Biocatalysis and Biotransformation, 2013; 31(3): 123–131

ORIGINAL ARTICLE

Enantioselective bioreduction of cyclic alkanones by whole cells

of Candida Species

RACHIT PATIL1, LINGA BANOTH1, AMIT SINGH1, YUSUF CHISTI2

& UTTAM CHAND BANERJEE1
1Department of Pharmaceutical Technology (Biotechnology), National Institute of Pharmaceutical Education and
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Research, Punjab, India and 2Department of Biochemical Engineering, School of Engineering, Massey University,
Palmerston North, New Zealand

Abstract
α-Tetralone, β-tetralone, and 1-benzosuberone were asymmetrically reduced to their corresponding alcohols by whole
cells of Candida viswanathii. The cells had the maximum ketoreductase activity when grown aerobically in shake flasks
(200 rpm) for 36 h in a medium consisting of fructose (30 g/L), malt extract (20 g/L), CaCl2 (1 mM), and β-tetralone
inducer (1 mM) at 25°C and pH 7. The optimal bioreduction conditions with the washed cells were a pH of 6.5 (phos-
phate buffer, 50 mM), a temperature of 30°C, and in the presence of isopropanol (4%, v/v) as a cosubstrate. A 100%
reduction with an enantiomeric excess of ⬎ 99% was achieved for β-tetralone (90 mM) and 6-bromo-β-tetralone (90 mM)
using 160 g/L fresh cell concentration in 30 h. Use of a higher fresh cell concentration (250 g/L) afforded ⬎ 80% conver-
sion of 15 mM α-tetralone in 30 h and 100% conversion of 1-benzosuberone (15 mM) in 30 h.
For personal use only.

Keywords: Ketones, bioreduction, Candida viswanathii

Introduction
Homochiral cyclic alkanols, such as (S)-indanol, (S)-
α-tetralol, (S)-β-tetralol, and (S)-1-benzosuberol,
are important precursors for the synthesis of many
useful compounds (Swizdor & Kolek 2009; Manitto
et al. 1995; Tschaen et al. 1995; Hiroshi et al. 1999;
Ferraz et al. 2003; Kouji et al. 2005). Several
approaches have been reported for producing opti-
cally active cyclic alkanols. For example, asymmetric
chemical reduction of prochiral ketones can be used
(Carballeira et al. 2004), but this is not particularly
efficient and requires the use of expensive chiral
chemical catalysts (Carballeira et al. 2004). Enantio-
pure cyclic alkanols can be produced by enzymatic
kinetic resolution of a racemic mixture of alcohols
(Yadav et al. 2009). In such a process, only one of the
enantiomers is converted to the desired product while
the other enantiomer remains unaffected. This allows
for the separation of the optically pure alcohol from
the unreacted alcohol, and the unwanted enantiomer
constitutes a waste product. Kinetic resolution,
therefore, can achieve a maximum yield of 50%.
Inexpensive and efficient methods are needed for
producing bulk quantities of enantiomerically pure
cyclic alkanols. Asymmetric reduction of prochiral
ketones to chiral alcohols via whole cell biocatalysis
offers an alternative route to chiral alcohols. Whole
cells can be inexpensive compared with purified
enzymes and are often more stable than the isolated
enzymes (Yan et al. 2010). The metabolic machinery
of the cells can regenerate cofactors; therefore,
expensive cofactors do not need to be added to prog-
ress the reaction, as is generally required for reduc-
tions involving purified enzymes (Kroutil et al. 2004;
Andrés et al. 2012). A model substrate, 1-benzosub-
erone (C11H12O, 6,7,8,9-tetrahydro-5H-benzocy-
clohepten-5-one), was used for screening various
microbial strains for the ability to reduce cyclic
alkanones to alcohols with a high yield and enanti-
oselectivity and the yeast Candida viswanathii being
identified as potentially most efficacious for the
desired reduction. Previously, C. viswanathii has
been used effectively for the stereoselective reduc-
tion of aryl–alkyl ketones (Soni & Banerjee 2006;

Correspondence: Uttam Chand Banerjee, Department of Pharmaceutical Technology (Biotechnology), National Institute of Pharmaceutical Education and
Research, Sector-67, S.A.S. Nagar-160 062, Punjab, India. Tel: ⫹ 91-172-2214682-687, Ext. 2142. Fax: ⫹ 91-172-2214692. E-mail: ucbanerjee@niper.ac.in

(Received 19 September 2012 ; revised 10 December 2012 ; accepted 18 February 2013)

ISSN 1024-2422 print/ISSN 1029-2446 online © 2013 Informa UK, Ltd.
DOI: 10.3109/10242422.2013.778252
 124 R. Patil et al.
Himani & Banerjee 2009) and heteroaryl–alkyl
ketones (Soni & Banerjee 2005; Soni et al. 2005);
however, the cells grown in previously reported
media formulations showed little activity toward
cyclic ketones (Soni et al. 2007). A new medium
formulation was developed to grow the cells with an
enhanced activity toward the target ketones. Suitable
reaction conditions were identified for the bioreduc-
tion using whole cells.
Bioreduction of α-tetralones to (S)-α-tetralols
has been reported using whole cells of Fusarium cul-
morum (Swizdor & Kolek 2009), Diplogelasinospora
grovesii (Carballeira et al. 2004), Mortierella isabellina
(Holland et al. 1987), and sprouted peas (Yadav
et al. 2009), but with poor to modest enantioselectiv-
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ity of the alcohol formed. Under optimized condi-
tions, the bioreductions demonstrated in the present
work achieved ee values of ⬎ 99% and conversion of
nearly 100%.
Materials and methods
Materials
α-Tetralone, β-tetralone, 6-bromo-2-tetralone and
1-benzosuberone, (RS)-1-indanol, and (RS)-α-
For personal use only.
tetralol were purchased from Sigma-Aldrich (Stein-
heim, Germany). The other standard alcohols,
6-methoxy-1-tetralol, β-tetralol (Tschaen et al.
1995), 6-methoxy-2-tetralol (Ferraz et al. 2008),
6-bromo-2-tetralol (Tschaen et al. 1995), and 1-
benzosuberol (Bonvallet et al. 2002), were obtained
by reducing the corresponding ketones using meth-
ods reported in the literature. Media components
and buffer salts (K2HPO4 and KH2PO4) were pur-
chased from Hi-Media (Mumbai, India). The solvents
for HPLC analyses were purchased from Fisher-
Scientific (www.fishersci.com).
Analytical methods
The products were characterized by 1H Nuclear
Magnetic Resonance Spectroscopy (1H NMR, 400
MHz Bruker Avance DPX300 FT-NMR using
tetramethylsilane (TMS) as the internal standard),
FT-IR spectroscopy (Nicolet Impact 400 FTIR
instrument; Thermo Electron Corporation, Madi-
son, WI, USA), and high-performance liquid chro-
matography (HPLC, Shimadzu, Japan; consisting of
LC-10AT pump, SPD-10A UV-VIS detector).
The bioreduction of 1-benzosuberone was fol-
lowed by HPLC using a C18 column (4.6 ⫻ 250 mm,
5 μm; Phenomenex, USA). Acetonitrile and sodium
phosphate buffer (50 mM, pH 7) mixed in a ratio of 1:1
(v/v) were used for elution at a flow rate of 1 mL/min.
The absorbance was recorded at 224 nm. The
enantiomeric excess (ee) of the (S)-1-benzosuberol
was estimated using a Lux amylose 2 column
(4.6 mm ⫻ 250 mm, 5 μm; Phenomenex, USA) with
acetonitrile and water (30:70, v/v) as the eluent, at
a flow rate of 0.5 mL/min.
The previously mentioned HPLC system was used
for monitoring the other bioreductions. (S)-α-Tetralol
was eluted using a 60:40 (v/v) solvent mixture of
acetonitrile and phosphate buffer (50 mM, pH 7) at a
flow rate of 1 mL/min. The absorbance was measured
at 222 nm. The enantiomeric excess of the product
was determined on the same Lux amylose 2 column
using a mixture of acetonitrile and water (30:70 v/v)
as the eluent, at a flow rate of 0.5 mL/min.
For quantifying (S)-β-tetralol, the elution solvent
was a 60:40 (v/v) mixture of acetonitrile and phosphate
buffer (50 mM, pH 7) at a flow rate of 1 mL/min, with
a wavelength of 194 nm. The enantiomeric excess of
the product was determined using a CHIRALCEL
OD-H® www.chiraltech.com) HPLC column. The
elution solvent was a 98:2 (v/v) mixture of hexane
and isopropanol at a flow rate of 0.5 mL/min. For
quantifying (S)-6-bromo-β-tetralol, the HPLC elu-
ent was a 60:40 (v/v) mixture of acetonitrile and
phosphate buffer (50 mM, pH 7), at a flow rate of
1 mL/min. The detection wavelength was 220 nm.
The enantiomeric excess of the product was deter-
mined using a CHIRALCEL OD-H® HPLC column.
The elution solvent was a 98:2 (v/v) mixture of hexane
and isopropanol at a flow rate of 0.5 mL/min.
Bioreduction reaction
The bioreduction reaction mixture (10 mL) con-
tained 160 g/L of wet cell mass and the cyclic
alkanone substrate dissolved in 5% (v/v) isopropanol
in phosphate buffer (pH 6.5). The reaction mixture
was incubated under the specified conditions, and
1-mL samples withdrawn at specified time intervals.
The samples were centrifuged (10,000g, 5 min), and
the supernatant extracted with ethyl acetate (3 ⫻
1 mL). The ethyl acetate phase was recovered and
evaporated using a rotary evaporator. The residue
was dissolved in 500 μL of a specified solvent for
analysis using HPLC.
Screening of microorganisms for bioreduction
of 1-benzosuberone
Pure cultures grown in various liquid media were
harvested by centrifugation (7000 g, 15 min) and
washed twice by resuspending in distilled water.
Washed cells (0.16 g/mL fresh weight) were incubated
(30°C, 200 rpm, 120 h) with 1-benzosuberone
(5 mM in 5% v/v mixture of isopropanol in phosphate
 Enantioselective reduction of cyclic alkanones by Candida viswanathii 125
buffer, pH 6.5). Samples were withdrawn at various
time intervals and measured using HPLC, as speci-
fied above. One unit of the bioreduction enzyme
activity was defined as the amount of dry cells that
produced 1 μmole of the product alcohol per hour
from 5 mM 1-benzosuberone at 30°C, pH 6.5 (phos-
phate buffer), in the presence of 5% v/v isopropanol.
Screening of media for growth
C. viswanathii was grown at 30°C, with an initial pH
of 7, in various media (MacConkey, Luria broth,
GYP, Skim milk modified broth, Nutrient broth,
Yeast malt broth, YPD, GyPM, YMP and Sabouraud
dextrose). Cell mass was used for the reduction of
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ketones. After 36 h in the exponential growth phase,
1-benzosuberone was added to a final concentration
of 5 mM. Aliquots of 1 mL were collected every 6 h,
the cells were removed by centrifugation (7000 g,
15 min), and the supernatant was extracted with
ethyl acetate (3 ⫻ 1 mL). The extract was dried and
the residue analyzed using HPLC to quantify the
conversion of the substrate and the enantiomeric
excess of the product.
For personal use only.
Optimization of physicochemical parameters
for cell growth
Various physical parameters (inoculum age and size,
temperature, and initial pH) for the cultivation of the
C. viswanathii were examined by changing one
parameter at a time while keeping the others con-
stant. In each case, cell mass growth, bioreduction
of 1-benzosuberone, and enantiomeric excess of the
product were determined. Sabouraud dextrose
medium (10 g/L peptone and 20 g/L dextrose) was
the basal control medium used for growing the
C. viswanathii cells. In separate experiments, the car-
bon source and the nitrogen source of this medium
were replaced with the other specified sources. The
medium was then supplemented with specified metal
salts and inducers, in attempts to obtain a higher
concentration of the biomass with a high specific
activity of ketoreductases. The washed cells were
then used for the conversion of 1-benzosuberone.
The conversion of the substrate and the enantio-
meric excess of the product were quantified. The final
microbial biomass concentration at 36 h was mea-
sured as dry weight of the washed biomass pellet.
Optimization of cyclic alkanone bioreduction
by the cells
For bioreduction of α-tetralone and 1-benzosub-
erone by C. viswanathii, the reaction was carried out
at various specified combinations of pH, tempera-
ture, initial substrate, cell concentration, and cosub-
strate concentration. The conversion of the substrate
and the enantiomeric excess of the product were
determined using HPLC.
Bioreduction of cyclic alkanones
Bioreduction reaction mixture (100 mL) contained
250 g/L wet cells in phosphate buffer (pH 6.5),
15 mM α-tetralone, and 4% (v/v) isopropanol as a
cosubstrate. In separate experiments, α-tetralone
was replaced with 15 mM 1-benzosuberone or
90 mM β-tetralone. In all cases, the reaction mixture
was incubated at 30°C, 200 rpm, for 30 h. The cells
were then removed by centrifugation (10,000 g,
5 min) and the supernatant extracted with ethyl
acetate (3 ⫻ 1 mL). The solvent was removed using
a rotary evaporator. The crude product was dissolved
in acetonitrile and purified by PTLC using a hexane
and ethyl acetate solvent system (85: 15 v/v). The
products were quantified using HPLC and charac-
terized using NMR, IR, and MS.
Results and discussion
Reduction of 1-benzosuberone
The reduction of various ketones to their correspond-
ing enantiopure alcohols is shown in Scheme 1.
Freshly grown cell mass of Candida parapsilopsis,
Candida melibiosa, and C. viswanathii were used for
the reduction of 1-benzosuberone in shake flasks.
The highest yield (22%) of 1-benzosuberol was
achieved using C. viswanathii. The enantiomeric
(A) O OH
Candida viswanathii (S)
Phosphate buffer
α-tetralone (S)-α-tetralol
(B) O HO (S)
Candida viswanathii
Phosphate buffer
1-benzosuberone (S)-1-benzosuberol
(C) O Candida viswanathii
OH
(S)
Phosphate buffer
β-tetralone (S)−β-tetralol
(D) O Candida viswanathii (S)
OH
Br
Phosphate buffer
Br
6-bromo-2-tetralone (S)-6-bromo-2-tetralol
Scheme 1. Reduction of α-tetralone (A), 1-benzosuberone (B),
β-tetralone (C), and 6- bromo-2-tetralone (D).
 126 R. Patil et al.
excess of the product was ⬎ 99%. The yield of 1-
benzosuberol was 8 and 9% using C. parapsilopsis
and C. melibiosa, respectively. In view of these results,
C. viswanathii was used for all the subsequent exper-
iments. Enantioselective reduction of aryl–alkyl
ketones and heteroaryl–alkyl ketones using C. viswa-
nathii has been previously reported (Soni et al. 2005;
Soni et al. 2006). Acetophenone was used as an
enzyme inducer in these studies (Soni et al. 2005;
Soni et al. 2006). In the present study, 1-benzosub-
erone could be reduced by C. viswanathii cells grown
in a pre-optimized medium without the use of aceto-
phenone as an inducer. The conversion at 36 h was
26% and the enantiomeric excess exceeded 99%.
This suggested the involvement of a different ketore-
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ductase for the conversion of cyclic alkanones com-
pared with the enzymes involved in the earlier studies
(Soni et al. 2005; Soni et al. 2006). Optimization of
the cell growth medium (Sabouraud dextrose medium
contains (10 g/l) peptone and (20 g/l) glucose) was
indicated for enhancing the expression of the enzyme
involved in reducing cyclic alkanones.
Selection of medium for producing cells with enhanced
alkanone reductase activity
For personal use only.
Of the various defined media (MacConkey, Luria
broth, GYP, Skim milk modified broth, Nutrient
broth, Yeast malt broth, YPD, GyPM, YMP and Sab-
ouraud dextrose) screened for growing the cells with
a high ketoreductase activity, the Sabouraud dex-
trose medium (10 g/L peptone and 20 g/L dextrose)
was found to be the best for producing the cells with
the ability to reduce 1-benzosuberone. Conversion
of 62% 1-benzosuberone (5 mM) with an enantio-
meric excess of ⬎ 99% was observed in 84 h using
250 g/L of fresh cells. Cells grown in Luria broth
lacked the ability to reduce the ketones.
Enzyme production in Sabouraud dextrose medium
The alkanone reductase activity in the cells was
highest in the mid- to late stationary phase of growth.
The highest conversion of 1-benzosuberone (15 mM)
was obtained using cells harvested between 42 and
48 h of growth. It was found to be 72% and 76%
for α-tetralol and 1-benzosuberol, respectively, but
the enantiomeric excess of the product was highest
(⬎ 99%) with cells harvested at 36 h. Hence, for all
the subsequent experiments, cells were harvested at
36 h of growth. Inoculum size was always 2% (v/v).
An inoculum age of 18 h was the best for supporting
subsequent growth. Although the yeast could grow
and produce ketoreductase activity over a broad
pH range of 5–10, a pH of 7 afforded the highest
biomass concentration (5.7 g/L), and the biomass
was sufficiently active to achieve 72.4% conversion
of 1-benzosuberone (5 mM) in 36 h. Increasing the
culture’s pH up to 9 did not affect the final biomass
concentration much, yet sharply decreased the
enzyme activity of the cells. The effect of tempera-
ture on cell growth and enzyme activity was exam-
ined by growing the cells at temperatures ranging
from 20 to 40°C. The maximum cell concentration
and the specific enzyme activity at 36 h were obtained
at 25°C. Higher culture temperatures adversely
affected the biomass-specific enzyme activity. The
enantiomeric excess of the 1-benzosuberol produced,
using cells grown at 25°C, exceeded 99%. Various
levels of agitation (150, 175, 200, 225, and 250 rpm)
of shake flasks were tested for their effect on cell
growth and enzyme activity. An agitation speed of
200 rpm maximized the growth, as well as the
biomass-specific enzyme activity.
Optimization of nutrients for growing cells with
a high activity of alkanone reductase
The basal control medium was the Sabouraud dex-
trose medium (10 g/L peptone and 20 g/L dextrose).
In separate experiments, the carbon and nitrogen
sources in this medium were replaced with other
specified carbon and nitrogen sources. All growth
experiments were carried out at 25°C, 200 rpm, with
an initial pH of 7, in 500-mL shake flasks. The cells
were always harvested at 36 h. In all cases, the
specific activity was measured at 30°C using 1-
benzosuberone, as specified in “Screening of micro-
organisms for bioreduction of 1-benzosuberone.”
The cell fresh weight concentration used for the
bioreduction was 250 g/L.
Carbon source
In separate shake flask cultures, a variety of carbon
sources (sucrose, fructose, mannitol, glycerol, lac-
tose, and starch) were used in Sabouraud medium.
The initial concentration of each carbon source was
10 g/L. The final biomass concentrations and the
biomass-specific ketoreductase activities are shown
in Table I. The cells grown on fructose had the high-
est specific enzyme activity, while those grown
on sucrose had the highest biomass concentration
(Table I). Therefore, fructose was selected as the
carbon source for all future experiments.
Nitrogen source
Various organic and inorganic nitrogen sources were
evaluated separately for the production of biomass
 Enantioselective reduction of cyclic alkanones by Candida viswanathii 127
Table I. Effect of the carbon source on the final biomass Table III. Effect of metal ions on the final biomass concentration
concentration and in biomass-specific reductase activity of cells. and in biomass-specific reductase activity of cells.

Carbon Cell Specific Cell mass Specific activity

source concentration (g/L) activity (mmol/h g)a Metal ion concentration (g/L) (mmol/h g)a

Sucrose 5.9 ⫾ 0.1 0.23 Zn2⫹ 5.0 ⫾ 0.1 1.07

Fructose 5.7 ⫾ 0.1 0.64 Ca2⫹ 6.7 ⫾ 0.1 1.46
Glycerol 5.4 ⫾ 0.1 0.45 Fe3⫹ 5.8 ⫾ 0.1 0.39
Mannitol 5.8 ⫾ 0.1 0.27 Ni2⫹ 4.3 ⫾ 0.1 0.27
Lactose 3.2 ⫾ 0.1 0.33 Co2⫹ 4.5 ⫾ 0.1 0.33
Starch 3.4 ⫾ 0.1 0.13 Mg2⫹ 5.2 ⫾ 0.1 0.67
Controlb 5.9 ⫾ 0.0 0.51 Cu2⫹ 5.7 ⫾ 0.1 0.49
Na⫹ 5.4 ⫾ 0.1 0.52
aThe specific activity was measured using 1-benzosuberone. K⫹ 5.4 ⫾ 0.1 0.86
bThe control was Sabouraud dextrose medium. Controlb 6.8 ⫾ 0.1 1.24
aThe specific activity was measured (30 h, 30°C, 200 rpm) using
and for the biomass-specific enzyme activity. The 10 mM 1-benzosuberone in phosphate buffer (pH 7.0, 50 mM)
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final concentration of the nitrogen source in the containing 5% (v/v) isopropanol. The biomass concentration was
medium was always 5 g/L. The carbon source was 160 g/L.
bThe control was a medium with 30 g/L fructose and 20 g/L malt
always fructose at an initial concentration of 30 g/L.
extract.
The control medium contained 30 g/L fructose and
20 g/L peptone. Inorganic nitrogen sources sup-
ported the growth poorly, and the cells grown on Ca2⫹, all the metal ions had an inhibitory effect on
them were low in the enzyme activity (Table II).Yeast biomass growth and the alkanone reductase activity
extract supported good biomass growth (6 g/L), but of the biomass (Table III).
not the production of the enzyme (Table II). Malt
extract afforded a fairly high biomass concentration Inducers
(5.8 g/L), as well as the highest level of the ketore-
For personal use only.

ductase activity in the cells (Table II). In separate experiments, the effect of inducers on
growth and biomass-specific enzyme activity were
examined by adding specified alkanones (0.5 mM)
Metal ions to the growth medium at inoculation. α-Tetralone
The effects of metal ions on the final biomass and 1-benzosuberone did not induce the production
concentration and the biomass-specific alkanone of the enzyme. Both these compounds actually
reductase activity were examined by separately add- reduced the final concentration of the biomass, as
ing metal salts (ZnSO4, CaCl2, FeCl3, NiCl2, CoCl2, well as the biomass-specific enzyme activity relative
MgSO4, CuSO4, Na2HPO4, and K2HPO4) to the to control (Table IV). The composition of the control
culture medium at a salt concentration of 1 mM. medium was fructose (30 g/L), malt extract (20 g/L)
The basal medium consisted of fructose (30 g/L) and CaCl2 (1 mM). β-Tetralone was found to
and malt extract (20 g/L). With the exception of enhance the biomass-specific enzyme activity by
about 16% relative to control (Table IV). It was con-
sidered a good inducer, as it provided cells with a
Table II. Effect of nitrogen source on final biomass concentration
and in biomass-specific reductase activity of cells.
Table IV. Effect of inducers on final biomass concentration and
Cell Specific
in biomass-specific reductase activity of cells.
Nitrogen source concentration (g/L) activity (mmol/h g)a
Cell mass Specific activity
Peptone 5.8 ⫾ 0.1 0.52 Inducer (0.5 mM) concentration (g/L) (mmol/h g)a
Yeast extract 6.0 ⫾ 0.1 0.22
Soya peptone 5.2 ⫾ 0.1 0.45 α-Tetralone 6.0 ⫾ 0.1 1.26
Tryptone 5.4 ⫾ 0.1 0.28 β-Tetralone 6.0 ⫾ 0.1 1.77
Soybean meal 5.1 ⫾ 0.1 0.26 1-Benzosuberone 4.2 ⫾ 0.1 0.67
Malt extract 5.8 ⫾ 0.1 0.84 Acetophenone 7.1 ⫾ 1.1 0.35
Ammonium sulfate 1.9 ⫾ 0.1 0.16 Controlb 6.8 ⫾ 1.1 1.53
Ammonium chloride 1.9 ⫾ 0.1 0.14
Ammonium nitrate 1.9 ⫾ 0.1 0.18 aThe specific activity was measured (30 h, 30°C, 200 rpm) using
Controlb 5.8 ⫾ 0.1 0.72 10 mM 1-benzosuberone in phosphate buffer (pH 7.0, 50 mM)
containing 5% (v/v) isopropanol. The biomass concentration was
aThe specific activity was measured using 1-benzosuberone. 160 g/L.
bThe control was a medium with 30 g/L fructose and 20 g/L bThe control was a medium with 30 g/L fructose, 20 g/L malt

peptone. extract and 1 mM CaCl2.

 128 R. Patil et al.
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Figure 1. Effect of pH on the conversion and enantiomeric excess of the product for the conversion of α-tetralone to α-tetralol and
1-benzosuberone to 1-benzosuberol by C. viswanathii cells. [The initial substrate concentration was 5 mM. The fresh cell weight
concentration was 160 g/L. The reaction occurred at 30°C, 200 rpm for 30 h. The reaction mixture contained 5% (v/v) of
isopropanol.].

high biomass-specific enzyme activity without caus- had been found to be a good inducer of the enzymes
ing much reduction in the biomass concentration. for reducing aryl–alkyl ketones (Fatima et al. 2007).
Acetophenone exhibited a positive effect on cell Therefore, the present results suggest that the enzyme
For personal use only.

growth, but strongly reduced the biomass-specific system involved in the reduction of cyclic ketones is
enzyme activity (Table IV). Previously, acetophenone different from the enzyme system involved in the

Figure 2. Effect of temperature on the conversion and enantiomeric excess of the product for the conversion of α-tetralone to α-tetralol
and 1-benzosuberone to 1-benzosuberol by C. viswanathii cells. [The initial substrate concentration was 5 mM. The fresh cell weight
concentration was 160 g/L. The reaction occurred at pH 6.5, 200 rpm for 30 h. The reaction mixture contained 5% (v/v) of
isopropanol.].
 Enantioselective reduction of cyclic alkanones by Candida viswanathii 129

120 in buffers of various pH values in the range of 4.5–

α-Tetralone 9.0 (phosphate buffer, 50 mM). The incubation tem-
100 1-Benzosuberone perature was 30°C with an initial concentration of the
substrate at 5 mM. The best results (78% conversion
80
Conversion (%)

and ee ⬎ 99% for α-tetralone; 94% conversion and

ee ⬎ 99% for 1-benzosuberone) were obtained at pH
60 6.5 (phosphate buffer, 50 mM) (Figure 1). This com-
bination of pH and buffer was used in subsequent
40
work. At pH 6.5 (phosphate buffer, 50 mM), the
bioreduction was carried out at different incubation
20
temperatures in the range of 25 to 40°C, for otherwise
0
the same conditions as specified in this section. A tem-
2 4 6 8 10 perature of 30°C was found to be optimal (Figure 2).
Isopropyl alcohol (%, v/v) Therefore, a temperature of 30°C was used in all
subsequent studies.
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Figure 3. Effect of the isopropanol cosubstrate concentration on

the conversion of α-tetralone and 1-bezosuberone [The initial
substrate concentration was 5 mM. The fresh cell weight
concentration was 160 g/L. The reaction occurred at 30°C, pH 7
(phosphate buffer, 50 mM), 200 rpm for 30 h.].
reduction of aryl–alkyl ketones (Soni & Banerjee
2006) and heteroaryl–alkyl ketones (Soni &
Banerjee 2005; Soni et al. 2005). The medium
optimization effort resulted in a 3.4-fold increase in
the biomass-specific enzyme activity compared to
the cells grown in the Sabouraud dextrose medium.
For personal use only.
Effect of cosubstrate. A cosubstrate is commonly added
to a reaction media to assist with the regeneration of
the cofactors within microbial cells. Low molecular
weight aliphatic alcohols have been found to be good
cosubstrates for reactions involving dehydrogenases
(Shanshan et al. 2012). Therefore, several alcohols
(isopropanol, 2-hexanol, 2-heptanol, and 2-octanol,
2-decanol) were individually evaluated as cosub-
strates at 5% v/v level. The maximum substrate con-
version occurred with isopropanol. Various initial
concentrations (2–10%, v/v) of isopropanol were

The optimal growth medium was established to have

the following composition: 30 g/L of fructose, 20 g/L
of malt extract, 1 mM of CaCl2, and 1 mM of
β-tetralone.
Bioreduction of cyclic alkanones by cells
Effect of reaction pH and temperature. The effects of
pH on the conversion, as well as enantiomeric excess,
were examined by carrying out the reaction with the
cells suspended (160 g/mL fresh weight concentration)
then tested in attempts to further optimize its con-
centration. An isopropanol concentration of 4% (v/v)
was found to maximize the substrate conversion. At
this concentration, the conversions of α-tetralone
and 1-benzosuberone were 87.6% and 96.4%,
respectively. An isopropanol concentration of ⬎ 4%
(v/v) reduced the conversion, suggesting that it was
toxic to cells (Figure 3). Thus, 4% (v/v) of isopropa-
nol was used as a cosubstrate during bioreduction in
subsequent work.

100 100
α-Tetralone conversion
1-Benzosuberone conversion
β-Tetralone conversion
80 99
α-Tetralol ee
Enantiomeric excess ee (%)

1-Benzosuberol ee
β-Tetralol ee
Conversion (%)

60 98

40 97

20 96

0 95
0 5 10 15 20 25 30 35 40 45 50 55 60 65
Substrate concentration (mM)
Figure 4. Effect of the initial substrate concentration on the conversion of the substrate and the enantiomeric excess of the product [The
fresh cell weight concentration was 160 g/L. The reaction was carried out at 30°C, pH 6.5 (50 mM phosphate buffer), for 30 h.].
 130 R. Patil et al.

β-Tetralone conversion
100 6-Bromo-β-tetralone conversion

Conversion (%), enantiomeric excess ee (%)

β-Tetralol ee
6-Bromo-β-tetralol ee
80

60

40

20
Biocatal Biotransformation Downloaded from informahealthcare.com by McMaster University on 12/10/13

0
100 200 400 500 600 800 1000
Substrate concentration (mM)
Figure 5. Effect of the high initial substrate concentrations on the conversion of the substrate and the enantioexcess of the product [The
fresh cell weight concentration was 160 g/L. The reaction was carried out at 30°C, pH 6.5 (50 mM phosphate buffer), for 30 h.].

Effect of substrate concentration. The substrate toler- this, β-tetralone was well tolerated by the cells
ance of C. viswanathii toward α-tetralone, β-tetralone, (Figure 4). In fact, β-tetralone and 6-bromo-β-
and 1-benzosuberone was examined using an ini- tetralone could be reduced by resting cells at an
tial substrate concentration range of 5–60 mM in initial substrate concentration in the range of 100–
separate experiments. The conjugated cycloal- 1000 mM, but the time needed for complete conver-
For personal use only.

kanones α-tetralone and 1-benzosuberone were sion increased as the initial substrate concentration
less reactive, as expected, than the unconjugated increased. A too high substrate concentration
alkanone β-tetralone. For the α-alkanones, the adversely affected the ee value of the product. Using
conversion at 30 h was greatly reduced as the con- up to 90 mM β-tetralone and 6-bromo-β-tetralone,
centration increased (Figure 4). For example, at a 100% reduction was observed within 30 h with
an initial substrate concentration of 60 mM, the the enantiomeric excess value of the products
conversion was ⱕ 10% (Figure 4). In contrast to exceeding 99% (Figure 5).

Figure 6. Effect of cell mass concentration on the conversion of the substrate and the enantiomeric excess of the product [The reaction
was carried out at 30°C, pH 6.5 (50 mM phosphate buffer), for 30 h].
 Enantioselective reduction of cyclic alkanones by Candida viswanathii 131
Effect of cell concentration. In separate experiments,
the reduction of α-alkanones (15 mM) was carried
out using different concentrations of the C. viswa-
nathii cells. The conversion at 30 h (30°C, pH 6.5,
50 mM phosphate buffer) increased with increasing
the cell concentration up to a concentration of about
250 g/L (Figure 6). For 1-benzosuberone, a 100%
conversion could be achieved. For α-tetralone, the
maximum conversion was 82% (Figure 6). A cell
concentration of about 250 g/L was optimal for the
conversion.
The specific rotation [αD] value of the (S)-1-
benzosuberol (ee ⬎ 99%) was ⫹29.73° (c .01, CHCl3)
The [αD] value of the (S)-α-tetralol (ee ⬎ 99%)
was ⫹ 32.3° (c .01, CHCl3); (S)-β-tetralol (ee ⬎ 99%),
Biocatal Biotransformation Downloaded from informahealthcare.com by McMaster University on 12/10/13
⫺57.78° (c .01, CHCl3); and (S)-6-bromo-β-tetralol
(ee ⬎ 99%), ⫺52.3° (c .01, CHCl3).
Conclusion
A substrate conversion of 90–100% and product
enantiomeric excess of ⬎ 99% could be achieved for
most of the substrates examined with whole cells of
C. viswanathii. Several cyclic ketones (α-tetralone,
β-tetralone, 6-bromo-β-tetralone, and 1-benzosub-
erone) could be successfully converted to their
For personal use only.
corresponding alcohols. C. viswanathii is potentially
a good biocatalyst for the reduction of cyclic ketones,
but the cells need to be grown as specified here in
order to have a higher catalytic activity toward cyclic
ketones.
Acknowledgements
RP, LB, and AS gratefully acknowledge the financial
assistance of the Department of Biotechnology,
Government of India.
Declaration of interest: The authors report no
conflict of interest. The authors alone are responsible
for the content and writing of the paper.
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