Immobilization of Candida Antarctica lipase B on epoxy modified silica by sol-gel process

Article abstract of DOI:10.1016/j.molcatb.2016.02.014

An epoxy-functionalized silica support was prepared through 3-glycidoxypropyltrimethoxylsilane(KH560)modified silica sol in the presence of triblock copolymer F127 (F-560-S). The surface chemistry, micromorphology and pore structure of the supports were characterized by TG, FTIR, SEM and N₂ adsorption-desorption, which showed that the BET surface areas and pore volumes of epoxy-activated supports increased with the addition of F127. The Candida Antarctica Lipase B (CALB) was immobilized on the resulting carrier by covalent link. The lipase absorbed amounts were 58 mg/g support and 375 mg/g support on the normal silica (N-S) and modified silica (F-560-S), respectively. The immobilized lipases were examined as biocatalysts for transesterification of 1-phenethanol and vinyl acetate in nonaqueous medium. Solvents and temperature were investigated for influence of morphology and KH560 on the immobilized lipase. The catalytic activity of the CALB immobilized on F-560-S was improved in various solvents, especially in polar solvents. The immobilized CALBs maintained high activity, while the control experiment using free lipase gave very low ester production in the temperature range of 0-60 °C. Furthermore improved thermal stability of CALB immobilized on F-560-S compared to the CALB on N-S was observed. The CALB immobilized on F-560-S exhibited high operational stability in organic media which still retained 88.3% of its original activity for 12 h consecutive 7 runs.

Full text of DOI:10.1016/j.molcatb.2016.02.014

Journal of Molecular Catalysis B: Enzymatic 127 (2016) 76–81
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Immobilization of Candida Antarctica lipase B on epoxy modified silica
by sol-gel process
a
b
a
a
a,∗
Hong-Zhi Ma , Xiao-Wei Yu , Cong Song , Qing-lan Xue , Bo Jiang
a
Key Laboratory of Green Chemistry & Technology, Ministry of Education, College of Chemistry, Sichuan University, Chengdu 610064, China
Key Laboratory of Industrial Biotechnology of Ministry of Education, School of Biotechnology, Jiangnan University, 1800 Lihu Ave, Wuxi 214122, Jiangsu,
b
China
a r t i c l e i n f o
a b s t r a c t
Article history:
An
epoxy-functionalized
silica
support
was
prepared
through
3-
Received 12 September 2015
Received in revised form 23 February 2016
Accepted 24 February 2016
Available online 26 February 2016
glycidoxypropyltrimethoxylsilane(KH560) modified silica sol in the presence of triblock copolymer
F127 (F-560-S). The surface chemistry, micromorphology and pore structure of the supports were
characterized by TG, FTIR, SEM and N2 adsorption-desorption, which showed that the BET surface
areas and pore volumes of epoxy-activated supports increased with the addition of F127. The Candida
Antarctica Lipase B (CALB) was immobilized on the resulting carrier by covalent link. The lipase absorbed
amounts were 58 mg/g support and 375 mg/g support on the normal silica (N-S) and modified silica
Keywords:
KH560
Silica sol-gel
Enzyme immobilization
(F-560-S), respectively. The immobilized lipases were examined as biocatalysts for transesterification of
1
-phenethanol and vinyl acetate in nonaqueous medium. Solvents and temperature were investigated
for influence of morphology and KH560 on the immobilized lipase. The catalytic activity of the CALB
immobilized on F-560-S was improved in various solvents, especially in polar solvents. The immobilized
CALBs maintained high activity, while the control experiment using free lipase gave very low ester
◦
production in the temperature range of 0–60 C. Furthermore improved thermal stability of CALB
immobilized on F-560-S compared to the CALB on N-S was observed. The CALB immobilized on F-560-S
exhibited high operational stability in organic media which still retained 88.3% of its original activity for
1
2 h consecutive 7 runs.
©
2016 Published by Elsevier B.V.
1
. Introduction
The lipase (EC 3.1.1.3) is widely used in triglyceride hydroly-
Silica with large surface area and pore volumes exhibits excep-
tional adsorptive affinity with enzymes and the silica supports have
good biocompatibility, high specific surface area and good mechan-
ical properties [4–6]. Some researchers entrapped the enzyme in
pure silica matrixes to achieve immobilized enzyme by a sol-
gel process [7–11]. The sol-gel encapsulation techniques generally
give better enzymatic activity and stability. Nevertheless, there are
some disadvantages in the process of sol-gel immobilization [12].
One is that the enzyme may denature for the shrinkage of gel dur-
ing the process of condensation and drying. In addition, the slow
diffusion of molecules of substrate for the enzyme within the sol-
gel matrix hinders the catalytic activity of the immobilized enzyme
if the materials’ pore diameter is smaller than 20 Å [13].
Last decades, modification of silica gel with functional groups
has been performed. Surface modification is generally carried
out by silanization with organosilane compound. These surface
modified supports can strength the interaction with the enzyme
by a covalent binding, the stability and reusability have been
improved. There are two factors of silica support which greatly
influence the immobilized enzyme properties: the morphology and
sis, production of specialty esters and chiral substances split for its
high activity and specificity [1,2]. As a representative part, Can-
dida antarctica lipase B (CALB) has outstanding performance in
non-aqueous media and thus become one of the most widely used
biocatalysts in industrial applications, such as kinetic resolutions,
esterification, and transesterification [3].
However, like other enzymes, the application of free CALB is
limited because of some drawbacks like low stabilities, prohibitive
cost and hardness of separation from product. For avoiding these
defects, various methods for enzyme immobilization have been
carried out, such as physical methods(adsorption, entrapment,
encapsulation) and chemical methods (covalent bonding and cross-
linking).
∗ Corresponding author.
E-mail address: jiangbo@scu.edu.cn (B. Jiang).
http://dx.doi.org/10.1016/j.molcatb.2016.02.014
1
381-1177/© 2016 Published by Elsevier B.V.

H.-Z. Ma et al. / Journal of Molecular Catalysis B: Enzymatic 127 (2016) 76–81
77
O
OH
O
OH
Immobilizd CALB
+
O
O
(R,S)-1-Phenylethanol
(R)-1-Phenethylacetate
(S)-1-Phenylethanol
Scheme 1. Transesterification of 1-phenylethanol and vinyl acetate.
◦
the surface characteristics [11]. Previous studies are commonly
used post-synthesis modification to achieve surface functional-
ization silica supports. Bernal et al. [14] synthesized hierarchical
meso-macroporous silica and chemically modified by 3- glyci-
doxypropyltrimethoxylsilane(KH560). Mohammadi et al. [15,16]
reported several epoxy functionalized silica supports by KH560
grifting for immobilization lipase. However, post modification is
time consuming and it needs specific temperature and solvent con-
ditions with a chosen organosilane [17]. Contrary, as the presence
of silanol groups on the surface of silica sol can be easily modi-
fied by a variety of organic functional groups [18], functionalization
of mesoporous silica support can easily obtain when surfactant
organic molecules and organosilanes employed during the sol pro-
cess. Compared to previous method, adding the surfactant agent
and organosilanes during the sol process is more economical and
lower toxic. The enzyme loading capacity is also increased.
In our present work, an epoxy-fuctionalized silica was pre-
pared through KH560 modified silica sol in the presence of triblock
copolymer F127. Upon selective removal of the copolymer by
appropriate temperature, silica particles with large surface areas
and pore volumes were prepared. This method provides a simple
way to prepare fuctionalized silica support. The CALB was immo-
bilized on the F-560-S by covalent link. In our study, the influence
of the morphology and surface properties of host material on the
lipase absorbed amounts and enzymatic properties were investi-
gated in detail.
calcined at 230 C. The silica support (560-S) was prepared without
adding F127.
2
.3. Enzyme immobilization
8
ml of 15 mg/ml CALB solution and 0.24 g support were added
into 15 ml capped vials. The vials were shaken in the shaking bath
under 25 C for 12 h. Then the suspension was centrifuged and the
mother liquor was collected from the vial. The solid was washed
with the water 4 times, and lyophilized for 12 h. All liquors after
centrifuged were collected together in 100 ml volumetric flask and
dilute to volume for protein determination by the Bradford method
20]. The lipase absorbed amount was expressed as A value and
calculated as follows:
◦
[
A − A
120
1
2
A =
×
0.24
A
1
where A1 and A2 denoted the absorbance at 595 nm of the
CALB-Coomassie reagent solution before and after immobiliza-
tion respectively. All the experiments were repeated at least three
times.
2.4. General experimental procedure for enzymatic activity assay
2
. Experimental section
The activities of the free and immobilization lipase were
analyzed by measuring from synthesis of 1-phenylethyl acetate
through transesterification reaction of 1-phenylethanol and vinyl
acetate. In typical experimental procedure, 1.2 ml 1-phenylethanol
and 4.6 ml vinyl acetate were dissolved in the 4.2 ml reaction
medium (hexane or other organic solvents). 15 mg free CALB or
equivalent immobilization CALB was added. The mixture was
2
.1. Chemicals
Free CALB (EC 3.1.1.3; specific activity of 1.27 U/mg) and
triblock copolymer F127 (EO106PO70EO106
, Mn = 13400) were
purchased from Sigma. tetraethoxysilane (TEOS) and 3-
glycidoxypropyltrimethoxylsilane (KH560) were purchased
from Kermel Co (China). All organic solvents and other chemicals
used were of analytical reagent grade.
◦
shaken at 0 – 60 C and 250 rpm in a temperature controlled orbital
shaker. At regular time intervals, samples were withdrawn from
the reaction medium and analyzed by gas chromatography that
equipped with a flame ionization detector (FID) and capillary col-
◦
umn (Grace EC-WAX). The column temperature was kept at 60 C
2.2. Preparation of different silica supports
◦
◦
for 3 min and then raised to 240 C for 3 min with a rise of 20 C/min.
The temperature of the injector and detector was maintained at
The method of silica sol preparation was followed by previous
report [19]. Briefly, 16.4 g TEOS was mixed with 138.5 g ethanol,
◦
2
50 C. All the experiments were repeated at least three times.
0
.87 g ammonia water and 4 g deionized water. The solution was
◦
left in a closed glass container and stirred at 30 C for 2 h and aged
at 25 C for 2 weeks to obtain pure silica sol. The normal silica sup-
◦
2.5. Reusability of CALBs
port (N-S) was directly obtained from evaporation of ethanol of the
silica sol. The modification of the silica support were carried out as
follows: 0.3 g Pluronic F127 triblock copolymer, 1 g NH3 and 50%
After completion of the reaction described above, the immobi-
lized enzyme was carefully filtered washed with hexane 2–3 times
to remove any traces of substrates and product. The immobiliza-
tion CALBs were dried in air, and then reintroduced into the fresh
substrate solution for a next reaction.
(
w/w) KH560 were added in 100 ml silica sol prepared as above,
◦
the mixture was stirred at 30 C for 3 days. The modified silica sup-
port (F-560-S) was obtained by evaporating the excess ethanol and

7
8
H.-Z. Ma et al. / Journal of Molecular Catalysis B: Enzymatic 127 (2016) 76–81
Table 1
surface properties of silica support before and after modification.
−1
Pore volume(cm3
−1
)
Sample
N-S
ABET (m2
g
)
g
Pore size(nm)
538
455
725
0.563
0.545
0.709
3.9
3.2
10.3
560-S
F-560-S
respectively. These results showed that the epoxy group was not
◦
affected by the 230 C calcination.
3.1.2. Morphology characterization of the supports
Fig. 3 shows the surface morphology of N-S and F-560-S. The
porous structure of F-560-S was clearly observed.
To gain further insight into the morphology of supports, the N2
adsorption-desorption was used to investigate N-S, 560-S and F-
5
60-S (Table 1). As Table 1 shows, the pore size, pore volume and
surface area of N-S decreased slightly after KH560 modification.
Fig. 1. TG curves of 560-S (A); F-560-S (B).
2
−1
However, the F-560-S exhibited a surface area of 725 m g and
3
−1
pore volume of 0.709 cm g according to BET method. Obviously,
the increase was due to the presence of template F127 in the sil-
ica sol-gel process. Furthermore, the pore size of F-560-S has an
average diameter of 10.3 nm, which is compatible with CALB [21].
3.2. Lipase absorbed amounts on different supports
The immobilization on epoxy modified silica took place through
a two step mechanism: the enzyme was firstly adsorbed on the sup-
port, and then covalently attached to the epoxy groups presented
in the support surface [22]. Takahashi et al. [23] suggested that the
interaction between enzyme and mesoporous support was a critical
factor for immobilization enzyme. The immobilization capabilities
of supports were compared in Fig. 4. It was not surprising that N-
S showed the lowest adsorption of 58 mg/g for its week physical
absorption. As for the 560-S and F-560-S, the adsorption amount
was about 1.8 and 6.5 times larger than N-S, respectively. This result
may indicate that covalent link and morphology of supports have
combined impacts on immobilization CALBs. KH560 strengthens
the connection between silica support and CALB, which prevent
the lipase leaking in the washing process. For F-560-S with larger
surface area and pore volume, there are more entrances exposed
to CALB comparing to N-S and 560-S. The amounts of entrances
facilitate the absorption behavior of CALBs. Similar phenomenon
was observed in previous study by Lei et al. [11], which suggested
that relative entrance amount accessible to lipases determines the
adsorption amount when the structure of hosts (particle size and
surface properties) is kept unchanged.
Fig. 2. FT-IR spectra of silica before and after KH560 modification.
A:KH560; B:N-S; C: F-560-S.
3
3
3
. Result and discussion
.1. Characterization of support
.1.1. Surface epoxy assays by TG and FTIR
The thermal decomposition processes of 560-S and F-560-S are
given in Fig. 1. There are two mass loss occurred in F-560-S in
3.3. Synthesis of 1-phenylethyl acetate catalyzed by CALBs
◦
◦
the range of 200 – 240 C and 250 ∼ 300 C respectively. Compar-
◦
ing with the mass loss of 560-S started at 260 C, the loss below
50 C may be attributed to the pyrolysis of F127 in F-560-S. There-
fore, calcination temperature of 230 C was used for the purpose
of removing the F127 while keeping KH560 intact, especially the
epoxy group on the support.
The catalytic activities of free and immobilization CALBs were
evaluated in the transesterification of 1-phenylethanol with vinyl
acetate (Scheme 1).
◦
2
◦
As seen in Fig. 5, the conversion of transesterification was greatly
improved when CALB was immobilized on the silica supports as the
immobilization process prevents enzyme aggregation and induces
conformational changes [24]. Compared with the Novozym 435,
N-S and 560-S immobilized CALBs, the catalytic activity of F-560-
S immobilized CALB remained almost unchanged. This result may
indicate that the lipase activity is not affected by the KH560. In
another word, KH560 has good compatibility with CALB.
FT-IR spectroscopy was used to confirm the epoxy group on
the F-560-S. As Fig. 2 (B and C) shows, slightly decrease in inten-
−1
sity of 3450.6 cm was observed. The band can be ascribed to the
H stretching frequency of silanol groups. Moreover, the peak
O
−
1
−1
at 2943.3 cm and 2843.0 cm is related to the −CH3 and −CH2-
bond stretching, which indicates that SiO was modified by KH560.
2
Determination of epoxy on the F-560-S was performed by using
the reaction between epoxy groups and sodium thiosulphate, and
subsequent titration of the released hydroxide ion with hydrochlo-
ric acid. The result revealed that the amount of epoxy groups on the
surface of F-560-S and 560-S is about 225 ␮mol/g and 256 ␮mol/g
3.4. Reusability of immobilized CALBs
For industrial productions based on immobilized enzymes, the
reusability of the biocatalyst is beneficial. Fig. 6 illustrates the

H.-Z. Ma et al. / Journal of Molecular Catalysis B: Enzymatic 127 (2016) 76–81
79
Fig. 3. Low and high magnification SEM images of normal silica. (A and B): N-S; (C and D): F-560-S.
Fig. 4. Lipase absorbed amounts of immobilized lipases on difference supports. Error
bar indicate the relative standard deviation for n = 3 independent experiments.
Fig. 5. Progress curves of difference CALBs in synthesis of 1-phenylethyl acetate.
᭿) CALB immobilized on F-560-S; (᭹) N435; (᭿) CALB immobilized on 560-S; (᭹)
(
CALB immobilized on N-S; (ꢀ) free CALB. The reaction conditions: 15 mg free CALB
or equivalent immobilization CALB, 1.2 ml (R,S)-1-phenylethanol and 4.6 ml vinyl
acetate in 4.2 ml of hexane, at 40 C, 250 rpm. Error bar indicate the relative standard
deviation for n = 3 independent experiments.
change of the catalytic activity of immobilized CALBs on normal
and modified silica particles after multiple reuses.
◦
Covalent link is one of the most powerful tools for lipase immo-
bilization. Moreover, the reusability of immobilization lipase is
strongly dependent on the amount of reactive groups on the sup-
port [25]. The CALBs on the F-560-S were significantly more stable
than that on N-S or 560-S, and the CALBs immobilized on the F-
are easily stripped off the surface of the N-S by the multiple soak-
ing, separations and washing process during the recycling reaction.
The better operational stability of the F-560-S immobilized CALB
than the 560-S immobilized CALB is probably resulted from the
unique fluffy and flexural structure as well as large surface area and
pore volume. These factors would increase the affinity interaction
between the epoxy groups of modified silica and CALB, resulting in
a slower leakage during the reuse process [26].
5
7
60-S still retained 88.3% of its original activity for 12 h consecutive
runs while the N435 was 83.48%. The activity of CALBs immobi-
lized on the N-S started declining at the second run and maintained
only 50% of its original activities after 7 runs. The poor stability of
CALB immobilized on N-S is due to the weak interaction between
the normal silica support and enzyme molecules, in which CALBs

8
0
H.-Z. Ma et al. / Journal of Molecular Catalysis B: Enzymatic 127 (2016) 76–81
Fig. 6. Reusability of the immobilized CALBs: (᭿) CALB immobilized on F-560-S; (᭿)
N435; (᭿) CALB immobilized on 560-S; (᭿) CALB immobilized on N-S. The reaction
Fig. 8. Effect of temperature on catalytic activity of CALBs. (᭿) CALB immobilized
on F-560-S; (᭿) CALB immobilized on 560-S;(᭹) CALB immobilized on N-S; (ꢀ) free
CALB. The reaction conditions: immobilization CALBs that were equivalent to 15 mg
free CALB, 1.2 ml (R, S)-1-phenylethanol and 4.6 ml vinyl acetate in 4.2 ml of hex-
ane, at 250 rpm for 12 h. Error bar indicate the relative standard deviation for n = 3
independent experiments.
conditions: immobilization CALBs that were equivalent to 15 mg free CALB, 1.2 ml
◦
(
R,S)-1-phenylethanol and 4.6 ml vinyl acetate in 4.2 ml of hexane, at 40 C, 250 rpm
for 12 h. After each run, the immobilized CALBs were recovered by filtration, washed
with hexane and reused for a new reaction under the same conditions. Error bar
indicate the relative standard deviation for n = 3 independent experiments.
support which altering microenvironment of the immobilized
CALBs [28].
3.6. Thermal stability of free and immobilized CALBs
The effect of temperature on the catalytic activity of free
and immobilized CALBs was presented in Fig. 8. The immobi-
lized CALBs exhibit higher activity than free CALB in the range
◦
of 0–60 C.The result demonstrated the fact that immobilization
process effectively protected the CALB against thermal denatura-
tion. Meanwhile, the conversion of transesterification increased
with the increased of reaction temperature. This is due to the
change of enzyme stereochemistry caused by temperature [29].
The CALBs has higher activity and lower stereospecificity for (S)-
1
-phenylethanol in the higher reaction temperature
Improved thermal stability of CALB immobilized on F-560-S
compared to the CALBs immobilized on 560-S and N-S is also
observed. The F-560-S immobilized CALB keeps 14.5% at 0 C, while
the CALB immobilized on N-S is almost inactive. This result further
supports the proposal that the surface characteristics and specific
morphology of F-560-S have positive effect on catalytic activity of
CALB.
Fig. 7. Effect of solvents on catalytic activity of immobilization CALBs. The reaction
◦
conditions: immobilization CALBs that were equivalent to 15 mg free CALB, 1.2 ml
◦
(
R,S)-1-phenylethanol and 4.6 ml vinyl acetate in 4.2 ml solvents, at 40 C, 250 rpm
for 12 h. Error bar indicate the relative standard deviation for n = 3 independent
experiments.
3.5. Solvent stability of immobilized CALBs
4. Conclusions
The influence of reaction medium on catalytic activity of immo-
bilized CALBs was investigated in various organic solvents (Toluene,
chloroform, tetrahydrofuran, pyridine, DMSO, DMSO mixed with
Epoxy modified silica particles with large surface area and pore
volume were prepared via sol-gel process in the presence of triblock
copolymer F127 (F-560-S). Selective removal of the copolymer from
the silica particles can be realized by calcination at the tempera-
8
0% Toluene and DMSO mixed with 80% t-butyl alcohol). It has been
reported that the enzyme catalytic activity would affect by polar
solvents for the reason of secondary structure alteration [27]. In
general, extreme hydrophilic solvents may stripping off the essen-
tial water from the enzyme and thus reduce its catalytic activity. As
seen in Fig. 7, CALBs immobilized on F-560-S and 560-S maintain
higher conversions than that on N-S in all the solvents except for
DMSO. Moreover, CALB immobilized on F-560-S support had a bet-
ter catalytic performance than other supports, especially in polar
solvents. The improvement of solvent stability might be explained
by the joint effects of covalent attachment and morphology of
◦
ture of 230 C while keeping the epoxy group unchanged. Covalent
immobilization of Candida Antarctica Lipase B (CALB) on F-560-S
was carried out, and the immobilization efficiency was about 6.5
times higher than normal silica (N-S). CALB immobilized on F-560-
S support shows good enzymatic reusability. Improved solvent and
thermal stability were also observed. The significant improvement
in catalytic activities implied that F-560-S immobilized CALB can
be of great value in practical application.

H.-Z. Ma et al. / Journal of Molecular Catalysis B: Enzymatic 127 (2016) 76–81
[15] M. Mohammadi, Z. Habibi, S. Dezvarei, M. Yousefi, S. Samadi, M. Ashjari,
81
Acknowledgment
Improvement of the stability and selectivity of Rhizomucor miehei lipase
immobilized on silica nanoparticles: selective hydrolysis of fish oil using
immobilized preparations, Process Biochem. 49 (2014) 1314–1323.
[16] M. Babaki, M. Yousefi, Z. Habibi, J. Brask, M. Mohammadi, Preparation of
highly reusable biocatalysts by immobilization of lipases on
The authors gratefully acknowledge the financial support from
the National High Technology Research and Development program
of China (863 Program) (No. 2012AA022207).
epoxy-functionalized silica for production of biodiesel from canola oil,
Biochem. Eng. J. 2 (2015) 3–31.
References
[17] P. Zucca, E. Sanjust, Inorganic materials as supports for covalent enzyme
immobilization, Methods Mech. Mol. 19 (2014) 14139–14194.
[
[
18] L. Ye, X. Zhang, B. Xiao, H. Ye, B. Jiang, Chem. J. Chin. Univ. 33 (2012) 897–901.
19] X. Zhang, B. Xia, B. Ding Bin, Y. Zhang, J. Luo, B. Jiang, Ultra-fast surface
hydrophobic modification of sol-gel silica antireflective coating with
enhanced abrasion-resistance, Mater. Lett. 104 (2013) 31–33.
20] M.B. Marion, A rapid and sensitive method for the quantitation of microgram
quantities of protein utilizing the principle of protein-dye binding, Anal.
Biochem. 72 (1976) 248–254.
21] J. Uppenberg, M.T. Hansen, S. Patkar, T.A. Jones, The sequence, crystal
structure determination and refinement of two crystal forms of lipase B from
Candida antarctica, Structure 2 (1994) 293–308.
22] C. Mateo, R. Torres, G. Fernández-Lorente, C. Ortiz, M. Fuentes, A. Hidalgo, F.
López-Gallego, O. Abian, J.M. Palomo, L. Betancor, Epoxy-amino groups: a new
tool for improved immobilization of proteins by the epoxy method,
Biomacromolecules 4 (2003) 772–777.
[
[
[
1] K.E. Jaeger, T. Eggert, Lipases for biotechnology, Curr. Opin. Biotechnol. 13
2002) 390–397.
2] M. Kapoor, M.N. Gupta, Lipase promiscuity and its biochemical applications,
Process Biochem. 47 (2012) 555–569.
3] J. Cruz, K. Würges, M. Kramer, P. Pfromm, M. Rezac, P. Czermak,
Immobilization of enzymes on fumed silica nanoparticles for applications in
nonaqueous media, in: P. Wang (Ed.), Nanoscale Biocatalysis, Humana Press,
(
[
[
[
2
011, pp. 147–160.
[
[
[
[
[
[
4] W. Yen, X. Jigeng, F. Qiuwei, D. Hua, L. Muduo, X. Jigeng, F. Qiuwei, D. Hua, L.
Muduo, Encapsulation of enzymes in mesoporous host materials via the
nonsurfactant-templated sol-gel process, Mater. Lett. 44 (2000) 6–11.
5] M. Kramer, J.C. Cruz, P.H. Pfromm, M.E. Rezac, P. Czermak, Enantioselective
transesterification by Candida antarctica Lipase B immobilized on fumed
silica, J. Biotechnol. 150 (2010) 80–86.
6] Y. Jiang, Y. Wang, H. Wang, L. Zhou, J. Gao, Y. Zhang, X. Zhang, X. Wang, J. Li,
Facile immobilization of enzyme on three dimensionally ordered
macroporous silica via a biomimetic coating, New J. Chem. 2 (2015) 978–984.
7] L.M. Ellerby, C.R. Nishida, F. Nishida, S.A. Yamanaka, B. Dunn, J.S. Valentine,
et al., Encapsulation of proteins in transparent porous silicate glasses
prepared by the sol-gel method, Science 255 (1992) 1113–1115.
8] O. Or c¸ aire, P. Buisson, A. Pierre, Application of silica aerogel encapsulated
lipases in the synthesis of biodiesel by transesterification reactions, J. Mol.
Catal. B: Enzym. 42 (2006) 106.
[
[
23] H. Takahashi, B. Li, T. Sasaki, C. Miyazaki, T. Kajino, S. Inagaki, Catalytic activity
in organic solvents and stability of immobilized enzymes depend on the pore
size and surface characteristics of mesoporous silica, Chem. Mater. 12 (2000)
3301–3305.
24] C. Mateo, J.M. Palomo, G. Fernandez-Lorente, J.M. Guisan, R.
Fernandez-Lafuenten, Improvement of enzyme activity, stability and
selectivity via immobilization techniques, Enzyme Microb. Technol. 40 (2007)
1451–1463.
[
[
[
[
25] C.R. Rafael, A.G. Cesar, V. Giandra, A.Z.A. Marco, F.-L. Roberto, M.G. Jose,
Immobilization-stabilization of the lipase from Thermomyces lanuginosus:
critical role of chemical amination, Process Biochem. 44 (2009) 963–968.
26] G. Zhou, Y. Chen, S. Yang, Comparative studies on catalytic properties of
immobilized Candida rugosa lipase in ordered mesoporous rod-like silica and
vesicle-like silica, Microporous Mesoporous Mater. 119 (2009) 223–229.
27] K. Xu, K. Griebenow, A.M. Klibanov, Correlation between catalytic activity and
secondary structure of subtilisin dissolved in organic solvents, Biotech.
Bioeng. 56 (1997) 485–491.
9] S. Nassreddine, A. Karout, M. Christ, A. Pierre, Transesterification of a
vegetaloil with methanol catalyzed by a silica fibre reinforced aerogel
encapsulated lipase, Appl. Catal. A 344 (2008) 70–77.
[
[
[
10] A. Salis, M. Bhattacharyya, M. Monduzzi, V. Solinas, Role of the support
surface on the loading and the activity of Pseudomonas fluorescens lipase used
for biodiesel synthesis, J. Mol. Catal. B: Enzym. 57 (2009) 262–269.
11] J. Lei, J. Fan, C. Yu, L. Zhang, S. Jiang, B. Tu, D. Zhao, Immobilization of enzymes
in mesoporous materials: controlling the entrance to nanospace, Microporous
Mesoporous Mater. 73 (2004) 121–128.
12] X.J. Huang, A.G. Yu, Z.K. Xu, Covalent immobilization of lipase from Candida
rugosaoto poly(acrylonitrile-co-2-hydroxyethyl methacrylate) electrospun
fibrous membranes for potential bioreactor, Bioresour. Technol. 99 (2008)
28] A. Maryam, M. Mehdi, B. Rashid, Chemical amination of Rhizopus oryzae lipase
for multipoint covalent immobilization on epoxy-functionalized supports:
modulation of stability and selectivity, J. Mol. Catal. B: Enzym. 115 (2015)
128–134.
[
29] J.M. Patel, R.S. Phillips, Effects of hydrostatic pressure on stereospecificity of
secondary alcohol dehydrogenase from Thermoanaerobacter ethanolicus
support the role of solvation in enantiospecificity, ACS Catal. 4 (2014)
5
459–5465.
[
[
13] M.T. Reetz, A. Zonta, J. Simpelkamp, W. Könen, Efficient immobilization of
lipase by entrapment in hydrophobic sol-gel materials, J. Sol Gel Sci. Technol.
692–694.
4
9 (1996) 1397–1398.
14] C. Bernal, P. Urrutia, A. Illanes, L. Wilson, Hierarchical meso-macroporous
silica grafted with glyoxyl groups: opportunities for covalent immobilization
of enzymes, N. Biotechnol. 30 (2013) 500–506.