Immobilization of lipase on aminopropyl-grafted mesoporous silica nanotubes for the resolution of (R, S)-1-phenylethanol

Article abstract of DOI:10.1016/j.molcatb.2011.11.005

Mesoporous silica nanotubes and aminopropyl-grafted mesoporous silica nanotubes were prepared as supports to immobilize lipase from Candida sp. 99-125(ClLip) by physical adsorption. The immobilization conditions were investigated. Moreover, immobilized lipases on both kinds of supports were employed to catalyze olive oil hydrolization and resolution of 1-phenylethanol by esterification. The results showed that the hydrolization activity of the lipase immobilized on aminopropyl-grafted mesoporous silica nanotubes was almost twice of that on mesoporous silica nanotubes. In addition, the resolution of 1-phenylethanol catalyzed by the former catalyst also increased 22%. Circular dichroism spectra revealed a reduction of α-helix and an increase of β-sheet when lipase was adsorbed on aminopropyl-grafted mesoporous silica nanotubes, which suggested that parts of α-helix were extended and reformed to be β-sheet.

Full text of DOI:10.1016/j.molcatb.2011.11.005

Journal of Molecular Catalysis B: Enzymatic 76 (2012) 82–88
Contents lists available at SciVerse ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Immobilization of lipase on aminopropyl-grafted mesoporous silica nanotubes
for the resolution of (R, S)-1-phenylethanol
Wei Baia,c, Yun-Jie Yangb, Xia Taob, Jian-Feng Chenb,∗, Tian-Wei Tana,∗∗
a Beijing Key Laboratory of Bioprocess, College of Life Science and Technology, Beijing University of Chemical Technology, Beijing 100029, PR China
b Key Lab for Nanomaterials of the Ministry of Education, Beijing University of Chemical Technology, Beijing 100029, PR China
c Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, 32 Xiqidao, Tianjin Airport Economic Park, Tianjin 300308, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Mesoporous silica nanotubes and aminopropyl-grafted mesoporous silica nanotubes were prepared as
supports to immobilize lipase from Candida sp. 99-125(ClLip) by physical adsorption. The immobilization
conditions were investigated. Moreover, immobilized lipases on both kinds of supports were employed
to catalyze olive oil hydrolization and resolution of 1-phenylethanol by esterification. The results showed
that the hydrolization activity of the lipase immobilized on aminopropyl-grafted mesoporous silica
nanotubes was almost twice of that on mesoporous silica nanotubes. In addition, the resolution of 1-
phenylethanol catalyzed by the former catalyst also increased 22%. Circular dichroism spectra revealed
a reduction of ␣-helix and an increase of ␤-sheet when lipase was adsorbed on aminopropyl-grafted
mesoporous silica nanotubes, which suggested that parts of ␣-helix were extended and reformed to be
␤-sheet.
Received 19 February 2011
Received in revised form 14 October 2011
Accepted 1 November 2011
Available online 10 November 2011
Keywords:
Lipase
Immobilization
Mesoporous silica nanotubes
Aminopropyl-grafted
Phenylethanol
© 2011 Elsevier B.V. All rights reserved.
1. Introduction
functionalization can influence the final loading and activity of the
immobilized protein [6,7]. Covalent coupling with surface amine
The extracellular lipase from Candida sp. 99-125(ClLip), an
important industrial biocatalyst, has been widely used in esterifi-
cation and transesterification reactions, resolution of various chiral
compounds, and production of biodiesel because of its high reactive
activity. These reactions are heterogeneous and take place exclu-
sively at oil–water interfaces. The unique property of the activation
of lipase at an oil–water interface draws special demands of sup-
ports for enzyme immobilization, which should offer not only a
stable host but also the consistency with the catalytic environment.
Mesoporous silicates, synthesized using a surfactant templat-
ing method, have ordered porous structures with narrow pore size
distributions and thick walls, enhancing their stability. The large
regular repeating mesoporous structures of mesoporous silicates
offer the possibility of adsorbing or entrapping large biomolecules
within their pores as well as on the external surface area [1]. Since
the first report by Díaz and Balkus [2] in 1996, this research area
has grown rapidly [3,4]. To provide a strong interaction for immo-
bilization, mesoporous silicates have been functionalized with a
large variety of functional groups [5]. The method and extent of
functional groups on the support using glutaraldehyde, which can
bind to enzyme amine groups, can increase the stability of enzyme,
significantly reduce the amount of leaching, and allow the immobi-
lized enzyme to be reused. A range of enzymes, including penicillin
G acylase [8], glucose oxidase [9], ␣-amylase [9], Mucor javaniscus
lipase [10], glucose isomerase [11], trypsin [12], invertase and glu-
coamylase [13] has been covalently immobilized on mesoporous
silicates in this manner. However, the increased rigidity in a cova-
lently bound enzyme can cause a reduction in activity. On the other
hand, although examples of enzyme immobilizations on amine
functional mesoporous silicates by physical adsorption are rela-
tively rare, these materials also proved promising supports without
covalent bounding. Xu et al. reported lipase physically adsorbed
on amino-functionalized ordered mesoporous SBA-15 shows excel-
lent thermal stability and better recycle potential comparing with
SBA-15-PPL [14]. Based on the above discussions, we conclude that
mesoporous silicates with tailored particle size, morphology and
surface functional groups are of great significance to the develop-
ment of biocatalysis and enzyme engineering.
In this paper, we synthesized mesoporous silica nanotubes
(MSNTs) via a sol–gel route using needle-like CaCO3 nanopar-
ticles and modified them with aminopropyl groups to obtain
aminopropyl-grafted mesoporous silica nanotubes (NH2-MSNTs).
These two materials were employed as supports to immobilize
lipase from Candida sp. 99-125. After that, we investigated the
∗
Corresponding author. Tel.: +86 10 6444 6466; fax: +86 10 6443 4784.
Corresponding author. Tel.: +86 10 6441 6691; fax: +86 10 6444 8962.
E-mail addresses: chenjf@mail.buct.edu.cn (J.-F. Chen), twtan@mail.buct.edu.cn
∗∗
(T.-W. Tan).
1381-1177/$ – see front matter © 2011 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2011.11.005

W. Bai et al. / Journal of Molecular Catalysis B: Enzymatic 76 (2012) 82–88
83
2.4. Immobilization
Lipase solution was prepared in 0.02 mol/L phosphate buffer
with a protein concentration of 8.25 g/mL. The solution was then
added with some support followed by 10 min ultrasonic dispersion
to make it a visually homogeneous suspension.
Scheme 1. The amine-functionalization of MSNTs.
The conventional way of physical immobilization was acquired
in a shake flask with a suitable stirring speed at 20 ◦C, and after 12 h
the immobilized lipase was separated by centrifugation (5000 rpm,
5 min). To cast free lipase off, the solid collection was then washed
twice with the immobilizing buffer. The amount of adsorbed lipase
was calculated indirectly by determining the protein concentra-
tion of supernatant and the wash buffers according to the Bradford
method using bovine serum albumin as a standard [19].
preferences of immobilization conditions for the two different sup-
ports and applied these two immobilized lipases to the resolution
of (R, S)-1-phenylethanol. Some possible reasons in terms of the
secondary structure of the lipase were then discussed to explain
the different results.
The immobilized lipase was placed in a freezer at −20 ◦C for sev-
eral hours until frozen, and the frozen sample was then lyophilized
for 8 h to make it dry. The mass of buffer salts that may be contained
in the final sample is negligible due to its low concentration. The
immobilized lipase on MSNTs was named as MSNTs-lipase after-
wards, similarly, immobilized lipase on NH2-MSNTs was denoted
as NH2-MSNTs-lipase.
2. Experimental
2.1. Materials
Mono Q 5/50 GL column and ion exchange media were
from Amersham Biosciences (Uppsala, Sweden). Coomassie blue
G-250, Trishydroxymethylaminomethane (Tris), olive oil, and
polyvinyl alcohol (PVA) were obtained from Sanbo Biotech (Beijing,
China). Needle like CaCO3 templates were prepared by a unique
high gravity reactive precipitation (HGRP) technology as previ-
ously described [15]. 3-Aminopropyltriethoxysilane (APTS) was
purchased from Tokyo Chemical Industry. 1-Phenylethanol was
procured from Fluka (Sigma Aldrich, USA). All other chemicals used
in the experiments were obtained from commercial sources as ana-
lytical reagents without further purification. Milli-pore water with
a resistivity of 18.2 Mꢀ cm−1 was used throughout the study.
2.5. Characterization of supports and immobilized lipase
Contact angle measurements were performed in an OCA (Data
Physics Co., Germany) apparatus equipped with a digital camera.
The morphology and structures of the samples were character-
ized by scanning electron microscopy (SEM, Hitachi S-4700) and
transmission electron microscopy (TEM, Hitachi H-800).
2.2. Purification of crude lipase
2.6. Circular dichroism (CD) spectra
The lipase of Candida sp. 99-125 was purified after ammonium
sulphate–acetone precipitation [16] followed by anion exchange
chromatography [17]. However, because of fermentation differ-
ences between batches, the crude lipase activities varied.
The culture broth was centrifuged (4000 rpm, 15 min) initially to
remove culture medium and cells. Three volumes of ice-cold ace-
tone were then slowly added to the supernatant under constant
stirring during the addition of acetone and for 10 min afterwards.
The precipitate was collected by filtration and dried at room tem-
perature.
The anion exchange chromatography was run on ÄKTA basic
100 (Amersham biosciences). The crude lipase solution was loaded
on a Q Sepharose Fast Flow column (1.5 cm × 11.3 cm) equilibrated
with 20 mM Tris–HCl buffer (pH 8.0). The column was eluted with
0–400 mM NaCl linear gradient buffer at a flow rate of 1.0 mL/min.
The active fractions were collected, concentrated and desalted by
ultrafiltration with an Amicon cell using a PM 10 membranes. After
gradient washing the proteins bound to the column were washed
out with equilibration buffer containing 1 M NaCl.
Circular dichroism spectra were recorded on a JASCO J-810 spec-
tropolarimeter (JASCO) at 10 ◦C. The protein concentration and
optical path length was 0.2 mg/mL and 5 mm respectively. The sec-
ondary structure was analyzed using four component model (Helix,
Beta, Turn, and random coil) reference spectra.
To test free lipase, the sample was lipase solution in phos-
phate buffer at pH 7.5, while the blank sample is only buffer. To
test the sample of immobilized lipase, the sample was dispersed
ultrasonically previously in its optimized buffer. So was the blank
sample, which is prepared by the supports of the same concentra-
tion instead.
2.7. Hydrolization
Lipase activity was determined according to an olive emulsion
method [20]. The substrate solution consisting of olive oil (20 mL)
and PVA (60 mL) was emulsified in a homogenizer for 6 min at max-
imum speed. Then the enzyme solution or powder was added to
5 mL of substrate emulsion and 4 mL of 20 mM phosphate buffer,
pH 8.0 (K2HPO4–KH2PO4). Samples were incubated for 10 min at
40 ◦C. The reaction was stopped by adding 20 mL ethanol. Enzyme
activity was determined by titration of the fatty acid released
with 50 mmol/L sodium hydroxide. One activity unit of lipase was
defined as the amount of enzyme required to release 1 ␮mol of fatty
acid per min under assay conditions.
2.3. Fabrication of supports
MSNTs were fabricated according to the methods published by
Yang et al. [18], using needle-like CaCO3 templates [26].
The amine-functionalization of MSNTs made the supports
structurally different, which were named NH2-MSNTs after-
wards (Scheme 1). To obtain NH2-MSNTs, some MSNTs pow-
ders were dispersed in toluene before the addition of APTS
(NSi/NAPTS/Ntoluene = 5:1:500). The suspension was subsequently
heated under reflux at 125 ◦C in nitrogen, after 24 h the particles
were filtrated and washed respectively with toluene and ethanol
twice. At last, the particles were dried at 80 ◦C for 12 h to make
them the final product of NH2-MSNTs.
2.8. Esterification
The racemic compound 1-phenylethanol was resolved by ester-
ification when lipase showed highly enantioselectivity that the
R-enantiomer was consumed preferably. The process was schemed
as Scheme 2.

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W. Bai et al. / Journal of Molecular Catalysis B: Enzymatic 76 (2012) 82–88
Fig. 1. HPLC figure of the kinetic resolution of (R, S)-1-phenylethanol.
Scheme 2. Enzyme catalyzed kinetic resolution of (R, S)-1-phenylethanol.
Using caprylic acid as the acyl donor and lipase, the kinetic reso-
lution of 1-phenylethanol was achieved. In the presence of hexane,
the reaction progress went along efficiently at 35 ◦C.
The reaction results were determined by HPLC equipped with
a chiralcel OB-H column (0.46 cm × 15 cm) (DAICEL CHEMICAL).
The mobile phase was a mixture of n-hexane and isopropyl alco-
hol (V/V = 9/1), with the velocity 0.5 mL/min. Detection wavelength
was 211 nm (Fig. 1). Because of the high volatility of the solvent
hexane, the enantiomer excess ratio of the substrate (e.e.s%) was
considered the effective parameter indicating the reaction process.
The calculation of e.e.s% is given below, in which C stands for con-
centration.
CS-1-phenylethanol − CR-1-phenylethanol
CS-1-phenylethanol + CR-1-phenylethanol
e.e.s% =
× 100
3. Results and discussion
3.1. Characterization of supports
The SEM images of MSNTs and NH2-MSNTs (Fig. 2) showed their
outlooks were approximately the same. Using nano-sized needle
like CaCO3 as template, both kinds of supports possess mesoporous
tubular structure with openings, an inner diameter of 200–400 nm,
a length of 8–10 ␮m and a wall thickness of approximately 40 nm,
which is consistent with the report by our group [21]. The surface
area and pore distribution of the samples were determined by an
ASAP 2010 surface area analyzer. From Fig. 3, it can be seen in the
pore volume of small pores, 2–4 nm, decreases dramatically after
APTS modification, but the larger pores, 8–30 nm, are hardly influ-
enced, resulting in an increase in the average pore diameter and a
decrease in the BET surface area and pore volume of the nanotubes
as a whole (Table 1). The zeta potentials of MSNTs and NH2-MSNTs
were also listed in Table 1.
Fig. 2. SEM images of MSNTs (a) and NH2-MSNTs (b).
3.2. Properties of immobilizing conditions for two different
supports
3.2.1. Effect of the ratio of carries to free lipase
If pore size and surface area could take the full responsibil-
ity of lipase loading, NH2-MSNTs would be assumed an inferior
kind of supports. However, in practical it is quite the con-
trary (Figs. 4–10). The lipase loading proportion on NH2-MSNTs
seemed always higher than that on MSNTs, and NH2-MSNTs-lipase
always showed higher catalytic activity in both hydrolization and
Fig. 3. The distribution of pore diameter of two different supports.

W. Bai et al. / Journal of Molecular Catalysis B: Enzymatic 76 (2012) 82–88
85
Table 1
Characterization of MSNTs and NH2-MSNTs.
Surface area (m2/g)
Pore volume (cm3/g)
Pore size (nm) (d-BJH)
Zeta potentiala (mV)
Contact angle (◦)
MSNTs
NH2-MSNTs
638.45
285.43
0.78
0.72
8.2
8.67
−33.4
26
44
31.4
a
Zeta potential values given here are in pure water.
Fig. 4. The effect on hydrolization activity of ratios of two different supports to
lipase (the initial lipase contents were same for different samples).
Fig. 5. TEM images of immobilized lipase on MSNTs (a) and NH2-MSNTs (b).
esterification. Xu et al. [22] found that when lipase was immobilized
by a PVA/PTFE composite membrane, the maximum reaction rate
per unit of membrane area was much higher than that immobilized
by a hydrophobic membrane. The accessibility of substrate to active
sites was improved by a hydrophobic support, which is widely used
in organic synthesis. The incompatibility of hydrophobic support
and substrate with water-soluble lipase unavoidably leads to con-
tradiction between hydrolytic rate and stability of lipase activity
however. In order to take advantage of the virtues of hydrophobic
and hydrophilic support, amphiphilic support, obtained by surface
modification, was encouraged [25].
Besides catalysis performance, different supports also result in
different preferences on immobilizing conditions. Fig. 4 shows the
effect of ratio of carries to enzyme on the lipase loading process. It
was found that the superior ratio of MSNTs to lipase turned to 3:1
for the activity recovery of free lipase, while for NH2-MSNTs the
superior ratio decreased to 1:1, which indicated that, to immobi-
lize a certain amount of lipase, the NH2-MSNTs would be consumed
less vs. MSNTs. Furthermore, higher activity was retained on NH2-
MSNTs with specific activity of lipase rose up to 6000 U/g. Adopting
Fig. 6. The effect of pH value towards free lipase.

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W. Bai et al. / Journal of Molecular Catalysis B: Enzymatic 76 (2012) 82–88
Fig. 9. Changes in the esterification activity of lipase along repeated use.
Fig. 7. The effect of pH towards immobilized lipase on MSNTs (a) and NH2-MSNTs
(b) (the lipase protein content were same for different samples).
the optimized ratio respectively in each case, the loading situations
were shown in TEM images (Fig. 5). Obviously, the immobilized
lipase on MSNTs was clearer, while the one on NH2-MSNTs pre-
sented darker.
Fig. 10. Far-UV circular dichroism spectra of free and immobilized lipase.
3.2.2. Effect of pH value during the process of immobilization
The activities of free lipase catalyzing both hydrolization and
esterification were measured with pH ranging from 6.5 to 9 (Fig. 6).
For free lipase, the hydrolization activity reached the peak at
8.5 while its esterification activity reached the peak at 7.5 (the
lipase powder acquired pH memory when the lipase solution was
lyophilized). It indicates that the lipase molecular conformation at
pH 8.5 suits hydrolization of olive oil whereas the esterification of
1-phenylethanol will be accelerated if the enzyme shows itself the
conformation at pH 7.5.
However, the situation completely changed during the immo-
bilizing process. Due to the surface charges of the supports, the
optimal pH value for the immobilization system shifted (Fig. 7). For
the adsorption of lipase on MSNTs, the immobilized lipase would
show a higher activity for both hydrolization and esterification
under the condition that the buffer solution is acidic and hold at a
low pH. On the other hand, the immobilized lipase will show higher
activity if the buffer solution for adsorption of lipase on NH2-MSNTs
is alkaline. When the NH2-MSNTs-lipase obtained at different pH
value was adopted to catalyze the hydrolization of olive oil and the
resolution of 1-phenylethanol, it could be clearly observed that the
optimal pH value for hydrolization is 8.5 and 9.0 for esterification.
Fig. 8. The resolution process catalyzed by free and immobilized lipase (10 mmol/L
1-phenylethanol × 30 mL, lipase content 0.05 g, 35 ◦C, stirring speed 120 rpm).

W. Bai et al. / Journal of Molecular Catalysis B: Enzymatic 76 (2012) 82–88
87
Table 2
Secondary structure estimation from CD spectrometry.
Lipase
MSNTs-lipase
NH2-MSNTs-lipase
␣-Helix
␤-Sheet
␤-Turn
Other
25.4%
24.6%
19.2%
30.8%
23.7%
25.6%
11.5%
39.2%
18.7%
40.4%
14.8%
26.2%
Total
100.0%
100.0%
100.0%
Another notable phenomenon was that the highest hydroliza-
tion activity of immobilized lipase on MSNTs was 10,400.0 U/g
lipase, whereas the value almost doubled for that on NH2-MSNTs
with 19,429.5 U/g lipase. Moreover, with respect to esterification
MSNTs-lipase also performed 2/3 less. These data obviously proved
amino-modified support to be a superior support.
3.3. The catalyzed kinetic resolution of 1-phenylethanol
Fig. 11. The reformation of secondary structure when lipase was adsorbed to NH2-
MSNTs.
To achieve bio-catalyzed kinetic resolution of 1-phenylethanol,
lipase with high enantioselectivity should be applied. Therefore,
free lipase, immobilized lipase on MSNTs and NH2-MSNTs were
added respectively into three stirring tubes. In each tube, the
substrate, solvent, lipase protein contents and other reaction con-
ditions were controlled to be same. The reaction process is shown
in Fig. 8. Because of the conglomeration in the presence of hexane,
free lipase did not show high catalyzing activity in the beginning
and e.e. value of 1-phenylethanol remained low within 90 h. On the
other hand, immobilized lipase showed significantly higher activ-
ity, with e.e. value of 1-phenylethanol as 80% when the reaction
was catalyzed by NH2-MSNTs-lipase, while the data reached 58%
using MSNTs-lipase.
One important reason for immobilization is the convenience of
recovery. Although the porous structure enables an efficient and
rather stable retain of the enzyme, it is unfortunately a shortcoming
and also a challenge for nano-materials, because of its light weight
and nearly invisible size.
As was showed in Fig. 9, the immobilized lipase was recovered
by centrifugation and reused at 35 ◦C for several cycles, and both of
lipase-MSNTs and lipase-NH2-MSNTs exhibited above 90% retained
activities after the 7th cycle, of which lipase-MSNTs performed
better. Due to the hydrophobic property of aminopropyl modified
supports, lipase-NH2-HSNTs presented a cloudy disperse in organic
solvent and much more easily to cling to the reactor wall, which, on
the other hand, was not the case for lipase-MSNTs. After uses some
of the wispy powders were detected adhered to the plastic wall of
the stirring tube, and because of the solvent volatility the adhering
catalysts were being exposed outside the solution gradually, which
probably explained the activity loss.
which, on the other hand, increased obviously when lipase was
adsorbed to NH2-MSNTs. Such contrast must be the result of the
intermolecular force between the abundant –NH2 on the supports
surface and the lipase protein.
Hydrogen bond and the cumulative effect of many hydrogen
bonds within ␣-helix stabilize this conformation. ␤-Sheet and ␤-
turn structure can be considered as special structures of ␣-helix
reformed by the extending of two amino acid residues [24]. In this
case, –NH2 on the supports surface competed for the hydrogen
bonding, and obviously the inter-group forces between –NH2 and
–OH/–C O would be stronger than that between –NH– of the amino
acid residues and –OH/–C O. Therefore, ␣-helix was extended and
turned out to be ␤-sheet and ␤-turn (Fig. 11). The reformation of
secondary structure of lipase indicates another explanation about
the superiority of NH2-MSNTs-lipase. Besides the hydrophobic-
ity effect, the three-dimensional reformation of lipase molecule
geometry uncovered catalyzing sites and subsequently enhanced
its activity.
4. Conclusion
Mesoporous silica nanotubes and aminopropyl-grafted meso-
porous silica nanotubes were prepared and used for the
immobilization of lipase as new biocatalysts for the resolution of
(R, S)-1-phenylethanol. The results showed that NH2-MSNTs-lipase
acquired higher activity than MSNTs-lipase for both esterification
of 1-phenylethanol and hydrolization of olive oil. The reason is the
reformation of secondary structure of lipase after the immobiliza-
tion on aminopropyl-grafted mesoporous silica nanotubes.
3.4. Reformation of secondary structure of lipase through
immobilization
Acknowledgements
The enhancement of lipase activity for resolution of 1-
phenylethanol may be mainly due to the hydrophobic activation
of NH2-MSNTs. He et al. found that the hydrolytic activity for the
hydrolysis of insoluble or partly soluble substrates increases with
enhanced support surface hydrophobicity [23]. It is not surprising
that similar effect was also found for the resolution by esterifica-
tion in hexane. In addition, another effect was discovered when free
and immobilized lipase were subjected to CD in the far UV region
of 190–240 nm (Fig. 10). The secondary structure estimation of the
samples was performed by the JASCO Secondary Structure Estima-
tion Program. The distributions of secondary structure of free and
immobilized lipase on MSNTs were slightly different (Table 2), with
␤-structure of MSNTs-lipase a little less than that of the free lipase,
This publication was supported by National Basic Research Pro-
gram of China (973 program) (Nos. 2011CB710800, 2011CB200905,
2009CB724703), The National Nature Science Foundation of China
(Nos. 20876011, 21106005) and Beijing Municipal Science and
Technology Commission (Nos. 2009GJA00016).
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