Biotransformation of prochiral sulfides into (R)-sulfoxides using immobilized Gordonia terrae IEGM 136 cells

Article abstract of DOI:10.1016/j.molcatb.2015.10.014

Biotransformation of methyl phenyl sulfide (MPS) into optically active (R)-sulfoxide was optimized using Gordonia terrae IEGM 136 cells immobilized into cryogel of polyvinyl alcohol. Biotransformation time of 0.5 g/L MPS was shortened from 144 to 24 h in comparison with free cells. Immobilized Gordonia cells were resistant to high (up to 1.5 g/L) sulfide concentrations and catalyzed its complete bioconversion into (R)-sulfoxide (95% ee) within 72 h. Using a sequential addition method, the total load of MPS reached 4.25 g/L. At that, MPS bioconversion was 96.9% and the enantiomeric excess (ee) value of the target (R)-sulfoxide was as high as 96%. Possible reuse of immobilized cells for MPS biotransformation was experimentally proved. The obtained biocatalyst showed a high activity towards ethyl phenyl sulfide, benzyl methyl sulfide, methyl p-tolyl sulfide and formed corresponding (R)-sulfoxides with 77-95% ee. Putative pathways for sulfide oxidation by Gordonia cells were proposed.

Full text of DOI:10.1016/j.molcatb.2015.10.014

Journal of Molecular Catalysis B: Enzymatic 123 (2016) 8–13
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Biotransformation of prochiral sulfides into (R)-sulfoxides using
immobilized Gordonia terrae IEGM 136 cells
a
a,b,∗
c
a,b
T.I. Kylosova , A.A. Elkin
, V.V. Grishko , I.B. Ivshina
a
Perm State National Research University, 15 Bukirev Str., 614990 Perm, Russia
Institute of Ecology and Genetics of Microorganisms, Ural Branch of the Russian Academy of Sciences, 13 Golev Str., 614081 Perm, Russia
Institute of Technical Chemistry, Ural Branch of the Russian Academy of Sciences, 3 Acad. Korolev Str., 614013 Perm, Russia
b
c
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 9 July 2015
Received in revised form 14 October 2015
Accepted 27 October 2015
Available online 31 October 2015
Biotransformation of methyl phenyl sulfide (MPS) into optically active (R)-sulfoxide was optimized using
Gordonia terrae IEGM 136 cells immobilized into cryogel of polyvinyl alcohol. Biotransformation time of
0
.5 g/L MPS was shortened from 144 to 24 h in comparison with free cells. Immobilized Gordonia cells
were resistant to high (up to 1.5 g/L) sulfide concentrations and catalyzed its complete bioconversion
into (R)-sulfoxide (95% ee) within 72 h. Using a sequential addition method, the total load of MPS reached
4
.25 g/L. At that, MPS bioconversion was 96.9% and the enantiomeric excess (ee) value of the target
Keywords:
(R)-sulfoxide was as high as 96%. Possible reuse of immobilized cells for MPS biotransformation was
Biotransformation
Methyl phenyl sulfide
Optically active sulfoxides
Immobilization
experimentally proved. The obtained biocatalyst showed a high activity towards ethyl phenyl sulfide,
benzyl methyl sulfide, methyl p-tolyl sulfide and formed corresponding (R)-sulfoxides with 77–95% ee.
Putative pathways for sulfide oxidation by Gordonia cells were proposed.
© 2015 Elsevier B.V. All rights reserved.
Gordonia terrae
1
. Introduction
Enantiopure sulfoxides are useful building blocks and stereo-
using fungi have some drawbacks because of complex nutrient
media used, low concentrations of the starting substrates and
moderate bioconversion rates. Therefore, a search of efficient
biocatalysts for organic sulfide oxidation among non-pathogenic
bacterial strains with non-mycelial growth, metabolic systems
lability and without pathogenic properties is in great demand. In
recent years, nonpathogenic members of Gordonia, Pseudomonas,
Rhodococcus, Streptomyces have been employed in the direct syn-
thesis of optically active (S)- and (R)-sulfoxides [13–21].
Immobilization is one of the effective technologies in the
optimization of biotechnological processes. Enzyme systems or
individual enzymes, fragments or the entire metabolic system of
microbial cells are used for immobilization. Application of whole
cells is more convenient because it allows avoiding the tedious
enzyme isolation and purification steps, involvement of expensive
cofactors. In a living cell, complexly organized processes (e.g. regen-
eration of cofactors), which maintain the catalytic activity at a high
level, occur along with the formation of a target product. It is well
known that microorganisms immobilized onto different carriers
are highly resistant to xenobiotics and unfavorable environmental
factors [22–25].
directing groups in asymmetric synthesis to obtain biologically
active compounds [1–3]. Optically active (R)- and (S)-sulfoxides can
be obtained from prochiral sulfides by chemical methods which
require expensive catalysts or protection of other reactive func-
tional groups. High enantioselectivity of the reactions is difficult
to achieve using chemical approaches. Employment of specific
enzyme systems is a promising way for the direct production of
(
R)- and (S)-sulfoxides. Biocatalysis allows to obtain target products
with high regio- and stereoselectivity in a one-stage process under
eco-friendly reaction conditions, avoiding extremes of temperature
and pressure [4,5]. First studies on biotransformation of prochi-
ral organic sulfides were carried out with whole actinobacterial
1
cells of Corynebacterium equi (Rhodococcus hoagii) [6,7], oppor-
tunistic microorganisms of risk group 2 [8,9], as well as filamentous
fungi Aspergillus [10], Helminthosporium sp., and Mortierella isabel-
lina [11,12]. The described oxidative biotransformations of sulfides
Polyvinyl alcohol (PVA) cryogels are attractive carriers for whole
cell immobilization. Cryogel is a macroporous polymeric gel mate-
rial formed as a result of the cryogenic treatment (freezing –
storage in the frozen state – thawing) of a polyvinyl alcohol solu-
tion [26]. Such carriers possess advantages when compared to
∗ Corresponding author at: Institute of Ecology and Genetics of Microorganisms,
Ural Branch of the Russian Academy of Sciences, 13 Golev Str., 614081 Perm, Russia.
Fax: +7 342 280 9211.
E-mail address: an220@mail.ru (A.A. Elkin).
1
The currently accepted name of taxon is given in parentheses.
http://dx.doi.org/10.1016/j.molcatb.2015.10.014
1
381-1177/© 2015 Elsevier B.V. All rights reserved.

T.I. Kylosova et al. / Journal of Molecular Catalysis B: Enzymatic 123 (2016) 8–13
9
other hydrogels. PVA itself is mechanically and chemically stable,
biocompatible and non-toxic for the cells, and its macroporous
structure provides a non-hindered mass transfer of substrates and
metabolites [24,26,27]. All these properties are gaining consid-
erable attention to this material as a carrier for entrapping the
bacteria able to catalyze directed biotransformations of organic
compounds.
Studies on the asymmetric oxidation of organic sulfides with
immobilized microorganisms are few [28–30]. In the previous work
we have shown high efficiency of Rhodococcus rhodochrous IEGM
PVA cryogel-immobilized cells was determined using 0.2% aque-
ous solution of iodonitrotetrazolium violet (INT, Sigma–Aldrich) by
a modified method [32]. The modification involves the ethyl acetate
extraction of formazan reduced from INT in the presence of actively
respiring bacterial cells. The extraction procedure was performed
three times. The number of viable bacterial cells in the PVA cryo-
gel matrix was determined from the calibration curve depicting the
dependency between the optical density OD600 of the extracted col-
orant and the number of living cells in the suspension enumerated
by plating the cell suspension on meat-peptone agar. The residual
n-hexadecane concentration in the granules was determined gravi-
6
6 cells immobilized into polyvinyl alcohol cryogel for the bio-
◦
transformation of methyl phenyl sulfide up to 1.5 g/L (MPS) into a
corresponding (S)-methyl phenyl sulfoxide ((S)-MPSO) with optical
purity of 82% [19]. According to our data, the use of free cells of other
actinobacterial taxon G. terrae IEGM 136 for MPS biotransformation
metrically and made up 0.1%. Immobilized cells were stored at 5 C
until use.
2.4. Biotransformation of organic sulfides
(
(
0.5 g/L) resulted in the formation of (R)-methyl phenyl sulfoxide
(R)-MPSO) with ee 95% [20]. The objective of this research was to
Batch cultivations of immobilized cells were performed in
100 mL Erlenmeyer flasks containing 30 mL of the mineral medium
possibly optimize the MPS biotransformation using G. terrae IEGM
1
36 immobilized into PVA cryogel.
supplemented with sulfides or rac-MPSO on an orbital shaker
◦
(
5
160 rpm) at 28 C. The biocatalyst (60 granules corresponds to
.0 ± 0.6 × 10 cells mL , and 8.0 ± 0.5 g (wet weight)/L) was rehy-
6
−1
2
. Experimental
drated in 0.5% NaCl for 24 h before use. The biotransformation
process occurred for 24–72 h. As controls, sterile solutions of sul-
fides or rac-MPSO in a mineral medium, as well as PVA cryogel
granules containing no bacterial cells were used. Equal amounts of
free and immobilized cells were used in the comparative experi-
ments on biotransformation of organic sulfides.
2.1. Bacterial strain and culture conditions
G. terrae IEGM 136 strain isolated from oil-polluted soil and
maintained at the Regional Specialized Collection of Alkanotrophic
Microorganisms [31] was used in this work. Its 16S rDNA gene
sequence data are deposited in the GenBank database under the
accession number KF134399.
In some experiments, sequential additions of MPS to the fermen-
tation medium were applied. The initial MPS concentration was
0
.5 g/L. Each new portion of sulfide (0.5, 0.75 or 1.0 g/L) was added
Bacterial cells used for immobilization were pre-grown for
2 h in the mineral medium containing (g/L) KNO , 1.0; K HPO ,
every 24 h. Total load of MPS was 2.0, 4.25 or 5.5 g/L respectively.
n-Hexadecane at a concentration of 0.1% (v/v) was introduced to
the medium additionally after 96 h of incubation. To monitor the
sulfoxide production during MPS biotransformation, the samples
7
1
3 2 4
.0; KH PO , 1.0; NaCl, 1.0; MgSO ·7H O, 0.2; CaCl ·2H O, 0.02;
2
4
4
2
2
2
FeCl , 0.001. n-Hexadecane was used as a carbon source and
3
added at a concentration of 3% (v/v). The medium was supple-
mented with 0.1% yeast extract (Microgen, Russia) and 1 mL/L
trace element solution (1.5 g/L FeCl × 7H O, 0.1 g/L H BO , 0.01 g/L
(0.5 mL) were withdrawn from the culture broth under sterile con-
ditions at 24 h intervals over 168 h.
3
2
3
3
The long-term stability of immobilized cells was shown in
biocatalyst reuse MPS biotransformation studies. After each incu-
bation cycle (24 h/cycle), the used medium was decanted and PVA
cryogel-immobilized cells were washed three times with 0.5% NaCl
sterile solution and transferred into the fresh mineral medium
containing 0.5 g/L MPS and 0.1% (v/v) n-hexadecane. A biotransfor-
mation process was carried out and residual MPS and bioconversion
products were assayed in the post-fermentation medium.
ZnSO × 7H O, 0.05 g/L Co(NO ) × 6H O, 0.005 g/L CuSO × 5H O,
4
2
3
2
2
4
2
and 0.005 g/L MnCl × 4H O).
2
2
2.2. Chemicals
The following sulfides were used in this work: methyl phenyl
sulfide (99%, Sigma–Aldrich), ethyl phenyl sulfide (97%, Alfa
Aesar), benzyl methyl sulfide (98%, Acros), methyl p-tolyl sulfide
(
2
(
99%, Sigma–Aldrich), 2-chlorothioanisole (96%, Sigma–Aldrich),
-bromothioanisole (97%, Sigma–Aldrich), 4-methoxythioanisole
97%, Sigma–Aldrich). In some experiments, racemic methyl phenyl
2
.5. Analytical methods
Biotransformation products were extracted with ethyl acetate.
sulfoxide (rac-MPSO) obtained via chemical oxidation of MPS was
used as a substrate [5]. Enantiomeric purity and composition of
the racemic mixture was assayed by chiral HPLC. MPS (0.5, 1.0,
Combined organic extracts were dried over anhydrous sodium sul-
fate. The solvent was removed in a rotary evaporator (Heidolph).
For preliminary detection of biotransformation products, thin layer
chromatography (TLC) was carried out on silica gel plates with a
fluorescent indicator (Sigma–Aldrich), and the oxidation products
were observed under UV light (254 nm) by comparison with ref-
erence samples. The standard samples of sulfoxides and sulfones
were synthesized by a chemical oxidation method using hydro-
gen peroxide as an oxidant [5]. rac-MPSO was purified by flash
chromatography using a Buchi C-601/C-605 chromatograph. As an
eluent, a mixture of n-hexane and ethyl acetate (85:15) was used
at a flow rate of 5 mL/min.
1
.5, 2.0 g/L), sulfides (1.5 g/L), and rac-MPSO (0.5 g/L) were added
in the growth medium as solutions in isopropanol (1:10 v/v).
Chemical reagents (isopropanol, chloroform, acetone, ethyl acetate,
n-hexane and n-hexadecane) used were purchased from Merck,
Sigma–Aldrich and of analytical grade.
2
.3. Immobilization of whole bacterial cells
Macroporous heterophase cryogel of polyvinyl alcohol (Nevin-
nomysk, Russia) was used as a carrier. A bacterial suspension
The qualitative and quantitative analysis of the biotrans-
formation products was performed by a chromatography–mass
spectrometry analysis (LC–MS) using an Agilent 6890N gas chro-
matograph fitted with a HP-5MS SN US 15189741−1 quartz column
and an Agilent MSD 5973N quadruple mass spectrometer. The
products obtained by biotransformation were separated by col-
(
(
OD600 1.6) and a PVA solution (12% w/v) were mixed at 1:2 ratio
v/v). The resultant mixture was pipetted into 96-well polystyrene
round-bottom plates with a standard 8 × 12 configuration. Step-
wise freezing and thawing of the resultant composite were
performed according to the protocol [24]. The number of viable

1
0
T.I. Kylosova et al. / Journal of Molecular Catalysis B: Enzymatic 123 (2016) 8–13
Table 1
Biotransformation of MPS by immobilized G. terrae IEGM 136 cells.
MPS, g/L
Time (h)
48
24
72
Sulfide
c (%)
(R)-sulfoxide
ee (%)
Sulfide
c (%)
(R)-sulfoxide
Sulfide
c (%)
(R)-sulfoxide
ee (%)
c (%)
92.2
66.3
26.5
–
c (%)
ee (%)
c (%)
0.5
1.0
1.5
2.0
7.8
98
>99
92
–
–
27.9
85.4
100.0
100.0
72.1
98
>99
93
78
–
–
–
100
100
100
30.7
98
>99
95
33.7
73.5
100.0
–
14.6
69.3
88
c (%)—Relative amount of sulfide, sulfoxide in the reaction mixture. ee—Enantiomeric excess of (R)-sulfoxide. Sulfone was not detected in the reaction products.
O
R1
G. terrae IEGM 136
S
S
R1
R2
immobilized into PVA
R2
R)-2a-g
(
1
a-g
a: R
e: R
1
= Ph, R
= o-BrPh, R
2
= CH
3
; b: R
3
1
= Ph, R
= p-CH
2
= C
3
2
H
5
; c: R
2
1
= PhCH2, R
2
= CH
3
; d: R
1
= o-ClPh, R
= CH
2
= CH ;
3
1
2
= CH
; f: R
1
Ph, R = CH
3
; g: R = p-CH
1
3
OPh, R
2
3
Scheme 1. Biotransformation of prochiral sulfides using immobilized G. terrae IEGM
36 cells.
1
Table 2
Biotransformation of prochiral sulfides 1a–g to sulfoxides 2a–g by immobilized G.
terrae IEGM 136 cells.
Sulfide
Conversion (%)
Sulfoxide
Configuration and ee (%)
1
a
100.0
88.9
76.3
62.8
61.6
86.8
63.0
2a
2b
2c
2d
2e
2f
(R) 95
(R) 77
(R) 83
(R) 15
(R) 16
(R) 95
(R) 21
Fig. 1. Biotransformation of MPS (0.5 g/L) using immobilized G. terrae IEGM 136
cells.
1
b
1
c
1
d
umn chromatography on 0.06–0.20 mm silica gel (Merck) at a
substance-to-sorbent ratio of ca. 1:12. The mixture of n-hexane
and ethyl acetate gradient (5–40%) was used as an eluent. Spe-
cific rotation values of sulfoxides in chloroform, acetone or ethanol
were measured using a PerkinElmer 341 polarimeter at 589 nm.
The asymmetric center configurations of sulfoxides were proved
by comparing the obtained values [˛D] with the literature data
1e
1
f
1
g
2g
Sulfide concentration—1.5 g/L. Biotransformation time—72 h. ee—Enantiomeric
excess of sulfoxide.
for biotransformation of 0.5 g/L MPS to 24 h. The hourly dynamics
of the biotransformation process showed a sequential increase in
sulfoxide production in the incubation medium.
[
33–36]. The data of [˛D] and MS for the chiral sulfoxides are
shown in Supporting information. The sulfoxides obtained were
used as standards to determine the retention times of (R)- and
The enantiomeric excess of (R)-sulfoxide remained constant
when the reaction time was extended to 72 h (Table 1).
(
S)-sulfoxides by high performance liquid chromatography (HPLC)
using a LC Prominence chromatograph (Shimadzu) fitted with a
EuroCel 01 chiral column (250 mm × 4.6 mm) and a diode-matrix
detector. Conditions of HPLC for detection of sulfoxides are shown
in Supporting information. The HPLC system used a mobile phase
consisting of n-hexane/isopropanol, % (95:5, 98:2) at a flow rate
According to previously obtained data, the sulfide-oxidizing
activity of free G. terrae IEGM 136 cells decreased at higher (1.5 g/L)
MPS loading [20], whereas complete bioconversion of 1.0 and
1.5 g/L MPS into optically active (R)-sulfoxides with >99% ee and
95% ee was achieved within 48 and 72 h of incubation, respec-
tively, when immobilized cells were used (Table 1). The enhanced
MPS biotransformation by immobilized cells compared to free ones
was probably due to the protection of PVA cryogel-entrapped cells
from the toxic effect of high MPS concentrations, as well as due to
accelerated reaction rates caused by the high local cell density in
the immobilized matrix and the possible presence of a biosurfac-
tant [19,24]. When MPS concentration was increased up to 2.0 g/L,
the catalytic activity of immobilized Gordonia cells decreased. After
◦
of 1.0–2.0 mL/min. The thermostat temperature was kept at 40 C.
Enantiomeric excesses of (R)-sulfoxides were calculated as follows:
ee = [
R] − [S]
× 100%,
[
R] + [S]
where [R] and [S]—concentrations of (R)- and (S)-sulfoxides, respec-
tively.
7
2 h of biotransformation, 69.3% of residual sulfide was detected in
3
. Results and discussion
the incubation medium. Its complete biotransformation into (R)-
sulfoxide (93% ee) was achieved only after 168 h (data not shown).
3.1. Biotransformation of MPS
3.2. Biotransformation of sulfides 1a–g
The cell suspensions of G. terrae IEGM 136 obtained in the n-
hexadecane-supplemented medium and used for immobilization
contained 0.1% (w/v) residual n-hexadecane. We have demon-
strated before [20] that free Gordonia and Rhodococcus cells most
effectively catalyzed stereoselective oxidation of 0.5 g/L MPS within
Prochiral sulfides 1a–g with different substituents were used as
substrates in the experiments on biocatalyst stereospecificity. Since
the complete bioconversion of MPS was achieved at its maximal
concentration of 1.5 g/L, in our further studies we used sulfides at
the same concentration to obtain (R)-sulfoxides 2a–g according to
the scheme (Scheme 1).
1
44 h at a similar concentration of n-hexadecane in the medium. As
shown in Fig. 1, the immobilized cells allowed to reduce the time

T.I. Kylosova et al. / Journal of Molecular Catalysis B: Enzymatic 123 (2016) 8–13
11
Table 3
Time-course of MPS biotransformation by sequential addition of sulfide with immobilized G. terrae IEGM 136 cells.
Time (h)
0.5 g/L
0.75 g/L
1.0 g/L
Sulfide
c (%)
Sulfoxide
Sulfone
c (%)
Sulfide
c (%)
Sulfoxide
Sulfone
c (%)
Sulfide
c (%)
Sulfoxide
Sulfone
c (%)
Configuration
c (%)
Configuration
c (%)
Configuration
c (%)
and ee (%)
and ee (%)
and ee (%)
2
4
7
9
20
44
68
4
8
2
6
12.4
–
–
87.6
100.0
94.0
82.0
n/d
(R) 92
(R) 95
(R) 95
(R) 97
n/d
–
–
7.0
3.2
–
93.0
96.8
100.0
94.9
91.1
86.5
89.2
(R) 93
(R) 96
(R) 96
(R) 96
(R) 96
(R) 96
(R) 96
–
–
–
5.1
5.9
6.7
7.7
14.2
12.7
7.4
10.2
16.5
20.3
25.4
85.8
87.3
88.7
85.5
78.7
75.7
70.5
(R) 93
(R) 91
(R) 90
(R) 90
(R) 90
(R) 86
(R) 86
–
–
6.1
18
n/d
n/d
n/d
3.9
4.3
4.8
4.0
4.1
–
–
1
1
1
n/d
n/d
n/d
3.0
6.8
3.1
n/d
n/d
n/d
n/d
c (%)—Relative amount of sulfide, sulfoxide and sulfone in the reaction mixture. ee—Enantiomeric excess of (R)-sulfoxide. Initial MPS load was 0.5 g/L. Each new portion of
sulfide (0.5, 0.75 or 1.0 g/L) was added every 24 h giving total MPS load of 2.0, 4.25 or 5.5 g/L respectively. n/d—not defined.
fide concentrations limit the employment of this method to obtain
optically pure sulfoxides in industry applications.
3.4. Sequential additions of MPS into the fermentation medium
To reduce the toxic effect of high sulfide concentrations on liv-
ing cells and to increase the product yield, a sequential substrate
addition approach could be adopted. Recently its effective appli-
cation was demonstrated in the preparative synthesis of 118 mM
(S)-MPSO in a two-phase system using Rhodococcus sp. CCZU10
biomass (80 g/L) [21].
In the comparative experiments using a sequential substrate
addition approach, MPS was added into the fermentation medium
at 0.5, 0.75 and 1.0 g/L every 24 h during 3 or 5 days giving total MPS
load of 2.0, 4.25 or 5.5 g/L respectively. From the results shown in
Table 3, sulfide loading by portions of 0.5 g/L led to the accumula-
tion of 18% undesired sulfone in the reaction products already after
Fig. 2. Biocatalyst reuse in biotransformation of MPS (0.5 g/L).
As shown in Table 2, conversions for sulfides ranged between
1.6 and 100.0% and the enantiomeric excesses of target (R)-
9
6 h, thereby further experiment was impracticable. The increased
6
concentration of the added sulfide (up to 0.75 g/L) resulted in the
complete bioconversion of 2.0 g/L MPS into (R)-MPSO (96% ee)
within 72 h. Sulfone was not present in the reaction products. Sub-
sequent additions of MPS (0.75 g/L per portion) led to a gradual
accumulation of sulfone, with its level being 7.7% after 168 h of MPS
biotransformation. The non-reacted MPS (3.1%) was also present in
the post-fermentation medium. The total MPS load was 4.25 g/L
after 168 h, with the relative amount of the target (R)-MPSO (96%
ee) being 89%. When MPS was added by portions of 1.0 g/L, its total
load reached 5.5 g/L in the cultivation medium by the end of the
experiment. The relative amount of (R)-sulfoxide in the reaction
mixture was 70.5% after 168 h; at that, residual MPS (25.4%) and
its oxidation by-product sulfone (4.1%) were detected among bio-
transformation products. Further MPS biotransformation was not
expedient because of the bacterial cell leakage into the medium
owing to mechanically damaged PVA cryogel granules. As illus-
trated in Table 3, sulfide addition by 0.75 g/L per portion is most
suitable to produce (R)-sulfoxide (96% ee) in 85% chemical yield
sulfoxides were 15–95%. The presence of electron-accepting groups
Cl, Br) in the aromatic ring of the sulfide influenced negatively
(
the conversion (about 60%) and enantiomeric purity of sulfoxides
formed. Enantiomeric excess for sulfoxides 2d, 2e was only 15–16%.
With electron-donating groups present in the para positions of the
aromatic rings, the conversions of sulfides 1f and 1g were 86.8
and 63.0%, respectively. Sulfoxide 2f was produced with 95% ee.
Biotransformation of ethyl phenyl sulfide 1b and benzyl methyl
sulfide 1c proceeded with conversion of 88.9 and 76.3%, respec-
tively, yielding the corresponding (R)-sulfoxides 2b (77% ee) and 2c
(
83% ee). According to the reported data [16,37], biotransformation
by certain strains of microorganisms could lead to the formation of
both (R)- and (S)-sulfoxides depending on the structure of prochi-
ral sulfide used as a substrate. Based on our findings, all sulfoxides
produced in this study possessed the (R)-configuration of the asym-
metric center.
3.3. Reuse of immobilized G. terrae IEGM 136 cells
(data not shown).
Experimentally selected conditions allow performing the MPS
bioconversion at the minimum content of a hydrocarbon substrate
0.1%, w/v) and low biocatalyst concentration (in terms of wet
biomass 8.0 ± 0.5 g/L).
Sustained (84.9–90.5%) bioconversion of MPS (0.5 g/L) by immo-
bilized G. terrae IEGM 136 cells was observed during eight cycles,
this allowed to perform oxidation of 4.0 g MPS into (R)-sulfoxide
within 192 h using one portion of the biocatalyst (Fig. 2). The enan-
tiomeric excess of (R)-sulfoxide made up 96% during the first five
cycles. Further use of Gordonia cells entrapped into a PVA cryogel
matrix led to a gradual decrease in the enantiomeric excess of the
forming (R)-sulfoxide, and the ee value did not exceed 70% after the
eighth cycle. Moreover, the biocatalyst being used for four cycles,
the formation of a MPS oxidation by-product achiral sulfone (3–9%)
was observed. Thus, the decrease in enantioselectivity during the
biocatalyst reuse and low (no more than 2.5 g/L for five cycles) sul-
(
3.5. Biotransformation of rac-MPSO
The results obtained in the present study suggest that depend-
ing on the biotransformation conditions used, G. terrae IEGM 136
strain is able to catalyze the formation of enantio-enriched (R)-
sulfoxide via direct asymmetric oxidation of a prochiral sulfide,
and also to maintain high enantiomeric excess of (R)-sulfoxide

1
2
T.I. Kylosova et al. / Journal of Molecular Catalysis B: Enzymatic 123 (2016) 8–13
I
O
S
S
MPS
(R)-sulfoxide
O
O
S
IV
S
II
quick
slow
quick
O
S
O
(R)-sulfoxide
(R)-sulfoxide
S
S
O
MPS
O
Sulfone
MPS
III
S
S
slow
slow
quick
(
S)-sulfoxide
(S)-sulfoxide
O
O
S
V
S
quick
quick
S
n/s
O
S
O
O
S
O
(
R)-sulfoxide
(
R)-sulfoxide
S
MPS
O
Sulfone
quick
O
MPS
Sulfone
S
S
slow
slow
n/s
(S)-sulfoxide
(S)-sulfoxide
Fig. 3. Putative pathways for oxidation of alkyl aryl sulfides into sulfoxides adapted to (R)-MPSO. I. Chen et al. [15]; II. Adam et al. [38]; III. Modified from Porto et al. [28];
IV. Modified from Li et al. [16]; Zambianchi et al. [39]. n/s—not stereoselective.
Table 4
Currently, several putative pathways have been described for
microbial oxidation of prochiral sulfides to produce optically active
Biotransformation of rac-MPSO using immobilized G. terrae IEGM 136 cells.
Time (h)
0
Sulfoxide c (%)
Configuration and ee (%)
Sulfone c (%)
sulfoxides (Fig. 3). One-stage processes I and II of the oxidative sul-
fide biotransformation lead to the production of enantiopure or
enantio-enriched isomers with a (R)- or (S)-configuration of the
asymmetric center, respectively [15,38]. Two-stage processes III–V
occur with a moderate stereoselective differentiation accompanied
by the substrate overoxidation to form achiral sulfone. However,
microorganisms catalyzing efficient kinetic (III, IV) [16,28] or non-
kinetic (V) sulfoxide oxidation are also described [39].
100.0
97.8
89.9
83.9
74.1
–
(R) 3
(R) 11
(R) 19
(R) 35
–
2.2
10.1
16.1
25.9
24
48
72
1
68
c
(%)—Relative amount of sulfoxide and sulfone in the reaction mixture.
ee—Enantiomeric excess of (R)-sulfoxide. rac-MPSO concentration of 0.5 g/L.
According to our findings, synthesis of (R)-MPSO with a single
use of immobilized G. terrae IEGM 136 cells, and also at the ini-
tial stages of MPS oxidation (biocatalyst reuse reaction) or with
sequential sulfide additions, occurs according to path II (Fig. 3). This
is evidenced from the enantiomeric excess value (95%) of (R)-MPSO
formed and the lack of sulfone in the biotransformation products
mixture. At the next stages of the biocatalyst reuse or addition of
new portions of sulfide substrate, the high conversion of MPS into
(R)-sulfoxide is additionally supported by the kinetic resolution of
sulfoxides formed via sulfone production from (S)-sulfoxide (see
Section 3.5), and that corresponds to pathway IV.
The decrease in bioconversion and stereoselectivity of sulfox-
idation in the presence of 2 g/L MPS or at the last stages upon
repeated use of the biocatalyst are probably due to the toxic
effect caused by high sulfide concentrations and the limited num-
ber of immobilized biocatalyst to be reused. Similar results were
obtained in the MPS oxidation using Rhodococcus sp. ECU0066
[16] or kinetic resolution of ␥-lactam during repeated use of PVA
cryogel-immobilized Microbacterium hydrocarbonoxydans L29-9
[40], respectively.
formed by (S)-isomer oxidation to sulfone. In the experiments
using rac-MPSO as a substrate, the ratio of enantiomers was found
to change, with (R)-sulfoxide being predominant in the reaction
mixture (Table 4). Upon (R)-sulfoxide accumulation in the post-
fermentation medium, the sulfone content increased up to 25.9%.
The relative amount of (R)-sulfoxide (35% ee) was 74.1% after 168 h.
As evidenced from the obtained data, it is possible to enrich
one sulfoxide enantiomer via oxidation of the other one to sulfone.
Some researchers [16,18] consider this approach as an alternative
to produce enantiopure sulfoxides from racemates. However, in
case of G. terrae IEGM 136 strain, this way is technologically not
expedient because of the low oxidation rate of (S)-sulfoxide to sul-
fone.
3.6. Putative pathways of MPSO formation
Based on the results of MPS oxidation by immobilized G. terrae
IEGM 136 cells, it should be noted that Gordonia catalyze selective
R)-MPSO formation with a high (95%) enantiomeric excess.
(

T.I. Kylosova et al. / Journal of Molecular Catalysis B: Enzymatic 123 (2016) 8–13
13
4
. Conclusions
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allow (i) reducing (6 times) the duration of the MPS (0.5 g/L) bio-
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7
7 to 95%. Using sequential sulfide additions the toxic effect of MPS
[
[
[
[
[
[
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The research was partially supported by the grants of the
Russian Federation Ministry of Education and Science (State
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(
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