Resolution of 1,1,1-trifluoro-2-octanol by Pseudomonas sp. lipase encapsulated in aggregated silica nanoparticles

Article abstract of DOI:10.1039/c3ra47416j

Lipase from pseudomonas sp. (PSL) was encapsulated in stacked silica nanoparticles and used for resolution of 1,1,1-trifluoro-2-octanol. The effects of reaction conditions, such as solvent, temperature, water activity and type of acyl donor were investiga

Full text of DOI:10.1039/c3ra47416j

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Resolution of 1,1,1-trifluoro-2-octanol by
Pseudomonas sp. lipase encapsulated in
aggregated silica nanoparticles†
Cite this: RSC Adv., 2014, 4, 6103
ab
a
a
c
b
b
Zhuofu Wu, Wenzhao Li, Zhi Wang, Ling Qin, Yunling Liu, Qisheng Huo*
and Zhengqiang Li*a
Lipase from pseudomonas sp. (PSL) was encapsulated in stacked silica nanoparticles and used for resolution
of 1,1,1-trifluoro-2-octanol. The effects of reaction conditions, such as solvent, temperature, water activity
and type of acyl donor were investigated. Compared with the free lipase, the encapsulated lipase exhibits
Received 9th December 2013
Accepted 18th December 2013
ꢀ1
ꢀ1
better performance (enzyme activity, 20.32 mmol g min ; E value, 33.83) under optimum conditions.
Most importantly, almost no enzyme leakage phenomenon takes place during continuous batch
operation, which suggests that this new encapsulation method has great potential for application.
DOI: 10.1039/c3ra47416j
www.rsc.org/advances
2
1
under the stress of freezing and dehydration. Once silica
nanoparticles stack together, stacked silica nanoparticles can
Introduction
The approaches of enzyme immobilization can be broadly not disperse in water owing to the irreversible fusion of silica
classied into three types: physical adsorption, covalent nanoparticles. In our previous work, we designed a new
immobilization and encapsulation, each with its own advan- encapsulation method based on this phenomenon. Aer
1–4
tages and disadvantages. Physical adsorption is a simple, adding b-galactosidase into the silica nanoparticles solution,
cheap and effective method, but enzyme leakage may occur due the enzyme was entrapped into the interstices formed by
5
–8
to the weak physical binding in most of the cases. Covalent stacked nanoparticles during lyophilization. Aer lyophiliza-
immobilization can efficiently avoid enzyme leakage, but it is tion, most of b-galactosidase was entrapped into the inter-
9
–14
complex and irreversible.
Encapsulation method can avoid stices of silica nanoparticles matrices. Only a small quantity of
the negative inuence on the enzyme conformation, while the enzyme was adsorbed on the surface of the matrices and could
diffusion limitation of products or substrates is still a serious be easily removed by washing. Our research demonstrates that
1
3,15–17
problem.
Huo et al. originally synthesized a novel silica nanoparticle b-galactosidase in an aqueous environment are improved. In
the thermal stability and pH stability of the encapsulated
18
for delivering drug in blood pool. To prevent the protein addition, the encapsulated b-galactosidase presents good
adsorption and platelet adhesion, the surface of the silica reusability. However, this new encapsulation method has not
nanoparticle was modied by poly(ethylene oxide) (PEO) which yet been extensively studied in enzyme catalyzed resolution.
could repel protein from the surface by steric repulsion and Therefore more work in this area shall be done to widen its
19
reduce the protein adsorption. This kind of silica nanoparticle application.
possesses good biocompatibility and dispersibility in water.
In this study, we adopted the encapsulation method
Their results also demonstrate that the silica nanoparticles described above to immobilize lipase from Pseudomonas sp.
manifest its exibility and mobility in the aqueous environment. (PSL). Aer encapsulation and characterization, encapsulated
It's known that the cross-linking of silica species can take lipase was used for the resolution of 1,1,1-triuoro-2-octanol.
2
0
place during the drying process. The lyophilization process This reaction was a kinetically controlled transesterication.
can guarantee and accelerate the stack of silica nanoparticles According to Kasche's report, the enzyme used in kinetically
controlled synthesis should be withdrawn in time to obtain the
a
maximum yield, and therefore the immobilization was the best
Key Laboratory for Molecular Enzymology and Engineering of the Ministry of
Education, College of Life Sciences, Jilin University, Changchun 130012, China. strategy for solving this problem. Rodrigues and coworker
22
E-mail: lzq@jlu.edu.cn; Fax: +86-431-85155201; Tel: +86-431-85155201
proposed that the yield of kinetically controlled synthesis
depend on the enzyme performed in the reaction. So, the
effects of reaction conditions, such as solvent, temperature,
water activity and type of acyl donor had been investigated.
Furthermore, the reusability of encapsulated lipase had also
been measured under its optimal reaction conditions.
b
1
State Key Laboratory of Inorganic Synthesis and Preparative Chemistry, College of
Chemistry, Jilin University, Changchun 130012, China. E-mail: huoqisheng@jlu.edu.
cn; Fax: +86-431-85168602; Tel: +86-431-85168602
c
Biological Research Institute of Jilin, Changchun 130012, China
†
Electronic supplementary information (ESI) available. See DOI:
0.1039/c3ra47416j
1
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Characterization of silica nanoparticle
Experimental
Transmission electron microscopy was performed on a FEI
Tecnai G2 F20 s-twin (FEI, American) at 200 keV. The particle
size of the silica nanoparticles was measured by a Malvern
Zetasizer Nano-S instrument at room temperature using
Dynamic Light Scattering (DLS) principle with a HeNe laser
Materials
Lipase from Pseudomonas sp. was kindly provided by Amano
Enzymes Co. (Nagaya, Japan). Tetraethyl orthosilicate (TEOS),
Pluronic F108 and dimethoxydimethylsilane (Me Si(OMe) ,
2 2
DMDMS) were commercially available from Sigma-Aldrich (St.
Louis, Missouri, USA). KBr obtained from BDH Co. (Poole, UK)
was of spectral grade. R-(+)-1-Phenylethyl isocyanate (R-(+)-PEIC)
was purchased from Fluka (USA, 97%). 1,3,5-Trimethylbenzene
(
633 nm). FTIR spectrum was surveyed using Nicolet 5700 FTIR
ꢀ1
spectrometer with a resolution of 4 cm
method.
through the KBr
(
TMB) was purchased from Tianjin Guangfu Fine Chemical
Resolution of 1,1,1-triuoro-2-octanol catalyzed by the
encapsulated lipase
Research Institute of China. 1,1,1-Triuoro-2-octanol, vinyl
esters and other organic solvents of analytical grade were
purchased from Shanghai Chemical Reagent Company (China). The reaction was implemented in a round bottom ask con-
All other chemicals and reagents were of analytical grade. All tained 1,1,1-triuoro-2-octanol (2 mmol), vinyl acetate (5 mmol),
aqueous solutions were prepared with Milli-Q water.
n-hexane (10 ml), water activity (a
lipase (50 mg) at 50 C.
w
¼ 0.58) and the encapsulated
ꢁ
Synthesis of silica cross-linked micellar nanoparticle
Water activity (a
w
) control and measurement
) was controlled according to the method
In a typical preparation, the synthesis of silica nanoparticles
was achieved by the previous method with a slight modi-
Water activity (a
described by Tian. All the reaction mixture components were
previously dried in a vacuum of 1 mmHg for 12 h. Following
that, all reaction mixtures with specic water activity (a ) were
prepared by adding a specic amount of water. The resulting
samples were pre-equilibrated at desired temperature for 24 h
in a sealed vial before being subjected to a
Water activity (a ) was measured by Hygrolab Humidity
Detector (Rotronic, Switzerland).
w
2
3
cation. Pluronic F108 (0.1 g) was dissolved in 10 g of HCl
solution (2 M) with stirring at room temperature, followed by
the addition of TMB (60 ml). The mixture was stirred at room
temperature for 0.5 hour. Then 0.33 g of TEOS was added to
the above solution. The mixture was stirred at room
temperature for 0.5 hour again, and then 0.08 g of DMDMS
was added. Stirring was continued at room temperature for
25
w
w
measurement.
w
0
.5 hour.
The HCl and EtOH (resulted from the hydrolysis of TEOS and
DMDMS) were removed by dialysis (Millipore, MW cutoff 8000–
2 000) against pure water with stirring at room temperature.
Reusability
1
The encapsulated lipase was used for successive batches. Aer
each cycle, reaction mixture was centrifuged at 12 000 rpm for
The water was refreshed every 2 hours. The dialysis process was
monitored by measuring the pH of solution and terminated at
ꢀ
1
3
min. The obtained precipitate was washed three times with
pH 7.0. Then, the silica nanoparticle solution (40 mg ml ) was
prepared.
n-hexane to remove any residual substrate or product and then
kept overnight in a vacuum oven for a complete drying. Dried
enzyme powder was used at next cycle under otherwise equiv-
alent conditions. The residual activity of recycled enzyme was
compared with the enzyme activity of the rst cycle (100%).
Encapsulating the lipase
Crude lipase was puried as follows: 2 g of crude lipase was
dissolved in 10 ml of PBS buffer (50 mM, pH 7.0) in ice bath. The
lipase solution was centrifuged at 3500g for 3 hours using
ultraltration device (Millipore, MW cutoff 10 000). The solu-
Determination of enantiomeric excess and E value
tion above the membrane was collected and then lyophilized. The enantiomeric excess and E value of encapsulated lipase was
ꢀ
1
26
The lipase solution (8 mg ml ) was prepared by redissolving measured according to previous method described by Yu.
ꢁ
the lyophilized lipase in the buffer at 4 C.
The lipase solution (5 ml, 8 mg ml ) was slowly added to gas chromatograph on
0 ml of the silica nanoparticle solution under stirring at 4 C. The (GC-14B) equipped with a FID detector and a column (EC-1000,
Samples were withdrawn from the vials and analyzed directly by
ꢀ
1
a Shimadzu gas chromatograph
ꢁ
1
ꢁ
resulting solution was frozen at ꢀ20 C for 30 min and then 30 m ꢂ 0.25 mm ꢂ 0.25 mm, Alltech). The temperatures of
ꢁ
lyophilized. The lyophilized product was repeatedly rinsed with injector and detector were 200 and 290 C, respectively.
ꢀ
1
Milli-Q water at room temperature, until no absorbance of protein Nitrogen was used as carrier gas at a ow rate of 60 ml min
in supernatant could be detected. Wet sample was lyophilized Temperature programming between 110 and 210 C with the
.
ꢁ
ꢁ
ꢁ
ꢀ1
again. Finally, lyophilized powder was stored at ꢀ20 C.
increment of 15 C min was used to determine the concen-
The protein content of enzyme solutions was determined by tration of 1,1,1-triuoro-2-octanol. (S)-1,1,1-Triuoro-2-octanol
24
Bradford method. Bovine serum albumin was used as stan- or (R)-1,1,1-triuoro-2-octanol was derived from R-(+)-PEIC
dard. The encapsulation yield (the amount ratio of entrapped (Scheme 1). The distinction of R from S enantiomers was ach-
lipase to lipase added to the encapsulated system) of lipase was ieved with temperature programming between 110 and 222 C
ꢁ
ꢁ
ꢀ1
93.8% in this experiment.
with the increment of 10 C min .
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Scheme 1 The formation of isomer for pre-column derivation.
The conversion (C) was determined via the decrease of 1,1,1-
ꢀ1
ꢀ1
triuoro-2-octanol. Enzyme activity (mmol min
g ) was
dened as the amount (in micromoles) of 1,1,1-triuoro-2-
octanol ester produced per minute per gram of encapsulated
enzyme. The enantiomeric excess of 1,1,1-triuoro-2-octanol
(
ee ) was determined by calculating the peak areas of two
S
derivatives eqn (1). The enantiomeric ratio (E value) was deter-
27
S
mined from C and ee by using eqn (2). The enzyme activity
and the E value of the enzyme were the average of three data
points. All the three data points were detected and calculated at
about 20% of conversion (in the range of 15–30%).
½
S ꢀ Rꢃ
S þ Rꢃ
ee
s
ð%Þ ¼
ꢂ 100
(1)
½
ln½ð1 ꢀ cÞð1 ꢀ ee
ln½ð1 ꢀ cÞð1 þ ee
s
s
Þꢃ
Þꢃ
E value E ¼
(2)
where S and R represent the concentrations of the (S,R)-diaste-
reomer and (R,R)-diastereomer, respectively.
Results and discussion
In this study, the silica nanoparticles were rstly synthesized.
Then, PSL was encapsulated in the interstices formed by
stacked silica nanoparticles during lyophilization (Fig. 1).
Fig. 2 TEM images (a) and light scattering measurement (b) of the
synthesized silica nanoparticles.
Synthesis and assembly of silica nanoparticles
In this experiment, the silica nanoparticles were synthesized
28
according to our previous method with a slight modication.
The silica nanoparticles were characterized by TEM (Fig. 2a) and
light scattering measurement (Fig. 2b). It can be found that a
uniform spherical nanoparticle is obtained and its average
diameter is about 45 nm.
The lyophilization can remove the water from the nano-
particles solution and guide the silica nanoparticles to stack
together. So, in this study, PSL was encapsulated in the matrices
formed by stacked silica nanoparticle through lyophilization.
Aer encapsulation and washing, immobilized enzyme was
characterized by TEM and FTIR, respectively.
TEM image of immobilized sample demonstrates that
aggregated silica nanoparticles are formed aer lyophilization
(
Fig. 3). FTIR spectrum of the silica nanoparticles, the lipase
Fig. 1 The encapsulation strategy for entrapping lipase in the inter-
stices of stacked silica nanoparticles. The black sphere and the white
ellipsoid represent silica nanoparticle and lipase, respectively.
and the encapsulated lipase are shown in Fig. 4. The stretching
vibration band of Si–O–Si can be found at 1100 cm (curve 1
ꢀ1
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Fig. 3 The TEM of the aggregated silica nanoparticles loaded with the
lipase.
Fig. 5 Effect of mass ratio (silica nanoparticles/lipase) on the encap-
sulation yield.
Optimizing the condition for resolution of 1,1,1-triuoro-2-
octanol
Temperature is an important parameter for an enzyme-cata-
30
lyzed resolution reaction. In this study, the relationship
Fig. 4 FTIR spectrum of the aggregation of the silica nanoparticles (1),
the aggregation of the silica nanoparticles loading with the lipase (2)
and pure lipase (3).
and curve 2 in Fig. 4). And the amide I and II bands of the lipase
ꢀ
1
ꢀ1
can be observed at 1643 cm and 1569 cm (curve 2 and curve
3
29
in Fig. 4). Moreover, our results verify that the aggregated
silica nanoparticles can not strongly adsorb the lipase (see ESI†)
The FTIR result demonstrates that the lyophilized product is
composed by silica-based matrices and the protein. These
results demonstrates that lipase from Pseudomonas sp. has been
successfully encapsulated in interstices formed by stacked silica
nanoparticles aer lyophilization.
The effect of mass ratio (silica nanoparticles/lipase) on the
encapsulation yield
The encapsulation yield depends on the mass ratio (silica
nanoparticles/lipase) used in this experiment. The mass ratio in
the range of 2–20 was investigated to obtain optimal encapsu-
lation yield (Fig. 5). The experimental result exhibits that the
encapsulation yield dramatically increases as the mass ratio
increases in the range of 2–10. With the further increase of the
mass ratio in the range of 10–20, the encapsulation yield rea-
ches a plateau. This result veries that almost all the lipase can
be encapsulated in stacked silica nanoparticles when the mass
ratio is above 10. Hence, the mass ratio of 10 was chosen in the
following experiment.
Fig. 6 Effect of temperature on the enzyme activity (a) and the
enantioselectivity (b) of the encapsulated lipase in resolution of 1,1,1-
trifluoro-2-octanol. Reactions were carried out in n-hexane (10 ml)
with 1,1,1-trifluoro-2-octanol (2 mmol), vinyl acetate (5 mmol), the
encapsulated lipase (50 mg) and water activity (0.58) at various
ꢁ
temperatures (30–65 C).
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Table 1 Effect of solvents on the activity and enantioselectivity of the used as acyl donor. Enzyme activity is the highest when vinyl
a
encapsulated lipase in resolution of 1,1,1-trifluoro-2-octanol
acetate is used as acyl donor and it becomes lower when the
carbon chain length of acyl donors increases. Considering that
Enzyme activity
(mmol g min
ꢀ1
ꢀ1
vinyl acetate is the cheapest acyl donor and it possesses the
lowest boiling point, which is helpful for its removing from
reaction system, vinyl acetate was selected as optimal acyl donor
in this study.
Solvent
log P
)
E value
n-Heptane
n-Hexane
Toluene
Cyclohexane
Acetonitrile
DMF
4.60
3.50
2.50
18.88
20.32
17.20
14.04
5.58
32.86
33.83
30.20
27.03
10.49
5.17
The hydration level of enzyme dominates protein exibility
and then inuences enzyme performance. The amount of water
1.20
ꢀ0.33
ꢀ1.00
ꢀ1.10
33
2.78
1.60
associated with enzyme can be controlled by water activity. As
shown in Fig. 7a, the enzyme activity gradually increases with
increasing aw in the range of 0.04–0.58, and then decreasing
1
,4-Dioxane
4.20
a
Reactions were carried out in different solvents (10 ml) with 1,1,1-
triuoro-2-octanol (2 mmol), vinyl acetate (5 mmol), the encapsulated sharply from a
w
0.58 to 0.91. At low a (0.04–0.13), low enzyme
w
ꢁ
lipase (50 mg) and water activity (0.58) at 50 C.
activity is observed owing to the absence of “necessary water” on
the surface of lipase which contributes to form favorable
conformation of enzyme for achieving transesterication reac-
tion. At moderate a values (0.13–0.58), water allows sufficient
w
enzyme exibility for catalytic activity. Along with the further
between temperature and enzyme performance of encapsulated
32
lipase in resolution of 1,1,1-triuoro-2-octanol was examined in
ꢁ
the range of 30–65 C. It can be found that enzyme activity
increase of a (0.58–0.91), the aggregation between lipases may
w
exhibits a bell shaped curve with changing temperature and
occur, which shall block the access of active site of lipase for
ꢁ
maximum enzyme activity is observed at 50 C (Fig. 6a). When
1,1,1-triuoro-2-octanol so as to reduce enzyme activity.
considering enantioselectivity, it can be found that E value
continuously decreases with the increase of temperature
Another possible explanation is as follows: when the “necessary
water” of lipase approaches to saturation, surplus water acting
as competing nucleophile attack acyl–enzyme complex to
(Fig. 6b), which is in agreement with Phillips' viewpoint that the
highest enantioselectivity can be gained at low temperature due
30
to enthalpic control. Because encapsulated lipase has the
ꢁ
ꢁ
highest activity and a good enantioselectivity at 50 C, 50 C was
selected as optimal reaction temperature for further study.
For non-aqueous enzymology, the enzyme performance is
31
oen affected by the polarity of organic solvents. In this study,
the activity and enantioselectivity of encapsulated lipase in
organic solvents with different log P (ꢀ1.1–4.6) were investi-
gated to optimize the reaction condition. As shown in Table 1,
the activity and enantioselectivity of the encapsulated lipase
increase with the increase of log P. Generally speaking, the
hydrophilic solvent may disrupt optimal enzyme conformation
and affect enzyme performance by interacting with the active
site of enzyme directly or stripping off the essential water from
32
enzyme. In this study, the highest enzyme activity and
enantioselectivity of encapsulated lipase were obtained when
n-hexane was used as the organic media.
We also screened the optimum acyl donor in this study and
the results were listed in Table 2. The highest enantioselectivity
of encapsulated lipase was obtained when vinyl butyrate was
a
Table 2 Effect of acyl donor on enantioselectivity transesterification
Enzyme activity
ꢀ
1
ꢀ1
Acyl donor
(mmol g min
)
E value
Vinyl acetate
20.32
14.72
11.25
8.90
33.83
42.84
43.88
40.40
35.43
Vinyl propionate
Vinyl butyrate
Vinyl valerate
Vinyl caproate
Fig. 7 Effect of water activity on the enzyme activity (a) and the
enantioselectivity (b) of the encapsulated lipase in resolution of 1,1,1-
trifluoro-2-octanol. Reactions were carried out in n-hexane (10 ml)
6.73
a
Reactions were carried out in n-hexane (10 ml) with 1,1,1-triuoro-2-
octanol (2 mmol), the encapsulated lipase (50 mg), water activity with 1,1,1-trifluoro-2-octanol (2 mmol), vinyl acetate (5 mmol), the
ꢁ
ꢁ
(0.58) and different acyl donors (5 mmol) at 50 C.
encapsulated lipase (50 mg) and water activity (0.04–0.91) at 50 C.
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Table 3 Resolution of 1,1,1-trifluoro-2-octanol in repeated batch interaction between lipase and silica nanoparticles becomes
a
process by encapsulated lipase in the aggregated silica nanoparticles
inevitable. The hydrophobic interactions between hydrophobic
area of lipase and the hydrophobic group of the matrices may
Enzyme activity
(mmol g min
ꢀ1
ꢀ1
cause interfacial activation of encapsulated lipase which can
induce the formation of the active open-lid conformation and
increase the enzyme performance of lipase. The hydrophobic
group of the matrices mainly originates from the methyl group
of dimethoxydimethylsilane.
Batch
)
E value
1
2
3
4
5
20.32
20.32
20.31
20.23
20.18
33.83
33.83
33.50
33.34
33.17
a
Conclusions
Reactions were carried out in n-hexane (10 ml) with 1,1,1-triuoro-2-
octanol (2 mmol), vinyl acetate (5 mmol), the encapsulated lipase
ꢁ
(50 mg) and water activity (0.58) at 50 C.
In this study, the lipase from Pseudomonas sp. was encapsulated
into the aggregated silica nanoparticles by lyophilization and
successfully used for resolution of 1,1,1-triuoro-2-octanol.
Almost no leakage phenomenon takes place during continuous
batch operations. Furthermore, these high-quality silica nano-
particles can be easily synthesized. These results prove that the
new encapsulation method has a great potential for application.
inhibit acyl transfer reactions. The results also demonstrate that
E value is not signicantly inuenced by the changes of a
w
34
(Fig. 7b), which is similar to the results in published literature.
Reusability
Acknowledgements
It's known that one of the best advantages of the immobilized
enzyme is its low cost in practical application by reuse. There-
fore, the reusability of the encapsulated lipase deserves further
research. As shown in Table 3, the encapsulated lipase remains
We appreciate help from group members Yifan Wang, Xiang Li,
Xue Wang, Ye Zhang for the characterization and synthesis of
the silica nanoparticles. This work was supported by the
National High Technology Research and Development Program
of China (2006AA02Z232 and 2012AA022202B), the National
Natural Science Foundation of China (Grant no. 31070708,
99.5% of its initial activity as well as 98.2% of initial enantio-
selectivity even aer ve runs. Furthermore, almost no leakage
phenomenon has been detected during continuous batch
operations. The perfect performance may attribute to the size
matching between the lipase and the interstices in aggregated
silica nanoparticles matrices.
21171064 and 21071059).
Notes and references
Free lyophilized lipase vs. the encapsulated lipase
1
R. C. Rodrigues, C. Ortiz, A. Berenguer-Murcia, R. Torres and
R. Fernandez-Lafuente, Chem. Soc. Rev., 2013, 42, 6290–6307.
E. Magner, Chem. Soc. Rev., 2013, 42, 6213–6222.
As shown in Table 4, the enzyme activity and E value of
encapsulated lipase were 1.5 and 2 times as much as that of free
lipase, respectively. It's known that most lipases can show
interfacial activation which induce the appearance of the active
2
3
M. Hartmann and X. Kostrov, Chem. Soc. Rev., 2013, 42,
6
277–6289.
U. Hanefeld, L. Gardossi and E. Magner, Chem. Soc. Rev.,
009, 38, 453–468.
35
open-lid conformation. Furthermore, as suggested by Roberto
and coworkers, bimolecular structure of lipase from Pseudo-
monas uorescens (PFL) can also enhance the enzyme activity by
4
2
5
6
7
B. Saha, J. Saikia and G. Das, RSC Adv., 2013, 3, 7867–7879.
M. Hartmann, Chem. Mater., 2005, 17, 4577–4593.
A. Vinu, V. Murugesan and M. Hartmann, J. Phys. Chem. B,
36
mutual interfacial activation. However, if larger enzyme
aggregation forms, the enzyme activity shall be inhibited. In
this study, PSL was encapsulated into the small interstice of
stacked silica nanoparticles aer lyophilization, which can
reduce the larger enzyme aggregation. Furthermore, the
2004, 108, 7323–7330.
8
9
A. Vinu, C. Streb, V. Murugesan and M. Hartmann, J. Phys.
Chem. B, 2003, 107, 8297–8299.
T. Y. Klein, L. Treccani, J. Thoming and K. Rezwan, RSC Adv.,
2
013, 3, 13381–13389.
0 M. Dong, Z. Wu, M. Lu, Z. Wang and Z. Li, Int. J. Mol. Sci.,
012, 13, 11443–11454.
Table 4 Enzyme activity and E value of lyophilized and encapsulated
lipase
1
1
1
a
2
´
1 R. C. Rodrigues, A. Berenguer-Murcia and R. Fernandez-
Lafuente, Adv. Synth. Catal., 2011, 353, 2216–2238.
2 C. Garcia-Galan, A. Berenguer-Murcia, R. Fernandez-
Lafuente and R. C. Rodrigues, Adv. Synth. Catal., 2011, 353,
Enzyme activity
ꢀ
1
ꢀ1
Enzyme
(mmol g min
)
E value
´
Free lipase
Encapsulated lipase
13.48
20.32
16.87
33.83
2
885–2904.
a
For encapsulated enzyme, the experimental data came from the 13 C. Mateo, J. M. Palomo, G. Fernandez-Lorente, J. M. Guisan
results of reusability; for lyophilized lipase, reactions were carried out
in n-hexane (10 ml) with 1,1,1-triuoro-2-octanol (2 mmol), vinyl
and R. Fernandez-Lafuente, Enzyme Microb. Technol., 2007,
4
14 B. Krajewska, Enzyme Microb. Technol., 2004, 35, 126–139.
0, 1451–1463.
acetate (5 mmol), lyophilized lipase (50 mg) and water activity (0.58)
ꢁ
at 50 C.
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1
1
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