Enhancing enzyme activity and enantioselectivity of Burkholderia cepacia lipase via immobilization on modified multi-walled carbon nanotubes

Article abstract of DOI:10.1039/c4ra10517f

Burkholderia cepacia lipase (BCL) was proved to be a potential catalyst in chiral resolution. However, it is not widely applied in industry because of the low catalysis activity and poor stability of the free lipase. In this study, BCL was immobilized on modified multi-walled carbon nanotubes to enhance its catalysis performance. The immobilization conditions were further optimized via single factorial experiments and response surface methodology (RSM). Under the optimum conditions, the enzyme activity attained was 50 200 U g^-1, 54 fold that of the free lipase in resolution of 1-phenylethanol, resulting in an immensely shortened reaction time from 30 h of the free lipase to 10 min of the immobilized one. SEM micrographs verified that CNTs were truncated and the closed ends were opened by concentrated H₂SO₄. EDS further confirmed the modification and successful immobilization of the lipase. FT-IR analysis demonstrated that improvement of enzyme activity and ee_s was correlated to the alteration of the secondary structure. Compared with other immobilized lipases, CNT-BCL exhibits great advantages and possesses promising potential in industrial application. This journal is

Full text of DOI:10.1039/c4ra10517f

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Enhancing enzyme activity and enantioselectivity of
Burkholderia cepacia lipase via immobilization on the modified
multi-walled carbon nanotubes
†
†
*
Caixia Ke , Xiang Li , Shuangshuang Huang, Li Xu, Yunjun Yan
(
Key Laboratory of Molecular Biophysics, the Ministry of Education, College of Life
Science and Technology, Huazhong University of Science and Technology, Wuhan
4
*
30074, P. R. China)
Corresponding author: Yunjun Yan
Tel +86ꢀ27ꢀ87792213;
Fax +86ꢀ27ꢀ87792213;
Email: yanyunjun@mail.hust.edu.cn
These authors contributed equally to this work.
†

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Abstract
Burkholderia cepacia lipase (BCL) was proved to be a potential catalyst in chiral
resolution. However, it is not widely applied in industry because of the low catalysis
activity and poor stability of the free lipase. In this study, BCL was immobilized on
the modified multiꢀwalled carbon nanotubes to enhance its catalysis performance. The
immobilization conditions were further optimized via single factorial experiments and
response surface methodology (RSM). Under the optimum conditions, the enzyme
activity attained 50,200 U/g, 54 fold that of the free lipase in resolution of
1
ꢀphenylethanol, resulting in an immensely shortened reaction time from 30 h of the
free lipase to 10 min of the immobilized one. SEM micrographs verified that CNTs
were truncated and the closed ends were opened by concentrated H SO . EDS further
2
4
confirmed the modification and successful immobilization of the lipase. FTꢀIR
analysis demonstrated that improvement of enzyme activity and ee was correlated to
s
the alteration of secondary structure. Compared with other immobilized lipases,
CNTsꢀBCL exhibits great advantage and possesses promising potential in industrial
application.
Keywords: Immobilization, Carbon nanotubes, Burkholderia cepacia lipase,
Resolution, 1ꢀphenylethanol, Response surface methodology (RSM)

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1
. Introduction
Lipase (EC3.1.1.3) has been extensively utilized in food, detergent, oil process,
1, 2
biodiesel preparation, and many other biosynthetic industries.
Recently, great
attention has been paid to the application in preparation of pharmaceutical
3
intermediates and chiral building blocks. Although many studies have been
conducted to improve the catalytic performance of lipases, there are still some
limitations in their industrial applications, such as poor stability, sensitivity of
environment, narrow pH range adaptability, etc. Moreover, it is also very difficult to
separate the free lipase from substrates and products. However, immobilized lipases
can overcome these aboveꢀmentioned limits and have demonstrated as one of the most
4
useful methods to enhance catalytic properties of free enzymes. So far, a variety of
materials has been used to immobilize enzymes and can improve catalysis
performance to some extent. Among them, a kind of newly emerging materialꢀꢀꢀ
5
carbon nanotubes (CNTs) has attracted more and more attention. CNTs are being
proved to be wonderful immobilization matrixes owning to their extraordinary
6, 7
mechanical, electrical, and thermal properties as well as biocompatibility.
It is
8
reported that CNTs and other nanomaterials exhibit an ability to stabilize protein.
9
10
Using CNTs as immobilize matrix, Mubarak et al. and Gòmez et al. successfully
immobilized cellulase enzyme and βꢀGlucosidase, and achieved high immobilization
efficiency and satisfying enzyme activities. Furthermore, it is important and urgent to
explain the probable mechanism for the enhancement of catalysis performance caused
by immobilization. On the other hand, though amount of studies have been done in

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immobilizing lipase on CNTs, yet most of them were focused on biosensors and
bioelectrochemical applications. There are few reports focused on immobilized
lipases via carbon nanotubes (CNTs) being used as biocatalyses.
Therefore, in this work, Burkholderia cepacia lipase (BCL) was chosen as the
target enzyme for immobilization because it is widely used in biofuel synthesis,
11
biorefinery and a wide variety of reactions in aqueous and nonꢀaqueous phases. The
BCL was produced from B. cenocepacia which was cloned, expressed and described
in our previously work. B. cenocepacia consists of lipA and its chaperone gene lipB
from a stable and high lipaseꢀproducing strain Burkholderia cepacia G63 (formerly
12
known as Pseudomonas cepacia). Till now, a number of immobilization methods
were utilized to improve catalysis of BCL, and many materials were chosen as
1
3
14
immobilization matrixes, such as macroporous resin NKA , silicaꢀmonolith ,
15
κꢀcarrageenan , etc. However, in term of enantioselective resolution, few methods
achieved good effects. Thanks to the good results of other kind of enzymes, herein,
CNTs were employed to immobilize BCL.
The reaction of enzymatic kinetic resolution of (R, S)ꢀ1ꢀphenylethanol was
s
utilized to evaluate the enzyme activity and enantioselectivity (ee ) as many reports on
enantioselective transesterification of 1ꢀphenylethanol with vinyl acetate are available
16, 17
in the literature, and may be regarded as a model reaction.
It is very easy to
compare the catalytic efficiency of the immobilized BCL with other enzyme towards
the same substrate and under similar reaction conditions. Moreover, the utilization of
1
ꢀphenylethanol as essential building block and synthetic intermediate has been

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18
applied in cosmetic, chemical industries and many other fields.
Thus, this model resolution reaction was used to evaluate the enzyme
activity/enantioselectivity (ee ) and to compare the catalytic efficiency between the
s
free and immobilized lipases in nonꢀaqueous medium so as to explore the BCLꢀCNTs
immobilization strategy. The main purposes of this work were to investigate the
BCLꢀCNTs immobilization strategy, exploit the probable mechanism for the catalysis
enhancement of the immobilized lipase, and to make comparison between the
immobilized lipases and the free form, and other immobilized lipases in nonꢀaqueous
medium for enantioselective resolution.
2
. Materials and methods
2
.1 Materials
The lipase of B. cepacia G63 was selfꢀproduced. B. cepacia G63 strain was fermented
12
in 10L bioreactor and purified by using the methods described in our previous study.
All the carbon nanotubes used in this work was purchased from Shenzhen Nanotech
Port Co., Ltd. (R, S)ꢀ1ꢀphenylethanol was bought from SigmaꢀAldrich Co., Ltd (St.
Louis, Missouri, USA). Potassium sulfate, ethanol, and other reagents of analytical
grade were obtained commercially from Sinopharm Chemical Reagent Co., Ltd,
Shanghai, P. R. China. Highꢀperformance liquid chromatography (HPLC) grade
organic solvents were purchased from TEDIA (USA).
2
.2 Modification of CNTs and characterization via SEM and EDS
2 4
The CNTs were pretreated by the concentrated H SO under sonication for 3 h to open

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the closed end and truncate the tubes. The nanotube suspension was then diluted and
washed with pure water by filtering through a 0.45 ꢁm polycarbonate membrane. The
samples were dried at 65 °C with a dryer. Then, the obtained samples were ground
into powder for the future use. Furthermore, the modified CNTs were analyzed by
SEM and EDS (Nova Nano SEM 450, FEI Company, and Eindhoven, Netherlands).
The samples were coated with gold using a sputter coating system and measured at an
acceleration voltage of 5 kV.
2
.3 Preparation of the immobilized lipase and characterization via EDS
The prepared CNTs were used as the support matrix for immobilization. The
properties of CNTs were listed in Table 1. A certain amount of lipase was dissolved in
5
ml buffer solution. The solution was then loaded into a 50 ml tube containing 0.2 g
prepared CNTs. The mixture was shaken at 37 °C, 200 rpm for several hours. Then,
the suspensions were centrifuged at 4 °C, 12,000 rpm for 5 min to remove the
supernatant. The derived immobilized lipase (CNTsꢀBCL) was dried in the
thermostatic vacuum drier and ground into powder for later use. The protein content
of the supernatant was determined by the Bradford method using bovine serum
19
albumin (BSA) as standard. The immobilization of BCL was further confirmed by
EDS.
During the immobilization procedure, the effects of pH (pH 3.0–10.0), free lipase
loading (2–15 %), and immobilized time (1ꢀ6 h) on the immobilization efficiency,
specific activity and lipase activity were specifically addressed. To study the effect of

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pH, three kinds of buffers were used for pH optimization. They were Na
2
HPO
4
–citrate
acid buffer (0.2mol/L, pH 3.0–5.0), phosphate buffer (0.2mol/L, pH 6.0–8.0), and
glycine–NaOH buffer (0.2mol/L, pH 9.0–10.0).
----------------------------Table 1----------------------------
2
.4 Lipase activity and protein content measurements
The resolution reaction mentioned above was utilized to measure the enzyme activity.
One unit (U) of enzyme activity was defined as the amount of enzyme which
produces 1 ꢁmol αꢀphenylethyl acetate in one minute under the assay conditions. The
protein content of the free and immobilized lipase was 0.58 wt% and 0.85 wt%
respectively. Immobilization efficiency (%) was estimated via Eq. (1).
immobilized protein
Immobilization efficiency(%) =
× 100
(1)
total loading protein
2
.5 Kinetic resolution of (R, S)-1-phenylethanol
The resolution reactions were carried out in 5 mL pure heptane, containing 1
mmol racemic 1ꢀphenylethanol, 4 mmol vinyl acetate and 0.05 g free or immobilized
BCL. The reactions were performed in a 50 mL stoppered flask at 50°C, 200 rpm for
1
0 min. During the resolution of (R, S)ꢀ1ꢀphenylethanol, effects of substrate molar
ratio (vinyl acetate/1ꢀphenylethanol), water content, reaction time, and reaction
temperature were examined and optimized. The standard assay conditions (molar ratio,
water content, reaction time, temperature) were used except when otherwise stated in

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the text. After the reactions, the free or immobilized lipase was removed by
centrifugation. The samples were filtered through a 0.44ꢁm filter and analyzed by
HPLC.
2
.6 HPLC analysis and calculation
2
0
As reported in our previously work , the samples were analyzed by HPLC (Model
300ꢀ525 SSI. Co., Ltd USA) using a Chiralcel ODꢀH column (4.6 mm×250 mm,
Daicel Chemical, Japan). The mobile phase consisted of hexane/2ꢀpropanol alcohol at
5/5 (v/v) with a flow rate of 1.0 ml/min. At 254 nm (Model 525 UV Detector SSI.
2
9
Co., Ltd USA), the substrate and product were detected. In the above condition, the
retention times of (R)- and (S)- 1ꢀphenylethanol in the Chiralcel ODꢀH column were
7
.28 and 8.23 min, respectively. All samples were run under the same conditions as
stated above.
According to method described by Chen et al., enantioselectivity was expressed
21
as E value and calculated by Eq. (2), substrate enantiomeric excess (ee
calculated by Eq. (3), and substrate conversion (C) by Eq. (4).
s
) was
ln[(1−C)(1−ee
ln[(1−C)(1+ ee
s
)]
)]
E =
(2)
s
S − R
S + R
ee
s
=
(3)
(4)
S
0
+ R
S
0
0
−(S + R)
+ R
C =
0
0 0
where, S and R respectively represented the concentrations of the (S)- and (R)-
enantiomers of 1ꢀphenylethanol before reaction, S and R were the concentrations of

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the (S)- and (R)- enantiomers of 1ꢀphenylethanol after reaction.
2
.7 Experimental design and statistical analysis
Box–Behnken design (RSM) was employed to further optimize the reaction
conditions. Experiments were designed to examine the interaction of three variables
(pH, loading lipase of free BCL, and temperature). SAS version9.0 was used for
regression analysis and analysis of variances (ANOVAs). The experimental data were
analyzed by the response surface regression (RSREG) procedure to fit the following
secondꢀorder polynomial equation (SAS 9.0).
(5)
where, Y was the response (ee %), β , β , β were constant coefficients, and X
1
s
0
i
ij
i
were independent variables.
2
.8 Characterization of the immobilized BCL via FT-IR spectroscopy
FTꢀ IR spectra were measured at 25 °C with a Vextex 70 FTꢀIR spectrometer
Bruker, Germany) equipped with a nitrogenꢀcooled, mercury–cadmium–tellurium
(
(
ꢀ1
MCT) detector, in the region of 4,000–400 cm . The spectrum acquisition (all
samples were overlaid on a zinc selenide attenuated total reflectance (ATR) accessory)
was from IR spectra, and the secondary structure elements based on the information
of amide I region and the band assignment were manipulated using software PeakFit
22
version 4.12 as per the method described by Yang et al.
3
. Results and discussion

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3
.1 Carrier selection, modification and characterization with SEM and EDS
Four types of CNTs (LꢀMWNTꢀ2040, LꢀMWNTꢀ4060, LꢀMWNTꢀ60100, and
MWNTꢀOH) with different properties (as listed in Table 1) were selected for
immobilization carriers. It is reported that the ideal pore diameter of immobilization
supports should be at least 4ꢀ5 fold of the immobilized protein so that the access
2
3
restrictions of the enzyme could be prevented. The LꢀMWNTꢀ60100 with the
maximum diameter of the four types achieved the highest enzyme activity and
2 4
immobilized efficiency (see Fig. 1) after oxidized by the concentrated H SO .
----------------------------Fig. 1----------------------------
SEM analysis was utilized to display the effect of oxidation. Compared to the CNTs
before oxidation, the ends of CNTs have already been opened and most CNTs were
truncated (see Fig. 2). As shown in Figure 2a, CNTs often assembled and closed.
However, the port of CNTs was opened after oxidation which could improve the
absorption ability of CNTs for lipase protein (Fig. 2b). The characteristic structure of
the modified CNTs, such as the surface defect sites and the open port, renders them
24
highly specific absorption property. In addition, there are four absorb positions :
inner space, outer surface, channels between adjacent nanotubes and channel formed
by single nanotube, which will further enlarge the surface of lipase immobilization.
----------------------------Fig. 2----------------------------

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In addition, EDS was used to further confirm the modification. As shown in
Figure 3a, only C was observed in pure CNTs, and C, O occurred after oxidation,
which indicated that CNTs oxidized by concentrated sulfuric acid led to –COOH,
–
OH and/or other active group formation at the defect sites in the end and side wall
(Fig. 3b).
----------------------------Fig. 3----------------------------
3
.2 Preparation of CNTs-BCL
3
.2.1 Effects of immobilization parameters
As it is well known, the immobilization conditions have significant effects on the
immobilization efficiency, specific activity and activity recovery of the immobilized
lipase. Such conditions with principal effect as pH, lipase loading and immobilization
time were examined in this study.
(1) Effect of pH on BCL immobilization
As known, pH value is a critical factor in many reactions as well as in enzyme
immobilization. As can be seen in Figure 4a, the highest enzyme activity and
immobilization efficiency were achieved when immobilization pH arrived at 7.
Actually, immobilization pH had little effect on immobilization efficiency which
always maintained at 90%, and the neutral and basic buffer had little effect on the
enzyme activity. However, the acidic buffer seemed to reduce the enzyme activity. For
the nonꢀpolar immobilized carrier, Panzavolta et al. have reported that pH value had

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little influence on immobilization efficiency while affected a lot on the esterification
25
activity. It is because enzymes in different pH are charged differently which
contributes to maintain the active conformation. According to the ‘pH memory’ theory,
enzymes maintain the ionization when they turn to organic phase from aqueous. Thus,
26
the difference of conformation was preserved.
(2) Effect of lipase loading
In the range of 0.1 to 1.0g BCL, the enzyme activity and immobilization efficiency
were gradually raised with the increase of lipase loading. Beyond 1.0g, the enzyme
activity and immobilization efficiency both declined. Meanwhile, though the activity
of enzyme changed a lot in different lipase loading, the immobilization efficiency
always remained a high level at > 90% (see Fig. 4b).
(3) Effect of immobilization duration
As shown in Figure 4c, the immobilization efficiency and enzyme activity were
gradually increased in the first 4h. After that, there is no increment in enzyme activity
and also no significant difference. Thus, the optimal immobilization duration was
selected as 4h in the following experiments. There is an equilibration process in the
hydrophobic absorption between CNTs and biomolecules. The adsorption capacity
increases with time lasting before the equilibration and maintains the maximum when
27
the adsorption reaches equilibrium. But if the equilibration attained, with time
lasting even longer, proteins often desorbed from the immobilized matrix slightly.
Therefore, the immobilization time should be so long enough to reach the equilibrium,
but should not be too long.

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----------------------------Fig. 4----------------------------
3
.2.2 Further confirmation of CNTsꢀBCL with EDS
Energy Dispersive Spectrometer (EDS) was utilized to further confirm the
successful immobilization of BCL. Exactly, as has been predicted, N occurred after
immobilization, which indicated that lipase protein had been successfully
22
immobilized on the oxidized CNTs , meaning CNTsꢀBCL has been formed.
----------------------------Fig. 5----------------------------
3
.2.3 Optimization of the immobilization conditions via RSM
According to the above single factorial experiments, the immobilization pH, lipase
loading and immobilization time were statistically significant to transesterification
efficiency. Considering the interactions between these parameters, RSM experiments
(Box–Behnken) were designed with three factors to further optimize the
immobilization conditions. Factors and level value of response surface analysis were
presented in Table 2. The fifteen experiments and the results were all listed in Table 3,
s
providing various levels of ee under different reaction conditions designed by SAS
version 9.0.
----------------------------Table 2, 3----------------------------
Based on the designed experimental results in Table 3, the experimental data

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were fitted to a secondꢀorder polynomial equation. The best fitting response surface
according to SAS software 9.0 for the ees value could be expressed as Eq. (6).
2
Y=52763.65+486.4583X
1
+3513.542X
2
+1096.458X
3
-64.47917X
1
+53.125X
1
X
2
2
2
+
5X X -2580.729X +143.75X X -78.85417X
(6)
and
had positive effects on ees value, while the
had negative effects on ees value. Analysis of variance of
1
3
2
2
3
3
1 2 3
As equation (6) shows, the model indicated that linear terms X , X , X
quadratic term X
1 2
X , X
1
X
3 2 3
, X X
2
2
2
quadratic term X
1
, X
2
, X
3
regression mode was shown in Table 4, from which it could be confirmed that the
coefficient of linear terms were bigger than the quadratic terms. Thus, it can be
inferred that the impact of single factors was stronger than that of binary terms and
interact terms.
----------------------------Table 4----------------------------
After the simplification and normative analysis of the equation, the response
surface curve was obtained and parts of which were shown in Figure 6. The optimum
conditions of the three independent factors were immobilization pH 7.96,
immobilization time 4.47h, and lipase loading 0.95g. Under the optimal conditions,
the predicted enzyme activity was 49,880 U/g. To verify the validation of the model,
an experiment was performed in triplicates to test the enzyme activity. The average
enzyme activity attained 50,200 U/g, which coincided with the predicted value, and
the associated ee was 98.5%.
s

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----------------------------Fig. 6----------------------------
3
.3 Enzymatic kinetic resolution of (R, S)-1-phenylethanol by CNTs-BCL
During the resolution of (R, S)ꢀ1ꢀphenylethanol, enantioselectivity was affected by a
variety of reaction parameters, and these parameters may interact with one another.
Effects of four principal parameters (substrate molar ratio, water content, reaction
time, and reaction temperature) were examined by single factorial experiments, and
the results were presented in Figure 7. As shown in Figure 7b, the addition of water
had a negative effect on conversion rate and ee , therefore, it is unnecessary to add
s
extra water during the reaction.
Based on the above factorial experiments, three statistically significant variables
(substrate molar ratio, reaction time, and reaction temperature) were selected for
further optimization using the response surface methodology. According to
BoxꢀBehnken design, the optimum parameters of the three independent factors were:
substrate molar ratio 6.57:1, reaction time 9.97 min, and reaction temperature
5
6.40 °C. Under the optimum conditions, the predicted ee
s
was 99.35%. To verify the
validation of the model, an experiment was conducted in triplicates. The mean ee was
s
9
9.52%, which coincided with the predicted value, and the corresponding conversion
rate was 49.77%.
----------------------------Fig. 7----------------------------

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3
.4 Characterization of CNTs-BCL via FT-IR
To further explore the probable mechanism for the performance enhancement of
the immobilized lipase, FTꢀIR was utilized to examine the secondary structure
alteration of the lipase. Peptide bond in different secondary structure can absorb
28
infrared in different wavelengths. As known, protein has strong absorbance
−
1
spectrum in the amide I region (1,700–1,600 cm ) because of the C=O bending
vibration. In this range, the main absorbance spectra of αꢀhelix, βꢀsheet, βꢀturn and
−1
−1
−1
random coil are 1,650–1,658 cm , 1,620–1,640 cm , 1,670–1,695 cm , and
−
1
29
1
,640–1,650 cm respectively. Calculating the peak area, the amount of the four
,
secondary structures was respectively obtained (see Table 5).
As shown in Table 5, CNTsꢀBCL has an increase in βꢀsheet and a decrease in
αꢀhelix, βꢀturn and random coil compared with free lipase. The enhancement of
30
enzyme activity was tightly related to the alteration of protein structure. As reported
31
by Barbe et al.,
BCL has a ‘lid’ upon its catalytic centre, and the ‘lid’ mostly
consists of αꢀhelix. When the combination between BCL and hydrophobic support
occurs, αꢀhelix composition of the ‘lid’ decreases, leading to its unfolding, this will
make the main catalytic area easier to access substrate. The increase of βꢀsheet is
probably related to the increase of lipase activity and flexibility of the protein. The
opening of ‘lid’ may be along with the formation of βꢀsheet which attributes to the
3
2
maintenance of this open and obviously increases the lipase activity.
----------------------------Table 5----------------------------

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3
.5 Comparison with other immobilized lipases
Compared with other immobilized lipases, the CNTsꢀBCL exhibited a much higher
33
catalytic efficiency and a satisfying reaction equilibrium time. Andrade et al., Wang
3
4
35
et al., and Hara et al. had immobilized BCL on superparamagnetic nanoparticles,
zirconia particles, and Sol–gels and crossꢀlinked aggregates, respectively. The best
results of their work were listed in Table 6. In addition, in our previous study, Li et al.
reported that the reaction equilibrium time was shortened to 0.5h by BCL
1
3
immobilized on macroporous resin NKA (MPRꢀNKA). Meanwhile, the BCL was
proved to be much better than the commercial lipases of Novozyme 435, Lipozyme
RM IM, and Lipozyme TL IM. (See Table 6)
----------------------------Table 6----------------------------
However, in this work, the reaction equilibrium could be reached in an even
shorter timeꢀꢀꢀꢀwithin 10 min, and the conversion and ee reached 49.77% and
9.52%, respectively. Moreover, the highest catalysis activity attained 50,200 U/g,
s
9
which was 54 folds that of the free lipase (924.1 U/g). Therefore, CNTsꢀBCL can
greatly improve production efficiency and effectively reduce production cost,
exhibiting a promising prospect in industrial application.
4
. Conclusion
In this study, the modified CNTs were utilized for BCL immobilization, resulting in a
significant enhancement on enzyme activity and enantioselectivity of the immobilized

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2 4
lipase. Preꢀtreated with concentrated H SO , CNTs were oxidized and the closed end
was opened to improve the absorption efficiency of lipase protein. Under the optimum
conditions (pH 7.96, time 4.47h, lipase loading 0.95g), enzyme activity of the
CNTsꢀBCL attained 50,200 U/g, 54 folds that of the free lipase. More importantly,
CNTsꢀBCL immensely shortened reaction time compared with other immobilized
BCL, suggesting CNTsꢀBCL exhibits a better catalytic efficiency and shows great
potential for industrial application.
Acknowledgments
This work is financially supported by the National Natural Science Foundation of
China (Nos. 31070089, 31170078 and J1103514), the National High Technology
Research and Development Program of China (Nos. 2011AA02A204 and
2
014AA093510), the Innovation Foundation of Shenzhen Government
JCYJ20120831111657864) and the Fundamental Research Funds for HUST (Nos.
014NY007, 2014QN119, 2012SHYJ004). The authors are grateful to Analytical and
(
2
Testing Centre of HUST for their valuable assistances in SEM, EDS and FTꢀIR
measurement.

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Table 1 Properties of CNTs used in the present study
Diameter
nm)
Length
(ꢁm)
Ash content Specific area
CNTs
Purity (%)
2
(
(%)
(m /g)
LꢀMWNTꢀ2040
LꢀMWNTꢀ4060
LꢀMWNTꢀ60100
MWNTꢀOH
20ꢀ40
40ꢀ60
60ꢀ100
20ꢀ40
5ꢀ15
5ꢀ15
5ꢀ15
＜2
＞97%
＞97%
＞97%
＞97%
＜3 wt%
90ꢀ120
40ꢀ70
＜3 wt%
＜3 wt%
＜3 wt%
40ꢀ70
100ꢀ120

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Table 2 Factors and level value of response surface analysis
ꢀ1
0
1
Independent variables
Symbols
(
Low Level)
(MidꢀLevel)
(High Level)
time/ h
Lipase loading/ g
pH
X
1
2
0.4
5
4
0.8
7
6
1.2
9
X2
X
3

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Table 3 RSM design and its experiment results
Y
X
1
X
2
X
3
Run
(enzyme activity
U/min/g protein)
48, 710
(
time/h)
(lipase loading/g)
(pH)
1
2
3
4
5
6
7
8
9
2.0
2.0
6.0
6.0
4.0
4.0
4.0
4.0
2.0
6.0
2.0
6.0
4.0
4.0
0.4
1.2
0.4
1.2
0.4
0.4
1.2
1.2
0.8
0.8
0.8
0.8
0.8
0.8
7
7
7
7
5
9
5
9
5
5
9
9
7
7
49, 200
48, 900
49, 560
48, 700
48, 980
48, 860
49, 600
48, 900
48, 970
49, 370
49, 520
49, 790
49, 770
1
0
1
1
1
1
1
2
3
4
1
5
4.0
0.8
7
49, 730

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Table 4 Analysis of variance of regression mode (α=0.01, confidence
coefficient=99%）
Source
DF
9
SS
MS
F
Pr>F
Model
2208302
1059925
1086652
61725
245366.9
353308.3
362217.2
20575
6518
37.64258
54.20225
55.56899
3.15648
0.0004
0.0003
0.0003
0.0124
Linear
3
Quadratic
Cross product
Error
3
3
5
32592
Lack of fit
Pure error
3
30725
10241
933
10.9732
0.0847
2
1866
2
R =98.55%
2
Adj.R = 95.93%

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Table 5 Quantitative estimation of the secondary structure elements of the free and
immobilized lipases
αꢀhelix (%)
28.3±1.09
20.4±3.21
βꢀsheet (%)
21.4±2.21
61.5±1.21
βꢀturn (%) Random coil (%)
Free BCL
25.8±0.91
6.2±0.81
24.5±0.21
11.9±0.71
CNTsꢀBCL

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Table 6 Comparison between the CNTsꢀBCL and other immobilized lipases
Immobilization
matrixes
Reaction Authors
time
Lipases
BCL
Conversion
50 %
ee
s
This study
CNTs
99 %
50%
99%
99%
10min
Superparamagnetic
nanoparticles
Andrade et al.
BCL
34%
48h
Wang et al.
Hara et al.
BCL
Zirconia particles
50%
48h
Sol–gels and
BCL
50%
25h
crossꢀlinked aggregates
Macroporous resin
NKA
Li et al.
Li et al.
Li et al.
Li et al.
BCL
50%
99%
0.5h
0.5h
Macroporous acrylic
resin
Novozym 435
43.3 %
75 %
Macroporous anion
exchange resin
Lipozyme RM IM
Lipozyme TL IM
2.6 %
4.8 %
24 %
15 %
0.5h
0.5h
Silica particles

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Figure captions
Fig.1 Enzyme activity and immobilization efficiency by different CNTs
Fig.2 SEM analysis of CNTs before (a) and after (b) oxidation.
Fig.3 EDS analyses of pure CNTs (a), oxidized CNTs (b).
Fig.4 Effects of immobilization conditions on BCL immobilization efficiency. (a)
Effects of pH; (b) Effects of lipase loading; (c) Effects of immobilization time.
Fig.5 EDS analyses of CNTsꢀlipase.
Fig.6 Response surface plot and contour plot of interaction between the three
independent factors on the enzyme activity. (a) Response surface plot for the
effects of X (immobilize time) and X (lipase loading); (b) Response surface
1
2
plot for the effects of X
1
3
(immobilize time) and X (pH); (c) Response surface
plot for the effects of X
for the effects of X (immobilize time) and X
contour plot for the effects of X (immobilize time) and X
contour plot for the effects of X (lipase loading) and X (pH).
2
(lipase loading) and X
3
(pH); (d) Response contour plot
(lipase loading); (e) Response
(pH); (f) Response
1
2
1
3
2
3
Fig.7 Effects of reaction conditions on enantioselectivity of resolution reaction. (a)
Effects of molar ratio; (b) Effects of water content; (c) Effects of reaction time;
(d) Effects of reaction temperature.

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Fig. 1