the
performance of the immobi-
À
Scheme 1. Synthesis of PMA-n-MS, where n refers to the molar percentage of PMA organosilane in the initial
mixture of the silane precursors.
lized lipases and supports with moderate hydrophobicity
have been proposed to be beneficial to the catalytic features
of lipase.[32–35] However, to the best of our knowledge, there
have been no reports on integrating the macromolecular
crowding effect and surface hydrophobicity into the fabrica-
tion of suitable mesoporous silicas to improve lipase immo-
bilization.
Herein, we report the accommodation of lipase molecules
into a series of mesoporous silicas with different amounts of
poly(methacrylate) (PMA) covalently anchored inside the
nanopores. This variation in polymer content facilitates an
adjustment of the degree of crowding and surface hydropho-
bicity of the enzyme environment. We found that the cata-
lytic efficiency and stability of lipase could be considerably
enhanced by profiting from the combined effects of macro-
molecular crowding and surface hydrophobicity.
PMA-n-MS (n>0) all exhibit the characteristic vibration of
PMA at 1740 cmÀ1, which arises from a C=O stretching vi-
À
bration, and peaks owing to the C H stretching vibration at
À1
À
2959–2836 cm , the C H bending vibration at 1450 and
À1
À1
À
1380 cm , and the C O stretching vibration at 1163 cm
(Figure 1A). These vibrations are not observed in the FTIR
spectrum of PMA-0-MS, thus suggesting the successful in-
corporation of the PMA polymer fragment into PMA-n-MS
(n>0). These PMA-n-MS (n>0) samples are thermally
stable up to 2508C in air (see the Supporting Information,
Figure S2). The first weight loss, below 1508C, is the loss of
physically adsorbed water. The second weight loss, which
occurs in the temperature range 300–4308C, is associated
with the degradation of the PMA polymer chains and the
third weight loss, which is observed in the range 500–6508C,
represents the complete decomposition of the polymeric
chains. The appearance of a distinct two-step weight loss
from 300 to 6508C implies that the PMA polymers that are
incorporated into PMA-n-MS possess relatively uniform
molecular mass.[38] Based on the TG results, the amount of
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Enzyme Entrapped in Polymer-Modified Nanopores
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À
PMA organosilane in the ini-
tial mixture, thereby providing
an opportunity for investigating
the macromolecular crowding
effect for immobilized enzymes.
The surface hydrophobicity/
hydrophilicity of PMA-n-MS
was investigated by water and
benzene adsorption experi-
ments (see the Supporting In-
formation, Figure S4). To ex-
clude the influence of textural
structure on the amount of ad-
sorption, the adsorbed water/
benzene molar ratio of PMA-n-
MS was calculated (Table 1).
PMA-0-MS, without PMA pol-
ymer, had much higher water/
benzene molar ratio than
PMA-0.6-MS, with PMA poly-
Figure 1. A) FTIR spectra and B) XRD patterns of PMA-n-MS. C) TEM image of PMA-3.6-MS.
PMA polymers in PMA-n-MS varied from 0 to 56.1 wt%
(Table 1). The existence of large amounts of PMA polymer
may provide a biocompatible microenvironment for the en-
zymes.
mer inside the nanopores (3.97 versus 1.55). This result indi-
cates that the incorporation of PMA polymer inside the
nanopores can increase the surface hydrophobicity. The
water/benzene molar ratio only decreases slightly (from 1.55
to 1.03) from PMA-0.6-MS to PMA-4.8-MS, thus suggesting
a gradual increase of the surface hydrophobicity of the
nanopores as the amount of PMA increases.
The XRD patterns of the PMA-n-MS samples all display
one intense (100) diffraction peak, as well as (110) and
(200) diffractions, which are indicative of a 2D hexagonal
P6mm symmetry; these results suggest that all of these sam-
ples have a highly ordered parallel cylindrical channel-like
mesostructure (Figure 1B). The TEM image of PMA-3.6-
MS clearly shows an ordered arrangement of the uniform
pores (Figure 1C), further confirming that the PMA-n-MS
samples have a highly ordered mesostructure. The PMA-n-
MS samples exhibit type-IV N2-adsorption/desorption iso-
therms with H1-type hysteresis loops, which are characteris-
tic of uniform mesopores with an open cylindrical geometry
(see the Supporting Information, Figure S3). The BET sur-
face area (SBET), pore diameter (DBJH), and total pore
volume (Vp) of PMA-n-MS are all inversely related to the
amount of PMA polymers that are incorporated inside the
samples (Table 1), presumably owing to the occupation of
the pore space by PMA polymers. The pore diameter (9.8–
7.1 nm) and pore volume (1.02–0.25 cm3 gÀ1) of the PMA-n-
MS samples could be adjusted by varying the amount of
Activity and stability of PCL immobilized inside PMA-n-
MS: Lipase from Pseudononas cepacia (PCL, about 5 nm)
was chosen as the model enzyme for our research. The load-
ing of PCL (LPCL) per unit weight of PMA-0-MS was mark-
edly lower than that on PMA-n-MS (n>0) that contained
comparable or much smaller textual parameters (Table 1),
thus suggesting that the PMA polymer in the nanopores
could increase the affinity between the enzyme and support
by benefiting from the enhanced hydrophobic interactions.
For PMA-n-MS (n>0), LPCL (in mggÀ1) was inversely relat-
ed to the polymer content, presumably owing to the smaller
pore diameter, surface area, and pore volume that accompa-
nied high polymer incorporation. The BET surface area,
pore volume, and pore diameter after PCL loading were ob-
viously lower than before PCL loading, thus indicating that
most PCL molecules were immobilized inside the nanopores
Table 1. Textural parameters, surface hydrophobic/hydrophilic properties, and PCL loading of PMA-n-MS.
[a]
[a]
[a]
[e]
Sample
DBJH
[nm]
SBET
[m2 gÀ1
Vp
[cm3 gÀ1
Polymer content
Adsorbed water/benzene
molar ratio[d]
LPCL
[b]
[c]
G
]
G
]
N
]
G
]
N
]
G
]
PMA-0-MS
9.8 (9.9)[f]
9.3 (9.0)
8.7 (8.2)
8.6 (8.1)
7.2 (6.9)
7.1 (6.1)
452 (359)
518 (190)
471 (187)
298 (106)
224 (85)
153 (30)
1.02 (0.84)
0.91 (0.40)
0.81 (0.33)
0.60 (0.22)
0.36 (0.13)
0.25 (0.07)
–
–
3.97
1.55
1.20
1.14
1.06
1.03
PMA-0.6-MS
PMA-1.2-MS
PMA-2.4-MS
PMA-3.6-MS
PMA-4.8-MS
214
353
525
627
694
166
286
402
490
561
16.15
15.53
12.91
13.11
10.38
17.74
19.17
21.51
36.42
41.52
[a] DBJH, SBET, and Vp refer to the pore diameter, BET surface area, and total pore volume, respectively. [b] The theoretical value of polymer content.
[c] Polymer content was determined by TG analysis. [d] Derived from the adsorption of water vapor and benzene vapor at 273 K. [e] PCL loading.
[f] Data in the parentheses refer to the samples that were loaded with PCL.
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C. Li, Q. H. Yang et al.
of PMA-n-MS (Table 1 and the Supporting Information,
Figure S3). The loading of PCL per unit volume of the
nanopores (LPCL, in mgcmÀ3), which was similar to the con-
finement molarity of an enzyme, as defined by Cornelissen
and co-workers,[24] was evidently positively correlated with
the polymer content (Table 1). This result is comparable to
the increase in the degree of crowding in enzyme solution
systems through the addition of crowding agents. Thus, the
degree of crowding of PCL in PMA-n-MS increased in the
sequence:
PCL-PMA-0-MS<PCL-PMA-0.6-MS<PCL-
PMA-1.2-MS<PCL-PMA-2.4-MS<PCL-PMA-3.6-MS<
PCL-PMA-4.8-MS (where PCL-PMA-n-MS refers to the
mesoporous materials with PCL loading).
The activities of PCL-PMA-n-MS were measured by the
hydrolysis of triacetin in aqueous solution at 308C. Figure 2
shows the relative activities of PCL-PMA-n-MS by taking
Figure 3. Residual activities of PCL that was accommodated inside
PMA-n-MS (PCL-PMA-n-MS) after incubation for 2 h at different tem-
peratures.
notable decline in activity upon incubation at 408C (activity
retention: 78%). On further increasing the incubation tem-
perature, the activity of PCL-PMA-0.6-MS gradually de-
creases and 60% of its initial activity is retained after treat-
ment at 708C. PCL-PMA-1.2-MS shows a similar downward
trend in response to heat treatment but affords slightly
higher residual activities under similar treatment conditions.
For PCL-PMA-2.4-MS, no remarkable loss in activity was
detected over the whole temperature range and an activity
retention of 94% was obtained, even after incubation at
708C. However, PCL-PMA-3.6-MS and PCL-PMA-4.8-MS,
which possessed higher polymer content inside the nano-
pores than PCL-PMA-2.4-MS, showed lower resistance to-
wards heat treatment, thus maintaining 81% and 79% of
the initial activities after treatment at 708C, respectively.
The thermostability of PCL-PMA-n-MS (n>0) were all dis-
tinctly higher compared with free PCL, which showed 23%
retention of its initial activity after incubation at 708C, ac-
cording to our previous report.[32] This high thermostability
of PCL-PMA-n-MS (n>0) (especially PCL-PMA-2.4-MS) is
very impressive and has scarcely been achieved by the im-
mobilization of PCL into mesoporous silicas, although it has
been widely reported that the introduction of hydrophobic
functionalities into a support leads to a rigidification of the
lipase structure against thermal denaturation.[43,44] Therefore,
it is reasonable to presume that, in our case, the macromo-
lecular crowding effect may play a significant part in im-
proving lipase thermostability, in addition to the contribu-
tion of surface hydrophobicity.
Figure 2. Relative activities of PCL that was accommodated inside PMA-
n-MS (PCL-PMA-n-MS) and the PCL loading per unit volume of each
sample.
the activity of free PCL as 100%. The activity of PCL nota-
bly decreased upon entrapment in PMA-0-MS (26% rela-
tive to free PCL), owing to the hydrophilic microenviron-
ment of the silica nanopores, which were incompatible with
the enzyme molecules and unfavorable for intriguing the
“interfacial activation”.[39–42] The relative activities of PCL-
PMA-n-MS (n>0, 34–121%) were higher than that of PCL-
PMA-0-MS, probably profiting from the sharply increased
hydrophobicity of the support with PMA polymers in the
nanopores. For the samples with polymer modification, their
relative activities gradually increased until the maximum rel-
ative activity (121%) was obtained for PCL-PMA-3.6-MS,
followed by a slightly lower relative activity (119%) for
PCL-PMA-4.8-MS.
Figure S5 shows the FTIR spectra of a solution of free
PCL and PCL-PMA-0-MS before and after incubation at
708C for 2 h. Without thermal incubation, these two samples
both display two characteristic adsorption bands, amide I
(1650 cmÀ1) and amide II (1540 cmÀ1), which are very sensi-
tive to the secondary structure of the protein (the amide I
band of free PCL is very broad, owing to the presence of
the aqueous solution).[45–48] After incubation, the FTIR spec-
The thermostability of PCL was assayed by measuring the
residual activity of PCL-PMA-n-MS after incubation at dif-
ferent temperatures for 2 h. As shown in Figure 3, PCL-
PMA-0.6-MS is sensitive to thermal treatment and shows a
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tra of the free PCL solution and PCL-PMA-0-MS (which
maintained 23% and 35% of their initial activity, respec-
tively, according to our previous investigation[32]) featured a
diminishing of the amide II band, thus reflecting the unfold-
ing of the protein.[45,47] Moreover, although a shift in the
amide I band of the free PCL was not clear, owing to the
overlapped adsorption of water, the amide I band of PCL-
PMA-0-MS clearly shifted from 1650 to 1638 cmÀ1 after
thermal incubation, which was generally considered to have
arisen from protein aggregation.[47,49,50] The FTIR spectra of
PCL-PMA-n-MS (n>0) after incubation showed no shift in
the amide I bands, which resembled those of native PCL
(Figure 4A), thus implying that there was no evident
more-crowded environment have a more-compact confor-
mation after thermal treatment and shows the interference
of the crowded microenvironment in the secondary structure
of an immobilized enzyme at high temperatures.[45,47]
Previous studies on proteins in solution, as well as en-
zymes entrapped in silica gels, have shown that the macro-
molecular crowding effect can shift the equilibrium in favor
of the more-compact native (folded) state of the enzyme by
decreasing the conformational entropy of the unfolded state,
as well as through “depletion-induced attraction”.[16,51–53] As
a consequence, the stability of the enzyme against heat and
denaturants could be significantly enhanced in crowded en-
vironments. Nevertheless, this promotion effect was not pro-
portional to the degree of crowding in the system, owing to
crowding-induced conformational changes in the enzyme,
which is another important aspect of the macromolecular
crowding effect.[20,22] According to our experiment results,
the macromolecular crowding effect generally exerts two op-
posing effects on the thermostability of PCL inside mesopo-
rous silicas: One is the preference for the folded conforma-
tion of the protein, which is beneficial for enzyme stability,
and the other is perturbation in the native secondary struc-
ture of the protein, which decreases the enzyme activity.
The competition of these two factors influences the final
thermostability of PCL inside the crowded environments.
On account of the good activity retentions for samples with
high degrees of crowding (PCL-PMA-n-MS, nꢀ2.4), it is ra-
tional to assume that, in our scenario, the negative influence
that results from alteration of the secondary structure is
subtle and the stabilization of the folded state of the
enzyme plays a dominant role in withstanding harsh incuba-
tion temperatures.
The effects of macromolecular crowding and nanopore sur-
face hydrophobicity on the catalytic performance of PCL
for the kinetic resolution of (R,S)-1-phenylethanol: The
effect of macromolecular crowding on the catalytic perform-
ance of the immobilized enzyme in dry organic solution has
not been reported previously. We investigated this effect by
considering the PCL-catalyzed kinetic resolution of (R,S)-1-
phenylethanol with vinyl acetate as the acyl donor and n-
hexane as the reaction medium at 308C (Table 2); because
the existence of water is known to retard the esterification
activity of lipase, the reaction with free PCL solution was
not performed. According to literature reports, the apparent
rate constant for the acylation of the R enantiomers of con-
versional lipases is four-to-six orders of magnitude higher
than that of the S enantiomers.[54] Therefore, it is not surpris-
ing that, in our experiments, (S)-1-phenylethanol was practi-
cally unreactive and immobilized PCL transformed the (R)-
1-phenylethanol into (R)-1-phenylethanol acetate with an
enantiomeric excess (ee) of >99% in all cases, independent
of the polymer content in the nanopores (Table 2). In terms
of the reaction rate of the samples, PCL that was inside the
polymer-tailored nanopores all outperformed that accom-
modated inside the nanopores without PMA modification
(Table 2 and the Supporting Information, Figure S6), proba-
Figure 4. A) FTIR spectra of fresh free PCL and PCL-PMA-n-MS (n>0)
after incubation at 708C; values are normalized by using the intensity of
the amide I band. B) Intensity ratios of the amide I/amide II bands for
PCL-PMA-n-MS (n>0) after incubation at 708C and the PCL loading
per unit volume of each sample.
enzyme aggregation during the treatment process. After in-
cubation, the amide II bands of PCL-PMA-n-MS (n>0)
were obvious and became increasingly distinguishable with
a gradually shift from 1540 to 1547 cmÀ1 as the degree of
crowding in the nanopores increased. The intensity ratio of
amide I to amide II, which was related to the relative con-
tent of the a-helical and b-sheet conformations in the pro-
tein, decreased from 16.3 to 1.7, in line with the degree of
crowding in the enzyme environment (Figure 4B). This
result suggests that enzymes that are accommodated in a
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C. Li, Q. H. Yang et al.
(9860 minÀ1) and kcat/Km (163.8 minÀ1 mmÀ1) were afforded
by PCL-PMA-3.6-MS, in agreement with the results in
Table 2 and the Supporting Information, Figure S6, which
showed that PCL-PMA-3.6-MS also afforded the highest ini-
tial velocity and conversion. The high catalytic efficiency of
PCL-PMA-3.6-MS is a combined result of the macromolecu-
lar crowding effect and surface hydrophobicity.
Table 2. Kinetic resolution of (R,S)-1-phenylethanol catalyzed by immo-
bilized PCL.[a]
As mentioned above, there have been several reports on
the correlation between the catalytic activity of lipase and
the hydrophobic/hydrophilic nature of the support. A gener-
ally accepted conclusion is that the hydrophobic support can
accelerate lipase catalysis, owing to, for most lipases (includ-
ing PCL), the presence of a “lid” that secluded the active
site from the medium; however, the lipase could undergo a
conformational rearrangement in the presence of the hydro-
phobic support, thus making the active site accessible to the
substrates (namely “interfacial activation”).[39–42] The higher
diffusion rate of the substrates into the nanochannels and
the enhanced affinity of the enzyme for the substrates,
owing to the increased surface hydrophobicity, could also
parallel the catalytic activity of the immobilized lipase.
In addition, based on the theoretical and experimental re-
sults that were derived from protein solution systems, that
is, that macromolecular crowding impacts the dynamics of
an enzyme and, hence, its catalytic activity, we can probe
the possible functions that the macromolecular crowding
effect could serve in the catalysis of PCL-PMA-n-MS (n>
0). In enzyme solution systems, high concentrations of mac-
romolecules occupy a large volume fraction of medium,
thereby decreasing the volume of solvent that is available
for the enzyme proteins, which, consequently, increases the
effective enzyme concentration. In our scenario, this result
could be represented by an increase in PCL loading per unit
volume of the nanopores (see above), which can shift the
Catalyst
Initial rate
Conv.
[%]
ee[b]
[%]
À1
[mmolminÀ1 mgprotein
]
PCL-PMA-0-MS
PCL-PMA-0.6-MS
PCL-PMA-1.2-MS
PCL-PMA-2.4-MS
PCL-PMA-3.6-MS
PCL-PMA-4.8-MS
0.005
0.028
0.033
0.083
0.098
0.088
8.2
19.5
24.9
41.9
50.0
48.8
99
99
99
99
99
99
[a] Typical reaction conditions: (R,S)-1-phenylethanol (0.5 mmol) and
vinyl acetate (2.0 mmol) were stirred in dry n-hexane (5 mL) at 308C for
6 h. [b] Determined in terms of the ester product.
bly owing to the dramatically increased hydrophobicity of
the PCL-PMA-n-MS (n>0) samples, considering the “inter-
facial activation” effect (see above). First, the enhancement
in polymer content of PCL-PMA-n-MS (n>0) had an en-
couraging impact on the catalytic efficiency of immobilized
PCL, with the highest initial rate (0.098 mmol
minÀ1 mgproteinÀ1) and conversion (50.0%) afforded by PCL-
PMA-3.6-MS, whereas PCL-PMA-4.8-MS, with higher poly-
mer incorporation, exhibited a minor drop in the initial rate
and conversion (48.8%), which was quite similar to the ob-
servations in terms of enzyme activity (see above).
A Michaelis–Menten kinetics study was also performed
on PCL-PMA-2.4-MS, PCL-PMA-3.6-MS, and PCL-PMA-
4.8-MS (Table 3 and the Supporting Information, Figure S7).
Increasing the polymer content in the PMA-n-MS samples
(n=2.4, 3.6, 4.8) from 402 to 561 mggÀ1 provoked a de-
À
equilibrium towards the formation of the enzyme substrate
complex, thereby accelerating the reaction velocity.[19,24,55]
Moreover, Jiang and Guo reported that, in protein solution
systems, macromolecular crowding could improve the intrin-
sic catalytic efficiency of an enzyme by enhancing its affinity
for the substrates, as reflected by an observable decrease in
the Michaelis–Menten constant (Km).[20] This result could
also contribute to increased enzyme activity in our heteroge-
neous system. However, the activity enhancement of macro-
molecular crowding is not tenable under all crowding condi-
tions. As is known in protein solution systems, elevating the
macromolecular crowding to a certain point causes the reac-
tion velocity to be slowed, owing to diffusion resistance;[24]
moreover, the high degree of crowding in the nanopores of
PCL-PMA-4.8-MS could also cause serious diffusion limita-
tions and lower the enzyme catalytic efficiency, as observed
in the much lower pore parameters of PCL-PMA-4.8-MS
than other samples (Table 1 and the Supporting Informa-
tion, Figure S3). Tailoring the nanopores of mesoporous
silica with PMA polymers can be described as killing two
birds with one stone. The enhanced support hydrophobicity
and the crowded environments around the immobilized en-
Table 3. Kinetic parameters for the kinetic resolution of (R,S)-1-phenyle-
thanol catalyzed by PCL-PMA-n-MS (n=2.4, 3.6, 4.8).[a]
Catalyst
Km
[mm]
kcat
[minÀ1
Kcat/Km
G
]
[minÀ1 mmÀ1
]
PCL-PMA-2.4-MS
PCL-PMA-3.6-MS
PCL-PMA-4.8-MS
77.8
60.2
55.2
3581
9860
6620
46.0
163.8
120.0
[a] The concentration of (R,S)-1-phenylethanol ranged from 100 mm to
10 mm with a fixed amount of PCL protein and 400 mm vinyl acetate.
crease in their textural parameters (especially for enzyme-
loaded samples) but an increase in their surface hydropho-
bicity and macromolecular crowding (Table 1). As shown in
Table 3, higher polymer incorporation into the nanopores
causes a decrease in their Km value from 77.8 to 55.2 mm,
thus reflecting an enhanced affinity between the active sites
of the enzyme and the substrate molecules. However, the
turnover number (kcat) and the catalytic efficiency (kcat/Km)
of the immobilized PCL did not increase linearly with poly-
mer content. Of the three samples, the highest kcat
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Enzyme Entrapped in Polymer-Modified Nanopores
FULL PAPER
zymes could both be favorable for increasing the catalytic
activity of the immobilized lipase.
with enzyme leaching, lead to remarkably lower conversion
with an increasing number of cycles.
A reusability assay of PCL-PMA-0-MS and PCL-PMA-
3.6-MS in the kinetic resolution of (R,S)-1-phenylethanol
was performed and the results are summarized in Figure 5.
The enantioselectivity remained almost the same (99%) for
both samples during the recycle process. The conversion of
PCL-PMA-0-MS decreased from 7% to less than 1% after
Conclusion
In summary, a series of mesoporous silicas with inert PMA
polymers covalently anchored inside their nanopores were
synthesized by the co-condensation of a mixture of sodium
À
silicate, TEOS, and PMA organosilane (which was prepared
by free-radical polymerization) in a HAc/NaAc buffer solu-
tion. These samples, abbreviated as PMA-n-MS (where n
À
refers to the molar percentage of the PMA organosilane in
the initial mixture of the silica precursors), possess different
levels of occupation of the nanopores by the PMA polymer
and altered surface properties. By using lipase from Pseudo-
monas cepacia (PCL) as the model enzyme, we found that
enhanced polymer content in PNIPAM-n-MS resulted in in-
creased crowding and surface hydrophobicity of the enzyme
environments. This increase tentatively affects a wide range
of the properties of the immobilized lipase, such as its cata-
lytic efficiency, thermostability, and reusability, as well as
the secondary structure of the enzyme. Accommodating en-
zymes inside polymer-modified mesoporous silicas allows us
to mimic the crowded environment for enzymes in vivo, as
well as modulating the surface properties of the nanopores.
Our research sheds light on the important role of the macro-
molecular crowding effect in enzyme immobilization and
provides a new approach for creating more-efficient and
stable heterogeneous biocatalysts for industrial applications.
Figure 5. A) Reusability of PCL-PMA-0-MS and PCL-PMA-3.6-MS.
B) FTIR spectra of fresh free PCL, PCL-PMA-0-MS, and PCL-PMA-3.6-
MS after five recycles (PCL-PMA-0-MS-recycle and PCL-PMA-3.6-MS-
recycle, respectively) for the kinetic resolution of (R,S)-1-phenylethanol.
five consecutive recycles, whereas PCL-PMA-3.6-MS still
presented a conversion of up to 32%, thus demonstrating
that modifying the nanopores with PMA polymer for immo-
bilized PCL causes improved enzyme reusability. The FTIR
spectra of PCL-PMA-0-MS and PCL-PMA-3.6-MS after
five recycles (PCL-PMA-0-MS-recycle and PCL-PMA-3.6-
MS-recycle, respectively), as well as that of the pristine PCL
solution (free PCL), are shown in Figure 5B. In comparison
with free PCL, the FTIR spectrum of PCL-PMA-3.6-MS-re-
cycle exhibits negligible differences in the amide I and ami-
de II bands, thus suggesting that the enzyme structure was
preserved during the recycle process and that enzyme leach-
ing (probably as a result of the insufficient hydrophobicity
of the support) was the principal reason for the slightly
lower conversion as the number of cycles increased. In con-
trast, a marked shift from 1650 to 1635 cmÀ1 for the amide I
band could be detected in the FTIR spectrum of PCL-
PMA-0-MS-recycle, which indicated that the conformation
of PCL-PMA-0-MS was considerably disturbed after the re-
usability test, probably owing to association between neigh-
boring enzyme molecules (see above). Therefore, the PMA-
polymer-modified nanopores can stabilize the conformation
of the enzyme, owing to the combined results of surface
functionalization and the crowded microenvironment. For
PCL-PMA-0-MS-recycle, enzyme denaturation, together
Experimental Section
Chemicals: Amano lipase PS from Pseudomonas cepacia (PCL,
30 UmgÀ1), methacrylate (MA, 98%), azobisisobutyronitrile (AIBN),
P123 (poly(ethylene oxide)-block-poly(propylene oxide)-block-poly(ethy-
lene oxide), EO20-PO70-EO20, Mw =5800), and (R,S)-1-phenylethanol
were purchased from Sigma–Aldrich. [3-(Methacryloxy)propyl]-trime-
thoxysilane (3-MOP, 98%) was purchased from ABCR GmbH & Co.
KG. Triacetin was commercially available from Alfa Aesar. Tetraethoxy-
silane (TEOS), sodium silicate solution (20% SiO2, 6% Na2O), vinyl ace-
tate, and other reagents were obtained from Shanghai Chemical Reagent
Inc. of the Chinese Medicine Group. All materials were of analytical
grade and used as received.
À
À
Synthesis of poly(methacrylate) organosilane (PMA organosilane): Pol-
À
À
y(methacrylate) organosilane (PMA organosilane) was synthesized by
the free-radical polymerization of [3-(methacryloxy)propyl]-trimethoxysi-
lane (3-MOP) and methacrylate monomer (MA), initiated by AIBN, ac-
cording to a literature procedure.[56] The MA monomer (13.3 g), 3-MOP
(3.3 g), and AIBN (60 mg) were dissolved in THF (300 mL). Argon was
bubbled into the reactor. The mixture was stirred for 1 h and the poly-
merization was carried out with stirring at 608C for 8 h under an argon
À
atmosphere. The resulting PMA organosilane was dried under reduced
pressure at RT and washed three times with Et2O. The number-average
À
molar mass (Mn) of PMA organosilane, as obtained by size-exclusion
chromatography, was 2780 gmolÀ1 and the chemical composition of
À
PMA organosilane was characterized by FTIR spectroscopy (see the
Supporting Information, Figure S1).
Synthesis of PMA-functionalized mesoporous silicas (PMA-n-MS): In a
typical synthesis, P123 (1.0 g) and EtOH (1.69 g) were dissolved in a
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C. Li, Q. H. Yang et al.
HAc/NaAc buffer solution (28 mL, pH 4.4, 0.52m HAc, 0.27m NaAc) at
258C under vigorous stirring. Sodium silicate (2 mL, 8.62 mmol) was
added and, after stirring at 258C for 20 min, a mixture of TEOS and
ative pressure (P/P0) of 0.99. The adsorption of water vapor and benzene
vapor was measured at 273 K on a Hiden Isochema Intelligent Gravimet-
ric Analyzer after the sample had been degassed at 353 K for 6 h. Ultra-
pure water and benzene that were used in the vapor-adsorption experi-
ments were both purified by three freeze-pump-thaw cycles prior to the
adsorption tests. FTIR spectra were recorded on a Thermo Nicolet
Nexus 470 Fourier transform infrared (FTIR) spectrometer by using KBr
À
PMA organosilane (total 8.62 mmol) in THF (2.0 mL) was added. The
À
molar composition of the mixture was sodium silicate/TEOS/PMA orga-
nosilane/P123/acetic-acid/sodium-acetate/THF/water=
50:50Àn:n:1.02:85.2:44.5:143.0:8921. The reaction mixture was stirred at
408C for 20 h and aged at 1008C under static conditions for an additional
24 h. After filtration, the as-synthesized materials were dried at 258C and
the surfactant was extracted by heating the as-synthesized sample (1.0 g)
at reflux in EtOH (200 mL) for 24 h. The extracted materials were denot-
ed as PMA-n-MS, where n (n=0, 0.6, 1.2, 2.4, 3.6, and 4.8) denotes the
pellets. UV/Vis spectra were recorded on
a SHIMADZU UV-2550
double-beam spectrophotometer by using a 1 cm quartz cell. X-ray dif-
fraction (XRD) patterns were recorded on a Rigaku RINT D/Max-2500
powder-diffraction system by using CuKa radiation (l=0.15406 nm).
TEM was performed at 120 kV. Thermogravimetric (TG) analysis was
performed under a flow of air on a Perkin–Elmer Pyris Diamond TG in-
strument within the temperature range 20–9008C with a heating rate of
58CminÀ1.)
À
molar percentage of PMA organosilane in the initial mixture of the
silane precursors.
Lipase immobilization, activity assay, and thermostability test: Crude
lipase from Pseudomonas cepacia (PCL) was purified and immobilized
onto solid materials as follows: Crude PCL (1 g) was dissolved in phos-
phate buffer solution (PBS, 20 mL, pH 8.0, 50 mm) and the solution was
shaken (160 rpm) for 3 h at 48C. The mixture was centrifuged to remove
any insoluble components. Then, the obtained supernatant (6 mL) was
added to the solid sample (30 mg) and the suspension was shaken
(160 rpm) at 48C for 12 h. The resulting product was centrifuged, washed
several times with PBS, and dried at 208C under vacuum. The lipase con-
tent in the supernatant and washings was determined by using the Brad-
ford method. The amount of PCL that had been accommodated inside
the solid support was calculated from the difference in concentration of
the lipase solution before and after immobilization. The lipase activity
was measured for the hydrolysis of triacetin. Triacetin (250 mL) and PBS
(10 mL, pH 8.0, 50 mm) were mixed at 308C under vigorous stirring for
5 min to prepare a uniform emulsion. Free lipase solution (100 mL) or a
lipase-loaded solid sample (10 mg) was added and the mixture was gently
stirred for another 10 min. Then, the mixture was titrated with a 0.05m
solution of NaOH in the presence of phenolphthalein indicator. A blank
experiment (without enzyme) was carried out according to the same pro-
cedure. Based on the consumption of NaOH, the amount of acetic acid
that had been produced was determined and the activity of free lipase or
immobilized lipase was calculated. To test the thermostability of the
lipase, free lipase solution and lipase-loaded solid samples were separate-
ly placed in screw-capped vials and incubated at a certain temperature
(40–708C) in a thermostatically controlled water bath for 2 h prior to the
activity measurements (at 308C). The residual activity of the thermally
treated PCL was calculated by taking its initial activity without thermal
incubation (regarded as kept at 208C) as 100%.
Acknowledgements
This work was supported by the National Natural Science Foundation of
China (20873144 and 20923001) and by the National Basic Research Pro-
gram of China (2009CB623503).
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Received: August 3, 2012
Published online: && &&, 0000
Chem. Eur. J. 2013, 00, 0 – 0
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C. Li, Q. H. Yang et al.
Entrapment: The effects of macromo-
lecular crowding and surface hydro-
phobicity on the properties of an
immobilized enzyme were explored by
accommodating lipase into a series of
mesoporous silicas that contained vari-
ous amounts of inert poly(methacry-
late) (PMA) covalently anchored
inside the nanopores.
Enzymes
J. Liu, J. Peng, S. Shen, Q. Jin, C. Li,*
Q. Yang* ......................... &&& — &&&
Enzyme Entrapped in Polymer-Modi-
fied Nanopores: The Effects of Macro-
molecular Crowding and Surface
Hydrophobicity
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These are not the final page numbers!