Resolution of 2-octanol via immobilized Pseudomonas sp. lipase in organic medium

Article abstract of DOI:10.3109/10242422.2010.551189

Pseudomonas sp. lipase (PSL) immobilization was performed using three different protocols. Lipase immobilized on Diaion HP20 (HP20-PSL) exhibited the highest catalytic activity and stability in the kinetic resolution of racemic 2-octanol. The reaction rate of HP20-PSL was approximately 20 times higher than that of free PSL and the residual activities of HP20-PSL and free PSL were respectively 84% and 19% after incubation in the reaction medium for 72 h. A study of the effect of different reaction parameters on HP20-PSL-catalyzed resolution of (R,S)-2octanol showed that the optimal water content of the immobilized matrix and the optimal molar ratio of vinyl acetate to 2-octanol were 60 ± 5% and 2.5:1, respectively. Under the optimized reaction conditions, (S)-2-octanol of high optically purity (enantiomeric excess > 99%) could be recovered at 53% conversion rate, and HP20-PSL could be reused for ten cycles without significant decrease in its activity and enantioselectivity.

Full text of DOI:10.3109/10242422.2010.551189

Biocatalysis and Biotransformation, March–June 2011; 29(2–3): 47–53
ORIGINAL ARTICLE
Resolution of 2-octanol via immobilized Pseudomonas sp. lipase in
organic medium
L. F. ZHAO1,2 & L.Y. ZHENG1
1
Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University,
2
Changchun, People’s Republic of China and Institute of Medicinal Plant Development, Chinese Academy of
Medical Sciences and Peking Union Medical College, Beijing, People’s Republic of China
Abstract
Pseudomonas sp. lipase (PSL) immobilization was performed using three different protocols. Lipase immobilized on Diaion
HP20 (HP20-PSL) exhibited the highest catalytic activity and stability in the kinetic resolution of racemic 2-octanol.
The reaction rate of HP20-PSL was approximately 20 times higher than that of free PSL and the residual activities of
HP20-PSL and free PSL were respectively 84% and 19% after incubation in the reaction medium for 72 h. A study of
the effect of different reaction parameters on HP20-PSL-catalyzed resolution of (R,S)-2octanol showed that the optimal
water content of the immobilized matrix and the optimal molar ratio of vinyl acetate to 2-octanol were 60 Ϯ 5% and 2.5:1,
respectively. Under the optimized reaction conditions, (S)-2-octanol of high optically purity (enantiomeric excess Ͼ 99%)
could be recovered at 53% conversion rate, and HP20-PSL could be reused for ten cycles without significant decrease in
its activity and enantioselectivity.
Keywords: Pseudomonas sp. Lipase, immobilization, 2-octanol, kinetic resolution
Introduction
tive acetylation with vinyl acetate as the acyl donor
Figure 1) (Wang et al. 2003). However, free lipase
(
Owing to their extreme versatility with respect to
substrate specificity and stereoselectivity, lipases
have been used in the past few years for the kinetic
resolution of racemic substrates (Jaeger et al. 1997;
Alcantara et al. 1998). They can catalyze hydrolysis
and synthesis reactions of the water-insoluble oily
substances that are encountered in many processes
has some disadvantages in the catalyzed resolution,
such as: (1) slow reaction rate (50% conversion
requires 5 days) and moderate enantioselectivity; (2)
insufficient stability (easily deactivated at elevated
temperatures and in organic media); and (3) difficult
recovery and reuse. Immobilization is an effective
method to overcome these problems.
(Stamatis et al. 1999; Jaeger & Eggert 2002).
Homochiral 2-octanol is an important organic
The extent of stabilization and the catalytic effi-
ciency of immobilized catalysts depend on the struc-
ture of the enzyme, the immobilization method and
the type of support. Many immobilization methods
have been used to produce heterogeneous enzyme
catalysts, usually as a means to enable the use of
continuous reactor systems to allow easy separation
of the biocatalyst from the reaction mixture and sub-
sequent recycling (Mateo et al. 2007). In the present
study two distinct immobilization methods were
used: adsorption on solid supports and cross-linked
enzyme aggregates.
intermediate that has been widely applied in the syn-
thesis of optically active compounds and functional
materials (Patel 2001). The homochiral alcohols are
currently produced via hydrolysis, transesterification
or esterification. Among the various methods, trans-
esterification catalyzed by lipase in an organic
medium is widely used today because of its conve-
nience (Akoh & Yee 1998; Högberg et al. 2000).
Enantiomerically pure (S)-2-octanol (enantiomeric
excess Ͼ98%) can be successfully obtained by
Pseudomonas sp. lipase (PSL)-catalyzed enantioselec-
Correspondence: L.Y. Zheng, Key Laboratory for Molecular Enzymology and Engineering of Ministry of Education, Jilin University, Changchun 130023,
People's Republic of China. Tel: ϩ86-431-88498972. Fax: ϩ86-431-88980440. E-mail: lyzheng@jlu.edu.cn
(
Received 9 September 2010; revised 15 November 2010; accepted 24 December 2010)
ISSN 1024-2422 print/ISSN 1029-2446 online © 2011 Informa UK, Ltd.
DOI: 10.3109/10242422.2010.551189

48 L. F. Zhao & L.Y. Zheng
Figure 1. Kinetic resolution of (R,S)-2-octanol catalyzed by PSL.
Adsorption is one of the simplest methods of
Experimental
physical immobilization, with the advantages of being
inexpensiveand‘mild’towardstheenzyme(Pencreac'h
et al. 1997; Wilson et al. 2006). This method profits
from the interactions between the support surface
and the outer shell of the enzyme. In order to prepare
biocatalysts with high enzyme loading and activity
retention, inorganic and organic porous materials
have been used as supports for PSL immobilization
Materials
PSL 2500 U g–1 was purchased from Amano Phar-
maceutical Co., Ltd (Nagoya, Japan). Tributyrin was
purchased from Acros Organics (Thermo Fisher Sci-
entific, Geel, Belgium). Diaion HP20 was donated by
Resindion SRL (Mitsubishi Chemical Corporation,
Milan, Italy). Molecular sieve SBA-15 was donated
by the Jilin University Hi-Tech Co., Ltd (Changchun,
China). Glutaraldehyde solution (25% v/v in water)
was purchased fromTianjin Chemical Reagent (Tian-
jin, China). R-(ϩ)-Phenylethyl isocyanate (R-(ϩ)-
PEIC) was obtained from Sigma (Beijing, China).
Racemic 2-octanol and vinyl acetate were purchased
fromThe First Reagent Factory of Shanghai (China).
Other reagents were all analytical grade or better, and
were used without further purification.
(Cao 2005). In such cases, immobilized lipases can
act on the fraction of substrate that is soluble in the
reaction medium and is able to penetrate the porous
structure of the immobilized biocatalyst. Moreover,
fully dispersed, immobilized lipase molecules cannot
undergo lipase–lipase or lipase–protein interactions.
Supporting lipases on porous materials offers an
additional advantage when operating in organic
media: the adsorption properties and alterable
hydrophilicity/hydrophobicity of the supports might
partly modulate the reaction microenvironment for
optimal enzymatic activity (Serralha et al. 2004;
Sheldon et al. 2005).
Cross-linked enzyme aggregates (CLEAs) are
self-supported insoluble biocatalysts that have shown
great potential in a number of biocatalytic applica-
tions (Cao et al. 2002). The preparation of CLEAs
is also a simple technique (Van Langen et al. 2005);
it has many advantages, such as highly concentrated
enzyme activity in the catalyst, high stability and
low production cost due to the exclusion of an addi-
tional carrier (Schoevaart et al. 2004). Another
important feature is that it is not necessary to start
with a purified enzyme preparation to make CLEAs.
However, the process results in a certain amount of
wasted enzyme material because a part of the enzyme
functions merely as a support and has no actual
catalytic activity.
Herein, three PSL derivatives (SBA-15-PSL,
HP20-PSL and CLEA-PSL) were prepared via dif-
ferent immobilization methods. The catalytic activ-
ity and stability of the different preparations were
measured in the kinetic resolution of 2-octanol.
Then, effects of reaction parameters on the HP20-
PSL-catalyzed resolution of 2-octanol, such as
water content and substrate molar ratio, were fur-
ther studied. Finally, the reusability of HP20-PSL
was investigated and compared with that of free
lipase.
Preparation of SBA-15-immobilized PSL
Lipase PSL (20 mg enzyme powder) was dissolved
in 5.0 mL phosphate buffer (25 mM, pH 8.0).The
support SBA-15 (0.3 g) was suspended in the
lipase solution. The mixture was stirred at 4°C
until adsorption equilibrium was established. The
solid materials were collected by centrifugation,
washed with five volumes of phosphate buffer (pH
8.0) and the final immobilized lipase was dried in air
or lyophilized.
Preparation of HP20-immobilized PSL
HP20 beads (0.1 g) were soaked in 75% (v/v) ethanol
for 12 h and then washed with ten volumes of deion-
ized water to remove residual ethanol. Lipase PSL
(20 mg powder) was dissolved in 2 mL phosphate
buffer (50 mM, pH 7.0). The treated HP20 beads
were suspended in the lipase solution and shaken for
2 h at 4°C. After incubation, the beads were washed
with phosphate buffer (pH 7.0) and the interparticle
water was eliminated by vacuum filtering.The result-
ing beads were stored at 4°C.
Preparation of cross-linked enzyme aggregates
Lipase PSL (30 mg powder) was dissolved in 1 mL
phosphate buffer solution (100 mM, pH 7.0) in a

Resolution of 2-octanol via immobilized lipase 49
centrifuge tube. Acetone (5 mL) was added drop-
wise to the lipase solution with gentle stirring at 4°C
for 2 h. Next a total of 50 μL glutaraldehyde
of the injector and the detector were 200 and 290°C,
respectively.The conversion was calculated from the
disappearance of the substrate and the enantiomeric
ratio (E) was calculated according to the equation
(Rotticci 2000):
(
25%(w/v) in water) was added to the mixture, which
was stirred continuously at 25°C for another 2 h and
then centrifuged at 5000 rpm for 10 min.The super-
natant was decanted and the residue washed with
acetone more than three times (5 mL each time)
until no more activity was detected in the superna-
tant.The final enzyme preparations were lyophilized
and stored at 4°C.
E ϭ ln[(1 Ϫ C)(1 Ϫ ee)]/ln[(1 ϭ C)(1 ϩ ee)].
Results and discussion
Immobilization of PSL
Lipase PSL was immobilized using carrier-bound
and carrier-free methods to give SBA-15-immobi-
lized PSL (SBA-15-PSL), HP20-immobilized PSL
(HP20-PSL) and cross-linked enzyme aggregates
of PSL (CLEA-PSL). The immobilization condi-
tions for the three procedures had been optimized
previously (unpublished results) and the immobiliza-
tion efficiency and activity retention of the immobi-
lized lipases were determined by the hydrolysis of
tributyrin (Table I).
Assay of lipase activity
The activity of the lipase was determined by titrating
the butyric acid produced by the hydrolysis of tribu-
tyrin (Secundo et al. 1999). A total of 100 μL tribu-
tyrin and 6.0 mL phosphate buffer solution (25 mM,
pH 7.5) were mixed and placed in a bath maintained
at 37°C. A total of 5 mg free PSL powder or 20 mg
immobilized PSL was added and stirred sufficiently
for 15 min.The extent of the reaction was monitored
by titrating the produced butyric acid with 0.05 M
NaOH using a pH-stat. Similarly, a blank experi-
ment without added enzyme was carried out using
the same assay procedure. Enzyme activity was
determined according to the amount of alkali con-
sumed. One unit (U) of lipase is defined as the
amount of enzyme required to produce 1 μmole of
free fatty acid per minute at 37°C.
The adsorption efficiency and activity of HP20-
PSL were evidently higher than those of SBA-
15-PSL; the highest immobilization efficiency and
activity retention were respectively 90.8% and
82.4%. The results might have a close relationship
with the pore sizes and surface characteristics of the
two supports. The pore diameter of SBA-15 was
close to the size of PSL (MW ϭ 31 kDa, average
molecular diameter about 5 nm). During immobili-
zation, the size of PSL is probably more than 5 nm
considering the shell of hydration and the conforma-
tional freedom; therefore, enzyme molecules were
possibly adsorbed on the outer surface of the sup-
port, resulting in low enzyme loading. Diaion HP20
is a macroporous styrene–divinylbenzene copolymer
having a hydrophobic matrix from the cross-linking
of hydrophobic monomers. The pore diameter of
HP20 was sufficiently large for PSL molecules to
enter the channels. PSL was immobilized on HP20
by hydrophobic interaction that, possibly, made the
lipase adopt a more active conformation. The lowest
activity retention was observed for CLEA-PSL, which
might be due to partial deactivation of the lipase
resulting from the addition of acetone and blockage
of the lipase active site caused by cross-linking during
CLEA preparation (Shah et al. 2006).
Lipase-catalyzed resolution of (R,S)-2-octanol
The same amount (200 U) of free PSL or immobi-
lized lipase was dispersed in 10 mL n-hexane con-
taining racemic 2-octanol (1 mmol), vinyl acetate
(2 mmol) and water activity (0.56). The reaction
vials were shaken at 160 rpm at 50°C and samples
were withdrawn periodically for analysis by GC.
Determination of conversion and enantiomeric excess
The degree of conversion (C) and the enantiomeric
excess (ee) of 2-octanol were determined by GC
using a 30 m ϫ 0.32 mm ϫ 0.25 μm Econo-CAP
SE54 capillary column in a Shimadzu gas chromato-
graph (GC-2014; Kyoto, Japan) with nitrogen as car-
–1
rier gas at 60 mL min and flame ionization detection.
The (R)-2-octanol or (S)-2-octanol was derivatized
with the chiral reagent (R-(ϩ)-PEIC) and distinction
of the R- from the S-enantiomer was achieved by tem-
perature programming between 110 and 222°C. The
ee of (S)-2-octanol was determined by calculating the
peak areas of the two derivatives. The temperatures
Catalytic performances of different PSL derivatives
In order to select the optimal PSL preparation for
production of (S)-2-octanol,the catalytic performance
of the different derivatives was analyzed under the
optimized conditions suggested byWang et al. (2004)

50 L. F. Zhao & L.Y. Zheng
Table I. Properties of the supports and a comparison of the immobilized lipases prepared with different methods.
Average pore
diameter (nm)
Specific surface
Pore volume
Particle size
range (μm)
Immobilization
efficiency (%)
Activity
retention (%)
2
Ϫ1
3
Ϫ1
Activity (U gϪ1
Sample
area (m g
)
(cm g
)
)
SBA-15
HP20
CLEA
6.2
50
–
840
600
–
1.2
1.3
–
50 – 100
250 – 400
–
387
1050
533
60.9
90.8
70.6
50.2
82.4
45.1
The lipase was immobilized under the following optimized conditions. SAB-15-PSL: PSL 20 mg support 0.3 g, 25 mM phosphate buffer,
pH 8.0; HP20-PSL: PSL 20 mg, support 0.1 g, 50 mM phosphate buffer, pH 7.0; CLEA-PSL: PSL 30 mg, acetone 5 mL, 50 μL
glutaraldehyde (25% w/v in water), 100 mM phosphate buffer, pH 7.0.
and Yu et al. (2007). The time courses of free and
immobilized lipases were tested in the kinetic resolu-
tion of (R,S)-2-octanol (Figure 2). PSL exhibited
higher catalytic activity after immobilization and the
times of reaching the balance point in the immobi-
lized lipase-catalyzed reaction were clearly shortened
compared with that of free lipase. HP20-PSL needed
only 1 day for conversions greater than 50%, whereas
during the whole process.This effectively reduced the
transport limitation of substrate and product, giving
HP20-PSL the highest catalytic efficiency.
Furthermore, the enantioselectivity of the dif-
ferent PSL preparations in the kinetic resolution
of (R,S)-2-octanol was determined (Table II). The
immobilized lipases exhibited similar enantioselec-
tivity to the free lipase. In the transesterification,
high enantiomeric excess of (S)-2-octanol (Ͼ98%)
could be obtained when conversions were greater
than 55%.
free lipase needed 5 days. The initial velocities (V ,
0
1
μmol minϪ ) of the various PSL preparations were
.08 (free PSL), 0.72 (SBA-15-PSL), 1.03 (CLEA-
0
PSL) and 1.58 (HP20-PSL); thus the rate of the
PSL-catalyzed reaction improved approximately
Stability of different PSL derivatives in the
reaction medium
20-fold after HP20 immobilization. This may
explained by the increased PSL dispersal through
immobilization, which decreases the chance of free
enzyme convergence in organic media and improves
the accessibility of the substrate to the enzyme's
active site.
SBA-15-PSL and CLEA-PSL displayed slight
aggregation over the course of the reaction, whereas
HP20-PSL was fully dispersed in the reaction medium
PSL-catalyzed resolution of (R,S)-2-octanol was
studied in different organic media; the highest activ-
ity and E value were achieved when n-hexane was
used as solvent (Wang et al. 2004; Yu et al. 2007).
The stability of free and immobilized lipases in
n-hexane was investigated for 72 h while periodically
measuring enzyme activity. Relative activity in this
experiment was calculated by taking the initial activ-
ity as 100%. As shown in Figure 3, the stability of
PSL derivatives diminished to a certain extent with
time and free PSL started losing activity after 5 h.
Nearly 50% activity was lost at 30 h, whereas the
activity of immobilized lipases was fully retained
for up to 18 h (HP20 retained total activity even at
7
6
5
0
0
0
4
0
24 h). After 72 h, free PSL had lost 81% of its activ-
ity, whereas HP20-PSL, SBA15-PSL and CLEA-
PSL retained 84%, 72% and 69% of their initial
activity, respectively. Thus the stability of PSL in
3
2
1
0
0
0
Table II. Resolution of (R,S)-2-octanol catalyzed by free and
immobilized lipases.
Reaction
time (h)
Conversion,
Sample
C (%)
eeS (%)
E value
0
0
30
60
Time (h)
90
120
150
Free
144
72
56
55.0
54.9
55.4
54.0
98.2
99.0
99.0
99.0
42.1
49.9
45.4
60.9
SBA-15
CLEA
HP20
Figure 2.The time course for kinetic resolution of (R,S)-2-octanol
catalyzed by free (— —) and immobilized PSL (—ᮀ—, SBA-15;
30
—
—, CLEA; —∗—, HP20). Reaction conditions: n-hexane, 10
mL; (R,S)-2-octanol, 1 mmol; vinyl acetate, 2 mmol; water activity,
.56; free or immobilized lipase, 200 U; temperature, 50°C.
Reaction conditions: n-hexane, 10 mL; (R,S)-2-octanol, 1 mmol;
vinyl acetate, 2 mmol; water activity, 0.56; free or immobilized
lipase, 200 U; temperature, 50°C.
0

Resolution of 2-octanol via immobilized lipase 51
1
1
20
00
60
90
8
7
0
0
50
40
30
8
0
0
0
0
60
5
4
0
0
6
4
2
2
1
0
0
30
2
1
0
0
0
0
0
0
10
20
30
40
50
60
70
80
1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0
Time (h)
Substrate molar ratio
Figure 3. Effect of incubation time in n-hexane on enzymatic
activity of free (— —) and immobilized PSL (—ᮀ—, SBA-15;
Figure 4. Effect of substrate molar ratio (vinyl acetate to 2-octanol)
on the activity (conversion; — —) and enantioselectivity (E value;
—
—, CLEA; —∗—, HP20). Relative activity was calculated by
—
•—) of HP20-PSL in the kinetic resolution of (R,S)-2-octanol.
taking the initial activity as 100%.
Reaction conditions: n-hexane, 10 mL; (R,S)-2-octanol, 1 mmol;
vinyl acetate, 1–5 mmol; HP20-PSL, 200 U; temperature, 50°C;
time, 24 h.
organic media obviously improved during immobili-
zation, especially for HP20-PSL, which exhibited the
highest stability in the reaction medium.After immo-
bilization a fraction of the enzyme is protected, which
reduces the deactivation of the enzyme caused by
organic solvents, resulting in enhanced stability.
The results indicated that Diaion HP20 was the
optimum support for PSL immobilization and that
HP20-PSL displayed the highest transesterification
activity and stability for the kinetic resolution of
dramatically influence the activity of the enzyme
(Valivety et al. 1992, 1993). In the present study,
water activity could be controlled by the addition of
salts or salt hydrates to the organic medium or sub-
strate, as described by Halling and co-workers (Kvit-
tingen et al. 1992; Halling 1994; Zacharis et al. 1997).
For lipase immobilized on the macroporous resin,
water activity of the reaction system could be modu-
lated by changing the water content of the immobi-
lization matrix. This not only simplified the reaction
procedure, but also avoided the negative influence of
heterogeneous compounds on catalysis. HP20-PSL
with different water content was used in the kinetic
resolution of (R,S)-2-octanol and the reaction course
was studied (Table III). The catalytic activity of
HP20-PSL improved significantly when the water
level was increased from 12% to 60%; the optimum
water content of HP-20-PSL was 60 Ϯ 5%. However,
excessive water in the immobilization matrix resulted
in an evident decrease of enzyme activity.The E value
of HP-20-PSL was maintained in almost all cases,
and the water content of immobilized PSL did not
markedly influence the enantioselectivity of the lipase
as previously reported (Bovara et al. 1993).
(R,S)-2-octanol. Thus HP20-PSL was selected for
further investigation.
Effect of reaction conditions on the HP20-
PSL-catalyzed resolution of (R,S)-2-octanol
Substrate ratio.The rate of an enzyme-catalyzed reac-
tion depends on the concentrations of enzyme and
substrate. The effect on the transesterification of
varying the molar ratio of vinyl acetate to 2-octanol
from 1:1 to 5:1 was studied (Figure 4). The results
showed that the catalytic activity of HP20-PSL
improved from 41.3% to 56.8% when the concentra-
tion of the acyl donor was increased and the opti-
mum substrate molar ratio was 2.5:1, which was
lower than that of free PSL (Wang et al. 2004; Yu
et al. 2007). This may reflect the changes in confor-
mation and microenvironment of the enzyme in the
reaction system after immobilization.The enantiose-
lectivity of HP20-PSL did not change significantly
with increasing molar ratio.
Under the optimized reaction conditions, the
residual (S)-2-octanol catalyzed by HP20-PSL was
recovered with high enantiomeric excess (Ͼ99%) at
53% conversion after 24 h.
Reuse of HP20-PSL
Water content of HP20-PSL. In non-aqueous enzy-
mology, water plays an important role because it can
Efficiency of reuse is an important factor for evaluat-
ing immobilized catalysts in industrial applications.

52 L. F. Zhao & L.Y. Zheng
Table III. Effect the water content of HP20-PSL on catalyzed
resolution of (R,S)-2-octanol.
The results indicated that HP20-PSL had potent
operational stability and reusability in the kinetic
resolution of 2-octanol, although a decrease of activ-
ity with increasing recycle instances was eventually
observed. This may be a result of deactivation and
leakage of the lipase during long-playing continuous
reactions in organic media.
Reaction Conversion,
Water content (%) time (h)
C (%)
eeS (%) E value
12 Ϯ 1
25 Ϯ 2
45 Ϯ 2
60 Ϯ 5
72 Ϯ 6
72
56
30
24
36
44.8
50.7
51.9
53.0
47.6
76.2
94.1
97.0
99.6
85.3
73.1
80.3
78.7
98.2
87.0
Conclusions
Reaction conditions: n-hexane, 10 mL; (R,S)-2-octanol, 1 mmol;
vinyl acetate, 2.5 mmol; HP20-PSL, 200 U; temperature, 50°C.
Pseudomonas sp. lipase was immobilized using three
different protocols, and HP20 was selected as the
optimal support. The resultant HP20-PSL was
used for the kinetic resolution of racemic 2-octanol
and the effects of reaction parameters on the cata-
lytic performance of HP20-PSL were further
investigated. The results show that catalytic activ-
ity, stability and reusability of HP20-PSL in non-
aqueous media are greatly superior to those of free
PSL; a slight increase of E value (from 42 to 98)
was also observed. Under the optimized reaction
conditions, enantiomerically pure (S)-2-octanol
Therefore, the reusability of HP20-PSL in the repeated
batch resolution of (R,S)-2-octanol was investigated
and compared with that of free lipase. Reactions using
the same amount (200 U) of free and immobilized
lipases in 10 mL n-hexane containing (R,S)-2-octanol
(1 mmol) and vinyl acetate (2.5 mmol) were carried
out at 50°C.The free or immobilized PSL in each case
was filtered and washed after each use, then re-
suspended in a fresh reaction mixture to measure
conversion and enantiomeric excess of (S)-2-octanol.
Residual activity was calculated by taking the enzyme-
catalyzed activity of the first cycle as 100%.The relative
activity of free and immobilized PSL after recycling for
ten times is given in Figure 5, which shows that the
durability of PSL was remarkably improved after being
immobilized on HP20. After four cycles, free PSL lost
its entire activity, whereas HP20-PSL retained its initial
activity until the seventh batch. HP20-PSL lost only
(
Ͼ99%) could be obtained at 53% conversion with
HP20-PSL as catalyst. In conclusion, HP20-PSL
has considerable promise as an immobilized bio-
catalyst for the production of (S)-2-octanol. How-
ever, the enantioselectivity of HP20-PSL may still
be improved by additives or modification of the
enzyme and support. Further work will be reported
in due course.
3.2% of its initial activity after ten cycles. Notably, the
enantiomeric excess of (S)-2-octanol was maintained
at 99% throughout the ten repeated reactions.
Declaration of interest: The authors are grateful
for financial support from the National Natural Sci-
ence Foundation of China (No.20432010,20672045
and 30570405), China Postdoctoral Science Foun-
dation (No. 20060400896) and Jilin Provincial Sci-
ence & Technology Sustentation Program (No.
1
1
20
00
2
0070553).The authors report no conflicts of inter-
8
6
4
2
0
0
0
0
0
est.The authors alone are responsible for the content
and writing of the paper.
References
Alcantara AR, De Fuentes IE, Sinisterra JV. 1998. Rhizomucor
miehei lipase as the catalyst in the resolution of chiral com-
pounds: an overview. Chem Phys Lipids 93:169–184.
Akoh CC, Yee LN. 1998. Lipase-catalyzed transesterification
of primary terpene alcohols with vinyl esters in organic media.
J Mol Catal B: Enzym 4:149–153.
Bovara R, Carrea G, Ottolina G, Riva S. 1993.Water activity does
not influence the enantioselectivity of lipase PS and lipoprotein
lipase in organic solvents. Biotechnol Lett 15:169–174.
Cao L. 2005. Immobilised enzymes: science or art? Curr Opin
Chem Biol 9:217–226.
1
2
3
4
5
6
7
8
9
10
11
Cycle number
Figure 5. Reusability of the free (— —) and HP20-immobilized
PSL (—•—). Relative activity was calculated by taking the initial
activity as 100%. Reaction conditions: n-hexane, 10 mL; (R,S)-
-octanol, 1 mmol; vinyl acetate, 2.5 mmol; free or immobilized
lipase, 200 U; temperature, 50°C.
Cao L, van Rantwijk F, Sheldon RA. 2002. Cross-linked enzyme
aggregates: a simple and effective method for the immobiliza-
tion of Penicillin acylase. Org Lett 2:1361–1364.
2

Resolution of 2-octanol via immobilized lipase 53
Halling PJ. 1994. Thermodynamic predictions for biocatalysis in
nonconventional media: theory, tests, and recommendations for
experimental design and analysis. Enzyme Microb Technol 16:
design to the study of an alcoholysis transformation promoted
by cutinase immobilized on NaY zeolite and Accurel PA6. J
Mol Catal B: Enzym 27:19–27.
1
78–206.
Shah S, Sharma A, Gupta MN. 2006. Preparation of cross-linked
enzyme aggregates by using bovine serum albumin as a proteic
feeder. Anal Biochem 351:207– 213.
Sheldon RA, Schoevaart R, van Langen LM. 2005. Cross-linked
enzyme aggregates (CLEAs): a novel and versatile method for
enzyme immobilization (a review). Biocatal Biotransformation
23:141–147.
Stamatis H, Xenakis A, Kolisis FN. 1999. Bioorganic reactions in
microemulsions: the case of lipases. Biotechnol Adv 17:293–318.
Valivety RH, Halling PJ, Macrae AR. 1992. Reaction rate with
suspended lipase catalyst shows similar dependence on water
activity in different organic solvents. Biochim Biophys Acta
1118:218–222.
Valivety RH, Halling PJ, Macrae AR. 1993.Water as a competitive
inhibitor of lipase-catalyzed esterification in organic media.
Biotechnol Lett 15:1133–1138.
Högberg HE, Lindmark M, Isaksson D, Sjödin K, Franssen
MCR, Jongejan H,Wijnberg JBPA, De Groot A. 2000. Forma-
tion of hemiacetal esters in lipase-catalysed reactions of vinyl
esters with hindered secondary alcohols. Tetrahedron Lett
4
1:3193–3196.
Jaeger KE, Eggert T. 2002. Lipases for biotechnology. Curr Opin
Biotechnol 13:390–397.
Jaeger KE, Schneidinger B, Rosenau F, Werner M, Lang D,
Dijkstra BW, Schimossek K, Zonta A, Reetz MT. 1997. Bacte-
rial lipases for biotechnological applications. J Mol Catal B:
Enzym 3:3–12.
Kvittingen L, Sjursnes B, Anthonsen T, Halling PJ. 1992. Use of
salt hydrates to buffer optimal water level during lipase cata-
lysed synthesis in organic media: a practical procedure for
organic chemists. Tetrahedron 48:2793–2802.
Mateo C, Palomo JM, Fernandez-Lorente G, Guisan JM, Fern-
andez-Lafuente R. 2007. Improvement of enzyme activity, sta-
bility and selectivity via immobilization techniques. Enzyme
Microb Technol 40:1451–1463.
Van Langen LM, Selassa RP,Van Rantwijk F, Sheldon RA. 2005.
Cross-linked aggregates of (R)-oxynitrilase: a stable, recyclable
biocatalyst for enantioselective hydrocyanation. Org Lett
7:327–329.
Patel RN. 2001. Biocatalytic synthesis of intermediates for the
synthesis of chiral drug substances. Curr Opin Biotechnol
Wang Z, Quan J, Weng L, Zheng LY, Liu M, Dong H, Cao SG.
2003. Improvement of properties of Pseudomonas sp. lipase in
resolution of 2-octanol. Chem Res Chin Univ 19:446–449.
Wang Z, Li ZQ, Yu DH, Weng L, Liu M, Zhang GR, Cao SG.
2004. Resolution of 2-octanol via lipase-catalyzed enantioselec-
tive acetylation in organic solvents. Chem Res Chin Univ
20:575–578.
Wilson L, Palomo JM, Fernandez-Lorente G, Illanes A, Guisan
JM, Fernandez-Lafuente R. 2006. Improvement of the func-
tional properties of a thermostable lipase from Alcaligenes sp.
via strong adsorption on hydrophobic supports. Enzyme Microb
Technol 38:975–980.
1
2:587–604.
Pencreac'h G, Leullier M, Baratti JC. 1997. Properties of free and
immobilized lipase from Pseudomonas cepacia. Biotechnol Bio-
eng 56:181–189.
Rotticci D. 2000. Understanding and engineering the enantiose-
lectivity of Candida antarctica lipase B towards sec-alcohols
[
PhD thesis]. Stockholm: Royal Institute of Technology.
Schoevaart R, Wolbers MW, Golubovic M, Ottens M, Kieboom
APG, Van Rantwijk F, Van der Wielen LAM, Sheldon RA.
2
004. Preparation, optimization, and structures of cross-linked
enzyme aggregates (CLEAs). Biotechnol Bioeng 87:754–762.
Secundo F, Spadaro S, Carrea G, Overbeeke PLA. 1999. Opti-
mization of Pseudomonas cepacia lipase preparations for catalysis
in organic solvents. Biotechnol Bioeng 62:554–561.
Serralha FN, Lopes JM, Lemos F, Ribeiro FR, Prazeres DMF,
Aires-Barros MR, Cabral JMS. 2004. Application of factorial
Yu DH,Wang Z, Zhao LF, ChengYM, Cao SG. 2007. Resolution
of 2-octanol by SBA-15 immobilized Pseudomonas sp. lipase.
J Mol Catal B: Enzym 48:64–69.
Zacharis E, Moore BD, Halling PJ. 1997. Control of enzyme
activity in organic media by solid-state acid–base buffers. J Am
Chem Soc 119:12396–12397.