Synthesis of both enantiomers of ethyl-4-chloro-3-hydroxbutanoate from a prochiral ketone using Candida parapsilosis ATCC 7330

Article abstract of DOI:10.1016/j.tetasy.2011.08.009

Candida parapsilosis ATCC 7330 when grown in a medium containing glycerol reduced ethyl-4-chloro-3-oxobutanoate to (R)-ethyl-4-chloro-3-hydroxybutanote (ee >99%, yield: 94%) while glucose and sucrose grown cells yielded (S)-ethyl-4-chloro-3-hydroxybutanote (ee >99%, yield: 96%). The activity of ethyl-4-chloro-3-oxobutanoate reductase was higher in glucose-grown cells (160 U/g protein) when compared to sucrose (158 U/g protein) and glycerol (22 U/g protein). Both the enantiomers of ethyl-4-chloro-3-hydroxybutanoate (ee >99%) can thus be obtained using Candida parapsilosis ATCC 7330 by altering the carbon source in the growth medium.

Full text of DOI:10.1016/j.tetasy.2011.08.009

Tetrahedron: Asymmetry 22 (2011) 1548–1552
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Tetrahedron: Asymmetry
journal homepage: www.elsevier.com/locate/tetasy
Synthesis of both enantiomers of ethyl-4-chloro-3-hydroxbutanoate
from a prochiral ketone using Candida parapsilosis ATCC 7330
Tarjan Kaliaperumal a, Sathyanarayana N. Gummadi b, , Anju Chadha a,c,
⇑
⇑
a Laboratory of Bioorganic Chemistry, Department of Biotechnology, Indian Institute of Technology-Madras, Chennai 600 036, India
b Applied and Industrial Microbiology Laboratory, Department of Biotechnology, Indian Institute of Technology-Madras, Chennai 600 036, India
c National Center for Catalysis Research, Indian Institute of Technology-Madras, Chennai 600 036, India
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 21 July 2011
Accepted 12 August 2011
Available online 15 September 2011
Candida parapsilosis ATCC 7330 when grown in a medium containing glycerol reduced ethyl-4-chloro-3-
oxobutanoate to (R)-ethyl-4-chloro-3-hydroxybutanote (ee >99%, yield: 94%) while glucose and sucrose
grown cells yielded (S)-ethyl-4-chloro-3-hydroxybutanote (ee >99%, yield: 96%). The activity of ethyl-4-
chloro-3-oxobutanoate reductase was higher in glucose-grown cells (160 U/g protein) when compared to
sucrose (158 U/g protein) and glycerol (22 U/g protein). Both the enantiomers of ethyl-4-chloro-3-
hydroxybutanoate (ee >99%) can thus be obtained using Candida parapsilosis ATCC 7330 by altering the
carbon source in the growth medium.
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
However, due to the presence of multiple dehydrogenases in the
whole cells, the enantiomeric purity of the products of the asym-
Enantiomerically pure ethyl-4-chloro-3-hydroxybutanoate is a
useful chiral building block for the synthesis of pharmaceuticals
and other bioactive molecules. (R)-Ethyl-4-chloro-3-hydroxybut-
metric reduction is not always high.3 The stereoselectivity of the
microbial reductions can be controlled by screening of the micro-
organisms,15,16 the addition of the substrate,17,18 modifying the
substrate,1 addition of specific inhibitors11,19–23 and thermal
treatment.11
anoate is a precursor of L
-carnitine1 and (S)-ethyl-4-chloro-3-
hydroxybutanoate [(S)-CHBE] is a key chiral intermediate in the
synthesis of Slagenins B and C,2 HMG-CoA reductase inhibitors
and 1,4-dihydropyridine type b-blockers.3 These chiral synthons
can be obtained via resolution of the racemate or the asymmetric
reduction of the prochiral ketone.4 Asymmetric reduction of pro-
chiral ethyl-4-chloro-3-oxobutanoate (COBE) has an advantage
over resolution because asymmetric reduction can give 100% yield,
unlike resolution, where the maximum yield is limited to 50%. The
asymmetric reduction of ethyl-4-chloro-3-oxobutanoate (COBE) by
chemical catalysts has already been reported on.5,6 The biocatalytic
asymmetric reduction of COBE to CHBE has been reported using
Baker’s yeast [rate 3.4 mmol/lh, ee (S) 97%], Candida magnoliae
[rate 9 mmol/lh, ee (S) 96%], Aureobasidium pullulans [ee (S) 97%],
Lactobacillus kefir [rate 85.7 mmol/lh, ee (S) 99.5%], Sporobolomyces
salmonicolor [ee (R), rate 8.2 mmol/lh] and Lactobacillus fermentum
[rate 0.5 mmol/lh, ee (R) 98%].4,7–14
Changes in cultivation conditions have also been shown to af-
fect the enantioselectivity and specific activity of whole cell cata-
lysed reductions. Ushio et al. demonstrated that resting-cell
suspensions of two methylotrophic yeasts could be persuaded to
reduce b-keto esters (including methyl 4-chloroacetoacetate) to a
higher level of enantioselectivity when grown on a methanol-
rather than a glucose-supplemented medium.24 In the reduction
of methyl acetoacetate by Candida parapsilosis DSM 70125, the
highest specific rate was achieved using cells grown on a medium
supplemented with decanoic acid rather than glycerol or glucose; a
marked effect on enantioselectivity was also observed.16 Growing
Pichia capsulata with xylose instead of glucose as the major carbon
source for growth resulted in an eight-fold increase in the specific
rate of ethyl (R)-4-chloro-3-hydroxybutanoate production. The
enantioselectivity was slightly reduced (41% ee) as compared to
that achieved with glucose-grown cells (61% ee).25
Recently, we reported an efficient method for the asymmetric
reduction of COBE to (S)-CHBE (ee >99%) using C. parapsilosis ATCC
7330 under optimized culture and reaction conditions.26 Herein we
report the synthesis of both enantiomers of ethyl-4-chloro-3-
hydroxybutanote in the homochiral form via a C. parapsilosis ATCC
7330 mediated asymmetric reduction and by changing the carbon
source in the growth medium.
Microbial asymmetric reductions have become increasingly
attractive due to the easy availability of the biocatalysts, and mild
reaction conditions in addition to being relatively inexpensive.
⇑
Corresponding authors. Fax: +91 044 2257 4102.
E-mail addresses:
gummadi@iitm.ac.in (S.N. Gummadi),
anjuc@iitm.ac.in
(A. Chadha).
0957-4166/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetasy.2011.08.009

T. Kaliaperumal et al. / Tetrahedron: Asymmetry 22 (2011) 1548–1552
1549
activity (activity: 22 U/g protein, 27 mmol/lh and yield 94%)
compared to cells grown in glucose (activity: 160 U/g protein, rate
163 mmol/lh and yield: 96%) and sucrose (activity: 1 U/g,
160 mmo/lh and yield: 95%) (Fig. 1b). This is in contrast to C. par-
apsilosis DSM 70125 grown in glycerol which exhibited 1.5 times
higher reductase activity than when grown in glucose.16 Further-
more, the glycerol grown cells exhibited maximum enantiomeric
excess (>99%) at stationary phase as opposed to the exponential
phase (ee 89%), whereas in the case of glucose and sucrose the
enantiomeric excess (>99%) did not vary with growth (Fig. 1c).
These results suggest that C. parapsilosis ATCC 7330 has multiple
COBE reductase enzymes, which are differentially expressed with
different carbon sources. In contrast, the Pichia capsulate grown
in glucose exhibited various (43–93% ee) enantioselectivities in
the asymmetric reduction of COBE with the harvest time.25
2. Results and discussion
2.1. Effect of carbon sources in the biocatalyst preparation (C.
parapsilosis ATCC 7330) on the asymmetric reduction of COBE
Carbon sources in the growth medium can markedly influence
the enantioselectivity and activity of the yeast mediated asymmet-
ric reduction.16,24 The effect of various carbon sources such as glu-
cose, sucrose, maltose, mannitol, starch or glycerol, during the
growth of C. parapsilosis ATCC 7330 on the asymmetric reduction
of COBE was studied. The organism accepted all of the carbon
sources for growth, with the exception of starch (Table 1). The
asymmetric reduction of COBE was observed in cultures grown in
sucrose, glucose and glycerol resulting in the formation of enanti-
omerically pure CHBE with an excellent enantiomeric excess
(>99%, Table 1). Maltose and mannitol supported the growth of C.
parapsilosis ATCC 7330 but the enantiomeric excess (ee) of the
product CHBE was 55% (S) and 97% (S), respectively. The C. parapsi-
losis ATCC 7330 grown in glucose exhibited the highest rate of
asymmetric reduction of COBE. (S)-CHBE (anti-Prelog) was the
product formed by C. parapsilosis ATCC 7330 for all of the carbon
sources tested, except in the case of glycerol where (R)-CHBE (Pre-
log product) was observed in excellent enantiomeric excess of
>99%. It should be noted that the C. parapsilosis DSM 70125 grown
in glycerol, when used for the reduction of acetoacetic acid methyl
ester showed no stereoselectivity (ee 0%).16 Our observations led
us to speculate that an (S)-specific COBE reductase (anti-Prelog en-
zyme) is expressed during the growth of C. parapsilosis ATCC 7330
with glucose and sucrose as carbon sources, whereas an (R)-spe-
cific COBE reductase (Prelog enzyme) is expressed when glycerol
is used as the carbon source.
2.2.2. Effect of inhibitors on the COBE reductase in C.
parapsilosis ATCC 7330
The enzymes responsible for the reduction of COBE in yeast are
aldehyde reductases,16 alcohol dehydrogenases28 and carbonyl
reductases.31 Alcohol dehydrogenase and aldehyde reductase
activities were assayed using the cell free extract of C. parapsilosis
ATCC 7330 grown on glucose, sucrose and glycerol in order to
investigate the enzyme responsible for the reduction of COBE.
The substrates used for the assay were p-NO2-benzaldehdye and
acetaldehyde. Both alcohol dehydrogenase and aldehyde reductase
activities were detected in the cell free extract of C. parapsilosis
ATCC 7330 grown on glucose, sucrose and glycerol (Fig. 2). Cell free
extracts from glucose and sucrose grown cells exhibited higher
alcohol dehydrogenase activity compared to the cell free extract
from glycerol grown cells, whereas aldehyde reductase activity re-
mained high irrespective of the carbon source used. Higher alde-
hyde reductase activity irrespective of the carbon source, is due
to the non-specific reduction of p-NO2-benzaldehdye, which is a
substrate used for the aldehyde reductase activity, by other aldo-
keto reductase enzymes present in the yeast C. parapsilosis ATCC
7330.30 The aldehyde reductase activity was seven times higher
than alcohol dehydrogenase in the glycerol grown cells and this
may be due to repression of the alcohol dehydrogenase I by a
non-fermentable carbon source (glycerol) in the yeast and induc-
tion of the alcohol dehydrogenase by a fermentable carbon source
(glucose).32 The above study indicated the presence of aldehyde
reductase enzymes and alcohol dehydrogenase enzymes in C. par-
apsilosis ATCC 7330 but the question of (S)- and (R)-specific COBE
reductases still remained unanswered.
2.2. Regulation of COBE reducing enzyme(s) in C. parapsilosis
ATCC 7330
In an attempt to better understand the COBEreducingenzymes in
C. parapsilosis ATCC 7330, experiments were carried out with the cell
free extracts of C. parapsilosis ATCC 7330 grown in glucose, sucrose
and glycerol. The COBE reducing enzyme from glucose and sucrose
grown cells preferred NADH, whereas those from glycerol grown
cells accepted both NADH and NADPH. The COBE reduction by glyc-
erol grown cells gave the (R)-alcohol irrespective of the cofactor.
2.2.1. Effect of harvest time on the asymmetric reduction of
COBE by C. parapsilosis ATCC 7330
In order to differentiate between the (S)-specific reductase in
the glucose and sucrose grown cells and the (R)-specific reductase
from the glycerol grown cells, the effect of o-phenanthroline (an
alcohol dehydrogenase inhibitor)32 and quercetin (an aldehyde/
carbonyl reductase inhibitor)27 was studied on the asymmetric
reduction of COBE by C. parapsilosis ATCC 7330. The asymmetric
The growth of C. parapsilosis ATCC 7330 in glucose, sucrose and
glycerol is almost identical (specific growth rate 0.33 hÀ1) (Fig. 1a).
Glucose and sucrose grown cells exhibited a maximum COBE
reductase activity at the mid log phase (14 h), while glycerol grown
cells a recorded maximum activity at the late log phase (24 h).
Furthermore, glycerol grown cells exhibited less COBE reductase
Table 1
Effect of carbon sources during the growth of C. parapsilosis ATCC 7330 on the asymmetric reduction of COBE
Carbon source
Rate of asymmetric reduction (mmol/lh)
Enantiomeric excess (%)
Absolute configuration
Glucose
Sucrose
Maltose
Starch
Mannitol
Glycerol
Without a carbon source
45
43
38
13
34
27
6
3
2
3
2
3
4
1
>99
>99
55
65
98
(S)
(S)
(S)
(S)
(S)
(R)
(S)
>99
>99
Culture conditions: C. parapsilosis ATCC 7330 was grown under the optimum conditions (inoculum age 12 h and 4% v/v of inoculum whose cell
density is 1.7 g/l) and the biomass was harvested at 22 h. Reaction conditions: 15 g cell dry weight/l, 50 g/l glucose, 0.12 g of COBE in 0.5 ml of
ethanol, 25 ml of potassium phosphate buffer (10 mM, pH 6.8), and incubated at 25 °C and 200 rpm.

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T. Kaliaperumal et al. / Tetrahedron: Asymmetry 22 (2011) 1548–1552
Figure 2. Effect of various carbon source during the growth C. parapsilosis ATCC
7330 on the alcohol dehydrogenase and aldehyde reductase activity. Culture
conditions: C. parapsilosis ATCC 7330 was grown under optimum conditions
(inoculum age 12 h and 4% v/v of inoculum whose cell density is 1.7 g/l) and
harvested time for a glucose-grown cell is 14 h and for a glycerol grown cell is 24 h.
Enzyme assay: The assay mixture contained Tris–HCl buffer (20 mM, pH 7.8),
cofactor (0.2 mM), substrate (8 mM) and 20 ll of the crude extract in a total volume
of 1 ml. The consumption of reduced NADH was followed spectrophotometrically
(V-530 UV/vis spectrophotometer) at 334 nm and 25 °C using molar absorption
coefficient of 6180 MÀ1 cmÀ1. Acetaldehyde and p-nitro benzaldehyde were used as
a substrate for the alcohol dehydrogenase assay and the aldehyde reductase assay,
respectively.
contrast quercetin did not inhibit this reaction (Table 2). The cell
free extract from the glycerol grown cells harvested at 24 h was
completely inhibited by quercetin but not inhibited by 1,10-phe-
nanthroline (Table 2). The (S)-specific reductase from the glycerol
grown cells expressed during the exponential growth phase
(Fig. 1c) was also inhibited by 1,10-phenanthroline. The results
from the inhibitor study indicated that the (S)-specific COBE reduc-
tase is an alcohol dehydrogenase I while the (R)-specific COBE
reductase is an aldehyde reductase in C. parapsilosis ATCC 7330.
In contrast to these results, the (R)-specific COBE reductase from
C. parapsilosis DSM 7012529 and C. parapsilosis IFO 139628,11 was
inhibited by 1,10-phenathroline. Extending this to the present
work, the (S)-specific COBE reductase from C. parapsilosis ATCC
7330 is an NADH alcohol dehydrogenase since it prefers NADH
and is inhibited by 1,10-phenanthroline, which is an alcohol dehy-
drogenase inhibitor, whereas the (R)-specific COBE is an aldehyde
reductase since it is inhibited by quercetin27,32 (Fig. 3). The de-
crease in activity of the (S)-specific reductase in C. parapsilosis
Table 2
Effect of various inhibitors on the COBE reductase from C. parapsilosis ATCC 7330
Inhibitors
COBE reductase activity U/g protein
Carbon source
Glucose
Sucrose
Glycerol
None
EDTA
1,10-Phenanthroline
Quercetin
160
160
0
2
1
157
157
0
3
2
22
22
21
0
3
1
1
Figure 1. Effect of the harvest time on the asymmetric reduction COBE by C.
parapsilosis ATCC 7330. Culture conditions: C. parapsilosis ATCC 7330 was grown
under optimum conditions (inoculum age 12 h and 4% v/v of inoculum whose cell
density is 1.7 g/l) and biomass was harvested at different times and their
corresponding effect on the growth, COBE reductase activity and enantiomeric
excess were determined. COBE reductase activity: The assay mixture contained
155
1
153
3
Culture conditions: C. parapsilosis ATCC 7330 was grown under the optimum con-
ditions (inoculum age 12 h and 4% v/v of inoculum whose cell density is 1.7 g/l) and
harvested time for glucose-grown cell was 14 h and for glycerol grown cell was
22 h. COBE reductase activity: The assay mixture contained Tris–HCl buffer (20 mM,
Tris–HCl buffer (20 mM, pH 7.8), NADH (0.2 mM), COBE (8 mM) and 20 ll of the
crude extract in a total volume of 1 ml. The consumption of reduced NADH was
followed spectrophotometrically (V-530 UV/vis spectrophotometer) at 334 nm and
pH 7.8), NADH (0.2 mM), COBE (8 mM) and 20 ll of the crude extract in a total
25 °C using a molar absorption coefficient of 6180 MÀ1 cmÀ1
.
volume of 1 ml. The consumption of reduced NADH was followed spectrophoto-
metrically (V-530 UV/vis spectrophotometer) at 334 nm and 25 °C using a molar
absorption coefficient of 6180 MÀ1 cmÀ1. Inhibitors and metal ions were added in
the cell free extract and incubated for 30 min before the assay.
reduction of COBE by cell free extracts from glucose and sucrose
grown cells was completely inhibited by o-phenanthroline; in

T. Kaliaperumal et al. / Tetrahedron: Asymmetry 22 (2011) 1548–1552
1551
OH
O
alcohol dehydrogenase
Cl
O
O
O
NAD+
(S)-ethyl 4-chloro-3-hydroxybutanoate
NADH
Cl
Candida parapsilosis
O
ATCC 7330
OH
O
ethyl 4-chloro-3-oxobutanoate
aldehyde reductase
Cl
O
(R)-ethyl 4-chloro-3-hydroxybutanoate
NAD+
NADH
Figure 3. Candida parapsilosis ATCC 7330 mediated asymmetric reduction of ethyl-4-chloro-3-oxobutanoate.
ATCC 7330 grown in glycerol may be due to the decrease in func-
tional alcohol dehydrogenase I mRNA in yeast when grown on a
non-fermentable carbon source.31 The (R)-specific COBE reductase
activity was induced only when C. parapsilosis ATCC 7330 was
grown in glycerol and was inhibited by the aldehyde reductase
inhibitor, thus implying that the (R)-specific COBE reductase is
the aldehyde reductase (EC.1.1.1.21) involved in the metabolism
of glycerol in the yeast.30
by sodium borohydride reduction of COBE. All other chemicals
were purchased from Merck (India).
4.2. Microorganism
C. parapsilosis ATCC 7330 was obtained from ATCC (Manassas,
VA 20108, USA) and maintained at 4 °C in yeast malt agar medium
that contained 5 g/l peptic digest of animal tissue, 3 g/l malt ex-
tract, 3 g/l yeast extract, 10 g/l dextrose and 20 g/l agar.
When compared to the reported whole cell yeast and fungal
biocatalytic systems for the asymmetric reduction of COBE,4,7–14
Candida parapsilosis ATCC 7330 as reported herein showed
improved enantiomeric excess and rate of asymmetric reduction.
Moreover, both enantiomers of ethyl-4-chloro-3-hydroxybutano-
ate in the homochiral form can be synthesized via asymmetric
reduction using Candida parapsilosis ATCC 7330 by changing the
carbon source in the growth medium. In addition, this method does
not require the pretreatment of cells and costly cofactors, such as
NADH or NADPH. This method, which uses the same biocatalyst
to synthesise both enantiomers of CHBE in the homochiral form
with high enantiomeric purity (ee >99%), a high rate of asymmetric
reduction [(S)-CHBE: 163 mmol/lh26 and (R)-CHBE: 27 mmol/lh]
and a high product concentration [(S)-CHBE: 230 g/l26 and (R)-
CHBE: 4.8 g/l] by simply changing the carbon source in the growth
medium, is suitable for further scale-up to an industrial level. This
method also provides a common platform for process development,
which is simpler than generating recombinant strains and using
them for scale-up.
4.3. Cultivation of microorganism
C. parapsilosis ATCC 7330 was precultured for 12 h at 25 °C with
orbital shaking at 200 rpm in a yeast malt broth medium that con-
tained 5 g/l peptic digest of animal tissue, 3 g/l malt extract, 3 g/l
yeast extract and 10 g/l dextrose. The precultured broth, 2 ml (4%
(v/v)) with a cell density of 1.7 g dry weight/l was transferred to
a 250 ml Erlenmeyer flask that contained 48 ml of growth medium
(5 g/l peptic digest of animal tissue, 3 g/l malt extract, 3 g/l yeast
extract and 10 g/l carbon source). The culture was grown on a rota-
tory shaker at 25 °C and 200 rpm for 22 h. The cultivated cells were
harvested by centrifugation (10,000 rpm, 10 min) at 4 °C and
washed three times with distilled water.
4.4. Asymmetric reduction of COBE
Cells harvested by centrifugation were washed and resus-
pended (19 g cell dry weight/l) in a 10 mM potassium phosphate
buffer, pH 6.8. Cell suspension (25 ml) was taken in a 150 ml Erlen-
meyer flask capped with a cotton plug and pre-incubated in a
water-bath shaker at 200 rpm and 25 °C for 15 min after adding
glucose (50 g/l). A solution of 0.12 g of COBE in 0.5 ml of ethanol
was then added to the reaction flask. Incubation was continued
for 15 min [for (R)-CHBE, the reaction time was 1 h] after the addi-
tion of COBE after which the reaction mixture was extracted with
ethyl acetate (2 Â 100 ml). The organic layer was dried over anhy-
drous sodium sulfate and the pure product was obtained by silica
gel chromatography using hexane/ethyl acetate (9:1) as a mobile
phase. In order to determine the rate of formation of CHBE, a
0.5 ml sample was taken every 5 min and centrifuged
(10,000 rpm, 10 min) at 4 °C to remove the cells. The supernatant
3. Conclusion
C. parapsilosis ATCC 7330 when grown in a medium containing
glycerol reduced ethyl-4-chloro-3-oxobutanoate to (R)-ethyl-4-
chloro-3-hydroxybutanote (ee >99%) whereas glucose and sucrose
grown cells yielded (S)-ethyl-4-chloro-3-hydroxybutanote (>99%).
In C. parapsilosis ATCC 7330, the (S)-specific COBE is an NADH alco-
hol dehydrogenase, whereas the (R)-specific COBE is an NADH
aldehyde reductase which is induced by glycerol. The main advan-
tage of this method is that both enantiomers of CHBE can be pre-
pared directly in high enantiomeric purity by using the same
biocatalyst by simply changing the carbon source in the growth
medium. This method also provides a common platform for pro-
cess development which is much simpler than the recombinant
techniques and does not require as much care as processes which
involve the recombinant strain.
was extracted with ethyl acetate (1 ml) of which 1 ll was injected
into the GC to determine the concentrations of COBE and CHBE.
4.5. Preparation of cell free extract
4. Materials and methods
4.1. Chemicals
The cell free extract was prepared after lysis of the cell suspen-
sion (40 g dry cell/l) in a 20 Mm phosphate buffer pH 6.8 by ultra-
sonication for 15 min (pulse of 1 s) at 4 °C. Cell debris was removed
by centrifugation (10,000 rpm for 30 min at 4 °C). The supernatant
(crude extract) was used to determine the enzyme activity.
COBE was purchased from Lancaster (Morecambe, England) and
distilled under vacuum before use. Racemic CHBE was synthesized

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T. Kaliaperumal et al. / Tetrahedron: Asymmetry 22 (2011) 1548–1552
4.6. COBE reductase activity
of Biotechnology, Government of India for funding and the Sophis-
ticated Analytical Instrumentation Facility (SAIF), IITM for the NMR
spectra.
The assay mixture contained 20 mM of Tris–HCl buffer pH 7.8,
NADH (0.2 mM), COBE (8 mM) and 20 ll of the crude extract in a
total volume of 1 ml. The consumption of reduced NADH was fol-
lowed spectrophotometrically (V-530 UV/vis spectrophotometer)
at 334 nm and 25 °C using a molar absorption coefficient of
6180 MÀ1 cmÀ1. One Unit (U) of COBE reductase is defined as the
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Biotechnol. 2010, 37, 159–165.
Conversion of the substrate (COBE) into product (CHBE) was
determined by GC using a TC Wax capillary column under the
following conditions: oven temperature 130 °C, injector and detec-
tor at 250 °C, carrier gas: He at 1 kg/cm2, detector: flame ionization
detector. Sample injection volume was 1 ll. The enantiomeric ex-
cess (ee%) was determined by a Jasco PU-1580 HPLC equipped with
a PDA detector. The chiral column used was Chiralcel OB-H (Daicel,
4.6 Â 250 mm) while the mobile phase used was hexane/isopropa-
nol (95:5) at a flow rate of 0.5 ml/min at 25 °C monitored at
220 nm. The 1H and 13C NMR spectra were recorded in CDCl3 on JEOL
GSX400 spectrometers operating at 400 MHz and 100 MHz respec-
tively. Chemical shifts are expressed in ppm values (d) using TMS
as the internal standard. Infrared spectra were recorded on a Shima-
dzu IR 470 Instrument. Optical rotations were determined on an
AutopolÒ digital polarimeter. Thin layer chromatography was per-
formed on Silica Gel 60 F-240 precoated silica gel aluminium sheets
to monitor the progress of the reaction. The mobile phase used for
the thin layer chromatography was 20% ethyl acetate in hexane.
Mass spectra were recorded on a Finnigan Mat 8230-GC–MS Spec-
trometer. Cell density in the medium was monitored by measuring
the optical density using a Jasco V-530 UV/vis spectrophotometer
at 600 nm. Dry cell weight was calculated from the established cal-
ibration (OD600 of 1 corresponds to 0.26 g dry cell weight/l). The spe-
cific growth rate was calculated by an exponential growth model.
27. Shimizu, S.; Kataoka, M.; Kita, K. J. Mol. Catal. B: Enzym. 1998, 5, 321–325.
28. Yamamoto, H.; Matsuyama, A.; Kobayashi, B. Biotechnol. Biochem. 2002, 66,
481–483.
Acknowledgments
29. Peters, J.; Minuth, T.; Kula, R. M. Biocatalysis 1993, 8, 31–46.
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One of us (Tarjan Kaliaperumal) thanks the Indian Institute of
Technology, Madras for a fellowship. We thank The Department