Functional expression of Serratia marcescens H30 lipase in Escherichia coli for efficient kinetic resolution of racemic alcohols in organic solvents

Article abstract of DOI:10.1016/j.molcatb.2014.04.012

A lipase from Serratia marcescens H30 was cloned and functionally expressed in E. coli in soluble form (more than 80%). The recombinant lipase activity reached 25,000 U/L after optimization. Different carriers were used to immobilize the recombinant S. marcescens lipase (SmL), and LH-EP was found to be the most ideal carrier. LH-EP immobilized SmL could catalyze enantioselective resolution of racemic alcohols. Using 4-phenyl-2-butanol as the model alcohol, the effects of temperature, organic solvent, water activity, acyl donor and substrate molar ratio on the LH-EP immobilized SmL catalyzed kinetic resolution were investigated. All of these factors influenced the resolution effect to some extent. At last, a high E value of more than 200 was achieved, with ee _S > 99% and a conversion of 49.9%. Further studies showed that the LH-EP immobilized SmL could also catalyze the kinetic resolution of other structure similar racemic alcohols. These results indicate that the SmL is useful for biocatalytic production of chiral alcohols. This work is the first attempt of using SmL in this field, and it further broadens the application field of SmL.

Full text of DOI:10.1016/j.molcatb.2014.04.012

Journal of Molecular Catalysis B: Enzymatic 106 (2014) 11–16
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Functional expression of Serratia marcescens H30 lipase in
Escherichia coli for efficient kinetic resolution of racemic alcohols in
organic solvents
Erzheng Sua,∗, Jingjing Xub, Pengyong Youb
a Enzyme and Fermentation Technology Laboratory, College of Light Industry Science and Engineering, Nanjing Forestry University, Nanjing 210037, PR
China
b State Key Laboratory of Bioreactor Engineering, New World Institute of Biotechnology, East China University of Science and Technology, Shanghai 200237,
PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
A lipase from Serratia marcescens H30 was cloned and functionally expressed in E. coli in soluble form
(more than 80%). The recombinant lipase activity reached 25,000 U/L after optimization. Different carriers
were used to immobilize the recombinant S. marcescens lipase (SmL), and LH-EP was found to be the most
ideal carrier. LH-EP immobilized SmL could catalyze enantioselective resolution of racemic alcohols. Using
4-phenyl-2-butanol as the model alcohol, the effects of temperature, organic solvent, water activity,
acyl donor and substrate molar ratio on the LH-EP immobilized SmL catalyzed kinetic resolution were
investigated. All of these factors influenced the resolution effect to some extent. At last, a high E value
of more than 200 was achieved, with eeS > 99% and a conversion of 49.9%. Further studies showed that
the LH-EP immobilized SmL could also catalyze the kinetic resolution of other structure similar racemic
alcohols. These results indicate that the SmL is useful for biocatalytic production of chiral alcohols. This
work is the first attempt of using SmL in this field, and it further broadens the application field of SmL.
© 2014 Elsevier B.V. All rights reserved.
Received 17 February 2014
Received in revised form 17 April 2014
Accepted 19 April 2014
Available online 28 April 2014
Keywords:
Serratia marcescens H30
Lipase
Immobilization
Kinetic resolution
Chiral alcohols
1. Introduction
the kinetic resolution of diastereomeric and enantiomeric mixtures
of racemic alcohols [8]. Conversely to oxido-reductases, lipases are
Enantiomerically pure compounds have become the focus of
research today. They are important building blocks in the produc-
tion of fine chemicals for pharmaceuticals, agrochemicals, and food
ingredients [1]. In recent years, chiral polymers and chiral electron-
ics are a fast-growing market with a brand new range of attractive
products and applications [2]. Chiral alcohols are especially widely
used as intermediates for the introduction of chiral information into
the product [3]. Chiral alcohols have been prepared by asymmet-
ric hydrogenation of prochiral ketones using ruthenium complexes
[4] or boron compounds [5]. The asymmetric addition of diethylz-
inc to aldehydes in the presence of chiral ligands has also generated
a large number of enantiopure alcohols [6]. But in the last decades,
the interest for creating stereogenic centers by applying biocat-
alytic methods has risen [7]. The oxido-reductases can catalyze the
stereoselective reduction of prochiral ketones for synthesis of enan-
tiopure alcohols [3]. Lipase-catalyzed reactions have been applied
to solve a number of various synthetic problems, one of which is
serine hydrolases that do not require cofactors nor catalyze side
reactions [9].
Lipases are the most widely applied enzymes for the regiose-
lective and enantioselective biotransformations, because they are
inexpensive, stable, and easy to recycle [8]. They also possess wide
substrate specificity and catalyze many reactions in aqueous and
non-aqueous media [10]. Lipase catalyzed kinetic resolution of
racemic alcohols can be performed via acylation with different
acyl donors such as carboxylic acids, esters and anhydrides. To
date, a large variety of lipases have been reported for such pur-
poses. In particular, lipase B from Candida antarctica is the most
robust enzyme with high catalytic efficiency in the resolution.
However, the high cost of this immobilized lipase severely lim-
its its use for commercial purposes [11]. Serratia marcescens can
secrete an extracellular lipase. This lipase is stable in some organic
solvents and applicable to the asymmetric synthesis of chiral com-
pounds [12]. One well-known example is the production of (2R,
3S)-3-(4ꢀ-methoxyphenyl) glycidic acid methyl ester [(−)-MPGM],
a key intermediate for the synthesis of diltiazem hydrochlo-
ride, from trans-3-(4ꢀ-methoxyphynyl) glycidic acid methyl ester
[( )-MPGM] [13–15]. The other example is its use for kinetic
∗
Corresponding author. Tel.: +86 25 85428906; fax: +86 25 85428906.
E-mail address: ezhsu@njfu.edu.cn (E. Su).
http://dx.doi.org/10.1016/j.molcatb.2014.04.012
1381-1177/© 2014 Elsevier B.V. All rights reserved.

12
E. Su et al. / Journal of Molecular Catalysis B: Enzymatic 106 (2014) 11–16
resolution of racemic flurbiprofen or ketoprofen, giving optically
pure (S)-flurbiprofen or ketoprofen with very high enantioselec-
tivtiy [16,17]. Jaeger et al. tried to use this lipase for the kinetic
resolution of other racemic esters such as isopropylideneglycerol
acetate in organic solvent but the reactions were failed [18]. How-
ever, there is no report about its application in kinetic resolution of
racemic alcohols until today.
The lipase genes from S. marcescens had been cloned and
expressed in E. coli [17,19,20]. But most of S. marcescens lipase
(SmL) genes were expressed in the form of inclusion bodies, which
accounted for as much as 70% of the target protein [18,21]. Recently,
we found the S. marcescens H30, a Gram-negative organism previ-
ously used for efficient production of 2, 3-butanediol in our lab
[22], could produce lipase during the culture. In the present study,
we cloned and functionally expressed the lipase from S. marcescens
H30 in soluble form in E. coli. The kinetic resolution of racemic alco-
hols was carried out using the immobilized recombinant SmL for
the first time to evaluate its potential for industrial application.
was added to a final concentration of 0.5 mM for inducing the SmL
expression, and cultivation continued for another 10 h at 25 ◦C.
Cells were collected by centrifugation at 6000 × g for 10 min, resus-
pended and washed twice with 25 mM Tris–HCl buffer (pH 8.0).
The cells were disrupted by the ultrasonic cell disruptor (Ningbo
Scientz, China) in an ice bath at the power controlled from 200 to
400 W, and each circle worked 4 s and intermitted 5 s, continued
20 min. The disrupted cells were collected by centrifugation at
12,000 × g for 10 min, and the supernatants were used for SmL
activity and SDS-PAGE analysis.
2.4. Purification and immobilization of the recombinant SmL
The supernatant of the recombinant E. coli cell lysate was loaded
onto Ni-NTA agarose affinity resin (Qiagen, USA) pre-equilibrated
with buffer A (200 mM NaCl; 25 mM Tris–HCl, pH 8.0; 10 mM imid-
azole). Weakly bound protein was eluted from the column by
washing twice with buffer B (200 mM NaCl; 25 mM Tris–HCl, pH
8.0; 20 mM imidazole). Pure protein was eluted with elution buffer
C (200 mM NaCl; 25 mM Tris–HCl, pH 8.0; 80 mM imidazole). The
pure protein fractions were combined and ultra-filtrated by Amicon
Ultra-15 (10 K) centrifugation devices for removing imidazole.
All the carriers were treated as the suppliers’ instruction before
immobilization. Generally, different carriers (5 g) were added to
50 mL purified lipase solution in 250 mL screw capped glass vial,
and the mixtures were stirred at 25 ◦C and 200 rpm in an orbital
shaker. The protein concentration of the supernatant was deter-
mined at intervals. The immobilized lipase particles were filtered
and washed firstly with deionized water for three times, then thor-
oughly with 25 mM pH 8.0 Tris–HCl buffer. The immobilized lipases
were taken to assay their activities. The immobilization yield and
activity recovery were calculated.
2. Materials and methods
2.1. Materials
Restriction enzymes, Taq polymerase premix and T4 DNA
ligase were supplied by MBI Fermentas (Germany). The plasmid
extraction and DNA gel extraction were purchased from Generay
Biotech (Shanghai) Co., Ltd. Racemic ␣-phenethyl alcohol, 1-
phenyl-2-propanol, 2-phenyl-1-butanol and 4-phenyl-2-butanol
were bought from Alfa Aesar. LH-HA and LH-EP were donated
by Shanghai Bairui Biotech. Co., Ltd (China). Ion-exchange resins
D201, D301 and D315 were donated by HuaChang Polymer Co., Ltd
(Shanghai, China). Glutaraldehyde solution (25%) was purchased
from Sinopharm Chemical Reagent Co., Ltd (Shanghai, China).
Methanol, n-hexane and 2-propanol for HPLC analysis was bought
from Tedia. All other chemicals and reagents were obtained com-
mercially and of analytical grade.
2.5. Determination of protein content, SDS-PAGE and lipase
activity analysis
The protein content in the samples was determined by Brad-
ford method using bovine serum albumin (BSA) as standard [23].
The protein expression was analyzed on SDS-PAGE as described by
Laemmli [24]. The lipase activity was determined by alkali titration
method using olive oil as the substrate [25].
2.2. Strains, plasmids and culture conditions
S. marcescens H30 was deposited in the China Center for Indus-
trial Culture Collection with the accession number of CICC 20066.
E. coli DH5␣ and BL21 (DE3) were deposited in our laboratory. The
plasmid pMD19-T was used for the cloning, and the pET-32a was
used for protein expression. S. marcescens H30 was grown at 30 ◦C
in a culture medium consisted of 3 g/L beef extract, 5 g/L peptone
and 5 g/L NaCl. E. coli cells were regularly cultured in a Luria-Bertani
medium (LB, 10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl) at 37 ◦C,
and an appropriate amount of ampicillin was added when needed.
2.6. General procedure for LH-EP immobilized SmL catalyzed
resolution of racemic alcohols
A certain molar ratios of racemic alcohol and acyl donor such as
vinyl butyrate in an organic solvent (5 mL) was mixed with appro-
priate amount of immobilized lipase in a 25 mL screw capped vial
in a thermostatted shaker. The reaction was monitored and the
ee assessed by HPLC analysis on the chiral columns as stated in
the HPLC Analysis section. Aliquots (50 ␮L) of the reaction mixture
were withdrawn at intervals, diluted with n-hexane/2-propanol
(9:1, 450 ␮L), centrifuged and the supernatant (10 ␮L) was then
injected onto the column.
2.3. Cloning and functional expression of Serratia marcescens
H30 lipase gene in E. coli
According to the reported S. marcescens lipase gene
sequence [17,19,20], two specific primers, forward 5ꢀ-
GGTTGAATTCGGCATCTTTAGCTATAAGGA-3ꢀ (EcoRI) and reverse
5ꢀ-CCTTGCGGCCGCTAGGCCAACACCACCTGATC-3ꢀ
(NotI) were
designed. PCR amplifications were performed at 94 ◦C for 4 min,
followed by 30 cycles of 94 ◦C for 30 s, 58 ◦C for 1 min, and 72 ◦C
for 1 min, with a final extension step of 8 min at 72 ◦C. The PCR
product was separated and cloned into pMD19-T and sent to
sequence. The obtained SmL gene and pET32a with a Trx tag were
digested with EcoRI and NotI restriction enzymes simultaneously,
and ligated to construct the expression vector pET32a-SmL, then
transformed into the E. coli BL21 (DE3). The recombinant E. coli
BL21 (DE3)/pET32a-SmL was inoculated into 100 mL of LB medium,
and grown at 37 ◦C, 200 rpm until OD600 reached 0.6–0.8. IPTG
2.7. HPLC analysis
Transesterification reactions were monitored at 210 nm and
254 nm by chiral HPLC on a Chiralcel OD column or OB-H column
(4.6 mm i.d. × 250 mm) (Daicel Chemical Industries, Ltd., Japan) by
using n-hexane/2-propanol as eluent. The liquid chromatograph
employed was an Agilent 1100 instrument equipped with a DAD
detector. The temperature of the column was maintained at 20 ◦C.
The enantiomers were separated well enough for the accurate
determination of the ee values on either of the columns by choosing

E. Su et al. / Journal of Molecular Catalysis B: Enzymatic 106 (2014) 11–16
13
Table 1
an appropriate proportion of n-hexane/2-propanol for each com-
pound (mainly 90:10, 95:5 or 97:3, v/v). The enantiomeric ratio
E was calculated using E = ln[(1 − c) (1 − eeS)]/ln[(1 − c)(1 + eeS)]
where c and eeS represent the extent of conversion and the ee of
the unreacted alcohol substrate, respectively.
Comparison of immobilizing recombinant Serratia marcescens H30 lipase on differ-
ent carries.a
Carrier
Immobilization
time (h)
Immobilization
yield (%)b
Specific
activity (U/g)
Activity
recovery (%)c
LH-HA
LH-EP
D201
D301
D315
10
16
5
5
5
63
76
32
73
45
103
214
62
125
87
23
51
17
39
25
3. Results and discussion
3.1. Cloning and functional expression of the Serratia marcescens
H30 lipase gene in E. coli
a
All assays were performed in triplicates and the average value was presented.
Immobilization yield = (1 − protein concentration of supernatant/protein con-
b
centration of free lipase solution) × 100%.
A band of 1848 bp length was amplified from the chromosomal
DNA of S. marcescens H30 using the specific primers. The full-length
lipase gene motif encodes a polypeptide of 615 amino acid residues
with a predicted molecular mass of near 65 kDa. The nucleotide
sequence and deduced amino acid sequence of S. marcescens H30
lipase share 95–98% identities and 97–99% identities with lipases
from S. marcescens Sr41, S. marcescens SM6, S. marcescens ES-2 and
S. marcescens ECU1010, respectively [17,19,20,26].
c
Activity recovery = (total activity of immobilized lipase/total activity of free
lipase solution) × 100%.
3.3. LH-EP immobilized SmL-catalyzed kinetic resolution of
racemic alcohols
As it may be useful for kinetic resolution of other chiral com-
pounds, we tried to use the LH-EP immobilized SmL for resolution
of racemic alcohols. Initially, 4-phenyl-2-butanol (1) was chosen
as a model alcohol, and the LH-EP immobilized SmL-catalyzed
transesterification of 1 with the acyl donor vinyl butyrate was
investigated as a model reaction (Scheme 1). The immobilized SmL
could enantioselectively catalyze the transesterification of racemic
4-phenyl-2-butanol to the corresponding (R)-butyl ester, whereas
no (S)-butyl ester was detected in the reaction media (Fig. S3). This
R-enantioselectivity is the same as that in hydrolysis of trans-3-(4-
methoxyphynyl) glycidic acid methyl ester [( )-MPGM] (17, 27),
and accords with that proposed by Kazlauskas rule [28]. This is the
first attempt of using lipase from S. marcescens for resolution of
racemic alcohols. The effects of prime reaction parameters on the
model reaction were investigated to further characterize the LP-EP
immobilized SmL.
S. marcescens lipase genes were often expressed in the form of
inclusion bodies, which need be refolded to give functionally active
lipase. In order to fulfill the soluble expression of S. marcescens
H30 lipase in E. coli, the pET32a with a Trx tag was chosen to
construct the recombinant plasmid pET32a-SmL. The recombinant
E. coli BL21 (DE3)/pET32a-SmL were cultivated in LB medium at
25 ◦C for 10 h after inducing by adding IPTG. The lipase expression
result was shown in Fig. S1. It could be found that the lipase was
mainly expressed in soluble form in the supernatant (Fig. S1, lane
4), which accounted for about more than 80% of the target protein.
Fusion expression of S. marcescens H30 lipase gene with the Trx tag
of pET32a obviously improved the solubility. The lipase activity in
the supernatant was also determined to be about 5000 U/L.
The recombinant lipase expression was affected by some phys-
ical and chemical factors such as inducing temperature, inducing
time, the concentration of IPTG and the concentration of Ca2+. These
factors were further optimized to enhance the production of lipase.
It was found that Ca2+ showed a profound effect, and 5 mmoL/L of
CaCl2 could increase the lipase activity for 1.6-fold (Fig. S2). After
optimization, the lipase activity was enhanced to about 25,000 U/L
when induced by 0.8 mmoL/L of IPTG at 25 ◦C for 12 h with addition
of 5 mmoL/L of CaCl2.
3.3.1. Effect of temperature
Enzymes being protein in nature are sensitive to tempera-
ture. Higher temperature would lead to lower enantioselectivity
of lipase, and thus the effect of temperature was deliberated. The
result was shown in Table 2. A relatively high conversion yield
occurred between 35 and 50 ◦C. A high E value was also maintained
at temperatures between 25 and 45 ◦C, yet decreased significantly
at higher temperature (50 ◦C). The optimal temperature for the
kinetic resolution of racemic 4-phenyl-2-butanol was 45 ◦C, which
gave a conversion yield of 47.9% and a higher ees of 95.7%.
3.2. Purification and immobilization of the recombinant SmL
Based on the terminal 6 × His affinity tag, the recombinant lipase
was purified with different concentrations of imidazole (50 mM,
80 mM, 100 mM, and 200 mM). More than 80 mM of imidazole
could produce high purity recombinant lipase (data not shown).
The main disadvantage of using free lipase in industrial applica-
tions is the cost of enzyme. Enzyme immobilization can overcome
this problem by reutilizing the enzyme. Many supports have been
used for immobilizing various lipases. Several methods for immo-
bilizing the lipase from S. marcescens had already been investigated.
However, low loading capacity and high cost of carrier associated
with these methods imposed restrictions on the practical use in
industry [27]. Five carriers, including D201, D301, D315, LH-HA,
and LH-EP which are all homemade and cheap, were chosen to
immobilize the purified recombinant SmL. As shown in Table 1,
LH-EP was the best carrier, which gave the highest activity recov-
ery of 51% with the highest specific activity of 214 U/g. The other
four carriers (D201, D301, D315 and LH-HA) showed lower activity
recovery and specific activity, although the immobilization yields
of D301 and LH-HA were also more than 60%. LH-EP is an epoxy
resin that is accessible and economically advantageous, which also
makes it to be fit for immobilizing the SmL.
3.3.2. Effect of reaction solvents
In nonaqueous biocatalysis, the reaction medium shows signifi-
cant influence on the lipase activity and enantioselectivity. Organic
solvents are the most often used nonqueous reaction medium. Sol-
vents with low log P values are more hydrophilic, and they tend
Table 2
Effect of temperature on the LP-EP immobilized recombinant Serratia marcescens
H30 lipase catalyzed resolution of 4-phenyl-2-butanol.a
Entry
Temperature (◦C)
Conversion (%)
ees (%)
E
1
2
3
4
5
6
25
30
35
40
45
50
15.3
29.2
41.1
45.6
47.9
54.7
18.1
41.2
69.7
81.2
95.7
94.3
>200
>200
>200
>200
190
52
a
All assays were performed in triplicates and the average value was presented.
Reaction conditions: 25 mg LH-EP immobilized SmL, 200 rpm, vinyl butyrate/4-
phenyl-2-butanol molar ratio of 2:1, 5 mL n-hexane as reaction medium, reaction
time 36 h.

14
E. Su et al. / Journal of Molecular Catalysis B: Enzymatic 106 (2014) 11–16
Scheme 1. Serratia marcescens H30 lipase catalyzed kinetic resolution of racemic alcohols.
to strip away the water molecules present on the surface of the
lipase, which will reduce the catalytic activity of lipase. Solvents
with high log P values can stabilize lipase to some degree. Consid-
ering these effects of solvents, eight organic solvents with different
log P were assessed as the reaction medium for the LP-EP immo-
bilized SmL catalyzed resolution of 4-phenyl-2-butanol. Both the
reaction rate and enantioselectivity were affected largely by the
solvents employed (Table 3). The reactions were retarded to a great
extent in solvents with lower log P such as dioxane and chloroform.
Hydrophobic solvents with log P more than 3 gave higher conver-
sion and E value. The best result was achieved in n-hexane, and thus
it was used for the rest of the study.
3.3.3. Effect of water activity
Water has many effects on lipase catalyzed enantioselective
reactions, as water could be simultaneously a competitive and an
enantioselective inhibitor, a competitive substrate and a lubricant
[29]. The dependence of water activity of the lipases is different. The
LH-EP immobilized SmL, the substrates and the reaction medium
were separately adjusted to the desired water activity (aw) in sealed
containers for at least 2 days with the vapor phase of saturated salt
solutions of known aw (0.97, 0.75, 0.53, 0.33, 0.23, 0.11). The LP-EP
immobilized SmL catalyzed transesterification resolution of 4-
phenyl-2-butanol was investigated at different initial aw. As shown
in Table 4, the reaction rate and enantioselectivity increased along
with the decrease of aw from 0.97 to 0.33, and attained the maxi-
mum at aw of 0.33, then decreased with the further decrease of aw
.
Table 3
Effect of organic solvents on the LP-EP immobilized recombinant Serratia marcescens
H30 lipase catalyzed resolution of 4-phenyl-2-butanol.a
Table 4
Effect of water activity (aw
) on the LP-EP immobilized recombinant Serratia
Entry
Solvents
Conversion (%)
eeS (%)
E
marcescens H30 lipase catalyzed resolution of 4-phenyl-2-butanol.a
1
2
3
4
5
6
7
8
Dioxane
Tetrahydrofuran
Chloroform
Tertiary amyl alcohol
Benzene
Carbon tetrachloride
Cyclohexane
n-Hexane
16.2
24.3
13.2
17.4
29.4
17.3
48.4
47.9
19.3
32.1
15.2
21.1
41.0
20.9
91.8
95.7
>200
>200
>200
>200
148
>200
161
190
Entry
aw
Conversion (%)
eeS (%)
E
1
2
3
4
5
6
0.97
0.75
0.53
0.33
0.23
0.11
13.2
27.4
35.1
48.7
32.5
26.9
35.2
37.7
65.1
97.6
47.7
98.8
13
20
65
>200
158
141
a
a
All assays were performed in triplicates and the average value was pre-
sented. Reaction conditions: 45 ◦C, 25 mg LH-EP immobilized SmL, 200 rpm, vinyl
butyrate/4-phenyl-2-butanol molar ratio of 2:1, 5 mL organic solvent as reaction
medium,reaction time 36 h.
All assays were performed in triplicates and the average value was pre-
sented. Reaction conditions: 45 ◦C, 25 mg LH-EP immobilized SmL, 200 rpm, vinyl
butyrate/4-phenyl-2-butanol molar ratio of 2:1, 5 mL n-hexane as reaction medium,
reaction time 36 h.

E. Su et al. / Journal of Molecular Catalysis B: Enzymatic 106 (2014) 11–16
15
Table 5
Table 7
Effect of different acyl donors on the LP-EP immobilized recombinant Serratia
LH-EP immobilized recombinant Serratia marcescens H30 lipase catalyzed kinetic
resolution of various racemic alcohols.
marcescens H30 lipase catalyzed resolution of 4-phenyl-2-butanol.a
Entry
Acyl donor
Conversion (%)
ees (%)
E
Substrate
Conversion (%)
ees (%)
E
1
2
3
4
5
Vinyl acetate,
Isopropenyl acetate
Vinyl butyrate
Vinyl hexanoate
Vinyl octanoate
43.5
35.1
48.7
48.4
50.0
74.6
51.3
97.6
86.5
95.1
148
65
>200
67
␣-Phenethyl alcohol
1-Phenyl-2-propanol
4-Phenyl-2-butanol
2-Phenyl-1-butanol
32.7
46.9
49.9
49.3
48.6
88.3
99.2
97.2
>200
>200
>200
>200
146
aAll assays were performed in triplicates and the average value was pre-
sented. Reaction conditions: 45 ◦C, 25 mg LH-EP immobilized SmL, 200 rpm, vinyl
butyrate/racemic alcohols molar ratio of 4:1, 5 mL n-hexane as reaction medium, aw
of 0.33, reaction time 36 h.
a
All assays were performed in triplicates and the average value was presented.
Reaction conditions: 45 ◦C, 25 mg LH-EP immobilized SmL, 200 rpm, acyl donor/4-
phenyl-2-butanol molar ratio of 2:1, 5 mL n-hexane as reaction medium, aw of 0.33,
reaction time 36 h.
not a very appropriate substrate with a conversion rate and ees of
only 32.7% and 48.6%, respectively (Table 7). These results showed
that the position of chiral center affect the resolution process to
some extent.
Excessive water may stimulate the competing hydrolysis reaction.
However, too little water may lead to the short of lipase flexibility.
3.3.4. Effect of acyl donors
The effect of the acyl donors on the reaction rate and enan-
tioselectivity of the acyl transfer was subsequently investigated.
Enol esters are currently the most widely used acylating agents for
resolving racemic alcohols due to their high reactivity, irreversibil-
ity and selectivity. For the acylation of racemic 4-phenyl-2-butanol
catalyzed by LH-EP immobilized SmL in n-hexane, five acyl donors
with different carbon chain length were investigated. The carbon
chain length of acyl donor influenced the conversion rate and enan-
tioselectivity of the kinetic resolution (Table 5). High conversion
rate could be reached with the chain length of acyl donor more
than C4 (Table 5). Vinyl butyrate and vinyl octanoate proved to be
good acyl donors for providing good conversion rate and high E
values (Table 5).
4. Conclusions
The lipase gene from S. marcescens H30 was cloned and
expressed in E. coli in soluble form successfully. After optimization,
the lipase activity was enhanced to 25,000 U/L from an initial level
of 5000 U/L. LH-EP carrier gave the best immobilization effect with
an activity recovery of 51%. The LH-EP immobilized SmL was then
used to catalyze resolution of racemic alcohols. The optimized reso-
lution conditions were 45 ◦C, n-hexane as reaction medium, initial
aw of 0.33, and the molar ratio of vinyl butyrate to 4-phenyl-2-
butanol of 4:1. Under these conditions, high E value of more than
200, eeS of more than 99% and a conversion of 49.9% were achieved.
LH-EP immobilized SmL could also catalyze the kinetic resolution
of other structure similar racemic alcohols. These results indicate
that the SmL is a useful biocatalyst for production of chiral alcohols.
3.3.5. Effect of substrate molar ratio
To ensure that the resolution reaction proceeds rapidly and
completely, the lipase should not be inhibited by substrate concen-
tration. Various substrate molar ratios (2:1–1:12) were performed
to test their effects on the kinetic resolution. As shown in Table 6,
the conversion increased with the increasing of molar ratio from
1:2 to 4:1, then dropped a little when molar ratio increased to
12:1. The changing trend of ees was the same as that of conver-
sion. The E value was always more than 200 at all of molar ratios.
Comparatively speaking, molar ratio of 4:1 gave the best results in
conversion, ees and E value.
Acknowledgments
This work was supported by “A Project Funded by the Priority
Academic Program Development of Jiangsu Higher Education Insti-
tutions, PAPD”, the “Scientific Research Fund of Nanjing Forestry
University for the Introduction of High-level talents” and the “Pro-
gram for the Top Young Talents of Nanjing Forestry University”.
Appendix A. Supplementary data
3.3.6. Kinetic resolution of various racemic alcohols by LH-EP
immobilized SmL
Supplementary data associated with this article can be found,
in the online version, at http://dx.doi.org/10.1016/j.molcatb.
2014.04.012.
In order to investigate the general applicability of the devel-
oped LH-EP immobilized SmL catalyzed resolution methodology, it
was used to resolve other structure similar racemic alcohols. The
2-phenyl-1-butanol and 1-phenyl-2-propanol were likewise con-
verted into the (R)-esters with high conversion rate and complete
enantioselectivity. However, the ␣-phenethyl alcohol proved to be
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Molar ratio of vinyl butyrate/
4-phenyl-2-butanol
Conversion (%)
ees (%)
E
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