New kinetic and thermodynamic approach of one-pot synthesis of chiral alcohol by oxidoreductase from Candida parapsilosis

Article abstract of DOI:10.1016/j.procbio.2010.08.012

The stereoinversion process involving two sequential stereoselective oxidation and reduction reactions by oxidoreductases was a typical and important one-pot method for chiral secondary alcohol synthesis which has great potential for industrial application. The process to produce (S)-1-phenyl-1,2-ethanediol with whole cell was chosen as a model reaction to investigate the kinetic and thermodynamic properties of key enzymes which catalyzed the single oxidation and reduction step of the whole reaction. The kinetic data from initial rate experiments in the absence of product prove that both oxidation and reduction reactions follow Theorell-Chance BiBi mechanism. Parameters of V_m and ΔS suggest that the oxidation reaction was thermodynamically unfavorable and the restrictive step of the one-pot process was inhibited at high concentration of substrate PED. To remove the inhibition of substrate, extractive biocatalytic methods were successfully applied to improve the substrate PED concentration from 15 g/l to 50 g/l with high optical purity of 91.6% and yield of 82.5%.

Full text of DOI:10.1016/j.procbio.2010.08.012

Process Biochemistry 46 (2011) 233–239
Contents lists available at ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
New kinetic and thermodynamic approach of one-pot synthesis
of chiral alcohol by oxidoreductase from Candida parapsilosis
Xiao Qing Mu, Yan Xu∗, Ming Yang, Zhi Hao Sun
Key Laboratory of Industrial Biotechnology of Ministry of Education & School of Biotechnology, JiangNan University, 1800 Lihu Avenue, Wuxi, Jiangsu Province 214122, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 June 2010
Received in revised form 19 July 2010
Accepted 23 August 2010
The stereoinversion process involving two sequential stereoselective oxidation and reduction reactions
by oxidoreductases was a typical and important one-pot method for chiral secondary alcohol synthesis
which has great potential for industrial application. The process to produce (S)-1-phenyl-1,2-ethanediol
with whole cell was chosen as a model reaction to investigate the kinetic and thermodynamic properties
of key enzymes which catalyzed the single oxidation and reduction step of the whole reaction. The kinetic
data from initial rate experiments in the absence of product prove that both oxidation and reduction
reactions follow Theorell-Chance BiBi mechanism. Parameters of Vm and ꢀS suggest that the oxidation
reaction was thermodynamically unfavorable and the restrictive step of the one-pot process was inhibited
at high concentration of substrate PED. To remove the inhibition of substrate, extractive biocatalytic
methods were successfully applied to improve the substrate PED concentration from 15 g/l to 50 g/l with
high optical purity of 91.6% and yield of 82.5%.
Key words:
Candida parapsilosis
Stereoinversion
(S)-1-phenyl-1,2-ethanediol
Kinetic
Extractive biocatalytic method
© 2010 Elsevier Ltd. All rights reserved.
1. Introduction
thermodynamic researches of single oxidation or reduction reac-
tion were published [8–10].
With the steadily increasing demands for high valuable
enantiopure alcohols as the important fine chemicals and phar-
maceutical ingredients, many new asymmetric synthesis methods
catalyzed by microorganism were established and studied. Among
these processes, stereoinversion had been world widely focused
and developed owing to its high theoretical yield and optical
purity both of 100% from a cheap racemic substrate [1–3]. This
special one-pot process technique was described as a two-step
inter-conversion reaction between two enantiomers [4–6] which
includes the stereoselective oxidation of one alcohol enantiomer
into the corresponding ketone and its simultaneous reduction to
the mirror-image alcohol. Although there were lots of literatures
of successful stereoinversion reactions, few plant processes were
established for the common drawbacks such as long reaction time
more than one day and low substrate concentration of 1% as
reviewed by Christian and coworkers [7].
Enantiopure (S)-1-phenyl-1,2-ethanediol (PED) was widely
used in many sectors such as pharmaceutical, material and fla-
vor fragrance industries [11,12]. Nie and coworkers reported the
stereoinversion process to product (S)-PED catalyzed by Candida
parapsilosis [13,14]. Itwas foundthatit gothigh optical purityof 99%
and yield of 83% at substrate concentration of 15 g/l and low optical
purity of 35.5% and yield of 67.7% at high substrate concentration
of 30 g/l [15]. In further researches, two key enzymes, NAD+-linked
(R)-specific alcohol dehydrogenase (CPADH) and NADPH-linked
(S)-specific carbonyl reductase (CPCR) were purified and charac-
terized [16,17]. Kinetic and thermodynamic studies indicated that
both the oxidation reaction and the reduction reaction follow the
Theorell-Chance BiBi mechanism in which the coenzymes first bind
to the free form of the enzyme and without the formation of central
ternary complexes.
2. Materials and methods
To improve the efficiency of stereoinversion, the catalytic char-
acteristics including kinetic and thermodynamic properties of the
key enzymes of inter-conversion should be investigated. It would
be helpful to find the reason of low efficiency and to improve it. For
the difficulty of purification of the key oxidoreductases and prepa-
ration of the unstable reaction intermediate, only few kinetic and
2.1. Materials
All chemicals used in this work are of analytical grade and commercially avail-
able. ␤-Hydroxy-hypnone is prepared according to the method described by Liese
[18] and determined by HPLC.
2.2. Microorganism and culture condition
C. parapsilosis CCTCC M203011 was obtained from China Center for Type Cul-
ture Collection, Wuhan, China. Medium for growth of C. parapsilosis contained 4%
(w/v) glucose, 0.3% yeast extract, 1% (NH4)2HPO4 and 0.5% MgSO4·7H2O. Microor-
∗
Corresponding author. Fax: +86 510 85 91 82 01.
E-mail address: yxu@jiangnan.edu.cn (Y. Xu).
1359-5113/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2010.08.012

234
X.Q. Mu et al. / Process Biochemistry 46 (2011) 233–239
Scheme 1. Stereoinversion of enantiomer A leads to its mirror-image counterpart B.
ganism was cultured at 30 ◦C for 48 h. After incubation, the cells were harvested by
centrifugation.
where v is the initial rate, V the maximum rate, [A] the concentration of NADPH,
[B] the concentration of ␤-hydroxy-hypnone, Kia the dissociation constant of CPCR-
NADPH, Ka the Michaelis constant for NADPH, and Kb the Michaelis constant for
␤-hydroxy-hypnone.
Eqs. (4) and (5), which correspond to the linear mixed-type inhibition model
and linear non-competitive inhibition rate equation, were fitted to the experimental
data of the initial rate experiments with product inhibition
2.3. Kinetic studies of CPADH
All details about the kinetic studies and some kinetic data of CPADH are
described in Ref. [19].
Vm[S]
v =
v =
kcat
(4)
(5)
(6)
Km(1 + ([I]/Ki)) + [S](1 + ([I]/Kii))
2.4. Purification of (S)-specific carbonyl reductase (CPCR) [17]
Vm[S]
CPCR, reductase which catalyzed the reduction reaction of stereoinversion, was
purified after being dissociated from an ultrasonic oscillator (Sonic Materials Co.,
USA), precipitated using ammonium sulfate (40–80%) precipitation, and eluted
by ion-exchange chromatography (DEAE sepharose Fast Flow column, Pharmacia
Biotech), hydrophobic interaction chromatography (phenyl sepharose-650S col-
umn, Pharmacia Biotech) and affinity chromatography (Blue sepharose Fast Flow
column, Pharmacia Biotech). Purity of all the enzymes, including CPADH and CPCR,
was analyzed by SDS-PAGE and the amount of pure enzyme protein was deter-
mined using Bradford reagents (Bio-Rad) with bovine serum albumin as a standard
[20] (Scheme 1).
(Km + [S])(1 + ([I]/Ki))
Vm
=
[E0]
where Vm is the maximum rate, [S] the concentration of substrates, Km the Michaelis
constant of ␤-hydroxy-hypnone, [I] the concentration of inhibitor, Ki the apparent
value of the dissociation constant of enzyme–inhibitor complex, Kii the apparent
value of the dissociation constant of enzyme-varied substrate inhibitor complex,
[E0] is the total concentration of the enzyme.
2.8. The thermodynamics of enzymes
2.5. Enzyme activity of CPCR
The thermodynamics of the initial velocity was investigated in the temperature
range 20–60 ◦C under conditions where the substrates were limited. The ther-
mal treatment was performed in a constant temperature water bath for a fixed
period of time and cooled immediately in ice water. The residual enzyme activ-
ity was then measured to get the specific reaction rate Kd according to half-lives
of first and second order reactions. The temperature dependence of the reaction
rate constant for the studied enzymes served as the basis for fitting to the data
into the Arrhenius equation [21], where k0 is the pre-exponential factor, Ed is
the activation energy, R is the gas constant and T is the absolute temperature in
Kelvin
The standard assay mixture for reduction comprised, in 250 ␮l, 1 mM NADPH,
4 mM ␤-Hydroxy-hypnone and 50 ␮l enzyme solution (99 U/ml) using a 20 mM
Tris–HCl buffer, pH 4.5. The reaction mixture was incubated for 2 min without the
enzyme at 25 ◦C, and then the reaction was initiated by the addition of the catalyst.
The decrease of the amount of the NADPH was measured spectrophotometrically at
340 nm. One unit of enzyme activity was defined as the amount of enzyme catalyzing
the oxidation of 1 ␮mol of NADPH per min under the assay conditions.
2.6. Enzymatic reaction for the kinetic research of CPCR
ln 2
kd
t1/2
=
(7)
(8)
The determination of the kinetic mechanism of the reduction reaction of ␤-
hydroxy-hypnone to (S)-PED catalyzed by CPCR was done by initial rate and product
inhibition studies with NADPH (0–0.3 mM), 20 ␮l of the enzyme stock solution, ␤-
hydroxy-hypnone (0–0.8 mM), NADP+ (0–0.06 mM) as product inhibitor, pyrazole
(0–0.1 mM) as dead-end inhibitor and rac-PED (0.5–20 mM) as variable substrate.
All the experiments were performed in different pH values of Tris–HCl buffer.
ꢀ
ꢁ
ꢀEd
RT
kd = k0 exp
−
2.9. Stereoinversion reaction conditions of different systems
The mono-phase reaction mixture in 10 ml system contained 0.1 mol/l potas-
sium phosphate buffer (pH 6.5), the aqueous–organic reaction system contained
50% n-hexane and 50% 0.1 mol/l potassium phosphate buffer (pH 6.5), the
aqueous–aqueous system contained 0.7 mol/l potassium phosphate buffer (pH 6.5)
and 500 g/l PEG 4000, and the liquid–solid system contained 0.1 mol/l potassium
phosphate buffer (pH 6.5) and 20 g/l resin NKAII. All these reaction systems con-
tained 100 g/l (w/v) wet cells and were performed at 30 ◦C for 72 h with shaking at
150 rpm.
2.7. Data processing [19]
All data in the figures represent the mean of experimentally determined initial
velocities (triplicates), and are used to determine the kinetic parameters. Estimates
of kinetic parameters, and of their asymptotic standard error, were obtained by
fitting the appropriate rate equation to experimental initial rate data obtained using
a non-linear least-squares computer program of MATLAB (The MathWorks, Inc. MA,
USA).
Eqs. (1) and (2) represent the rate equations for the sequentially ordered BiBi
mechanism and Theorell-Chance BiBi mechanism in the presence of product P. Eq.
(3) represents the initial rate equation in the absence of product of both mechanisms
V[A][B]
v =
(1)
KiaKb + Kb[A] + Ka[B] + [A][B] + (KiaKbKq[P]/KpKiq) + (KbKq[A][P]/KpKiq) + ([A][B][P]/Kip
)
V[A][B]
v =
(2)
(3)
K
iaKb + Kb[A] + Ka[B] + [A][B] + (KiaKbKq[P]/KpKiq) + (KbKq[A][P]/KpKiq
)
V[A][B]
v =
KiaKb + Kb[A] + Ka[B] + [A][B]

X.Q. Mu et al. / Process Biochemistry 46 (2011) 233–239
235
Table 1
The stereo-selectivities and cofactor dependent of CPADH and CPCR.
Enzyme
Substrate
Coenzyme
Reaction type
Product
Enzyme activity (U/mg)
CPADH
CPADH
CPADH
CPADH
CPADH
CPADH
CPCR
CPCR
CPCR
CPCR
CPCR
(R)-PED
(R)-PED
(S)-PED
(S)-PED
␤-Hydroxy-hypnone
␤-Hydroxy-hypnone
(R)-PED
(R)-PED
(S)-PED
NADP+
NAD+
Oxidation
Oxidation
Oxidation
Oxidation
Reduction
Reduction
Oxidation
Oxidation
Oxidation
Oxidation
Reduction
␤-Hydroxy-hypnone
␤-Hydroxy-hypnone
–
–
(R)-PED
(R)-PED
–
–
0.3
9.6
–
NADP+
NAD+
–
NADPH
NADH
NADP+
NAD+
0.2
12.6
–
–
–
NADP+
–
–
(S)-PED
␤-Hydroxy-hypnone
NAD+
–
NADPH
(S)-PED
101.4
CPCR
␤-Hydroxy-hypnone
NADH
Reduction
(S)-PED
0.7
3. Results and discussion
determined when the substrate concentrations varied. As shown
in Fig. 1(a and b), the initial rate of oxidation reaction reached
the maximum values and then decreased rapidly with the increas-
ing concentration of PED while it remained the same with the
increasing concentration of ␤-hydroxy-hypnone. The initial rate of
reduction also increased with increasing substrate concentration
and reached the maximum value when the concentrations of coen-
3.1. The stereo-selectivities and cofactor independent of CPADH
and CPCR
The stereo-selectivities and cofactor independent of key
enzymes which catalyzed the oxidation and reduction reactions of
stereoinversion were helpful to study the reaction kinetics. Accord-
ing to the data listed in Table 1, it was very interesting that the
oxidation and reduction reactions were quite different. For the oxi-
dation reaction was reversible and the key enzyme CPADH was
mainly NAD+-dependent while the reduction reaction was irre-
versible and the key enzyme CPCR was NADPH-dependent. And
the enantiomeric excess of substrate would keep increasing dur-
ing the oxidation reaction and keep a constant of 100% during the
reduction reaction.
zyme and ␤-hydroxy-hypnone were 0.42 mmol l−1 and 1 mmol l−1
,
respectively (Fig. 1c and d). The rate decreased rapidly with the
increasing concentration of coenzyme while it remained constant
with the increasing concentration of ␤-hydroxy-hypnone.
According to the reaction rate, it was proved that the oxidation
reaction has been inhibited at high concentration of PED which
decrease the efficiency of stereoinversion reaction. Removal of the
substation inhibition is one key factor to improve the reaction effi-
ciency.
3.2. Initial rate patterns of the oxidation and reduction reaction
3.3. Mechanism of reduction reaction catalyzed by CPCR
To observe the effect of the substrate on the reaction rate,
both the initial rates of the oxidation and reduction reaction were
The initial rate of the reduction reaction catalyzed by CPCR was
observed with fixed concentrations of ␤-hydroxy-hypnone and
Fig. 1. Effects of substrate concentration on the initial velocity of the oxidation and reduction reaction. (a) Effects of PED concentration on the initial velocity of the oxidation
with 0.5 mmol l−1 NAD+. (b) Effects of NAD+ concentration on the initial velocity of the oxidation with 25 mmol l−1 PED. (c) Effects of ketone concentration on the initial
velocity of the reduction with 0.3 mmol l−1 NADPH. (d) Effects of NADPH concentration on the initial velocity of the reduction with 1 mmol l−1 ketone.

236
X.Q. Mu et al. / Process Biochemistry 46 (2011) 233–239
Table 2
Comparison of estimates of kinetic parameters of oxidation and reduction reaction
catalyzed by CPADH and CPCR.
CPADH for oxidationa
CPCR for reduction
Vm (␮mol min−1 mg−1
Kia (mmol)
Kb (mmol)
Ka (mmol)
Kib (mmol)
)
10.7 0.5
0.16 0.01
5.0 0.4
0.06 0.01
1.875 0.2
107.5 6.5
0.391 0.03
2.774 0.22
1.161 0.17
0.934 0.08
a
Data from Mu et al. [19].
Table 3
Estimates of kinetic parameters derived from initial rate studies on product (␤-
hydroxy-hypnone) inhibition of the oxidation of 1-phenyl-1,2-ethanediol catalyzed
by dehydrogenase from Candida parapsilosis with PED as the varied substrate.
Estimates of kinetic
parameters
Mixed-type competitive
inhibition
Competitive
inhibition
Vm (␮mol min−1 mg−1
Km (mM)
Ki (mM)
)
101.6 5.1
3.1 0.3
1.74 0.1
182.1 112.3
100.7 5.9
3.2 0.3
1.69 0.09
–
Kii (mM)
Previous studies suggested that oxidoreductases mainly fol-
lowed the well-known sequential ordered BiBi mechanism [22,23].
Recent literature [24–26] showed that some of these enzymes will
follow a special form of ordered BiBi mechanism, the Theorell-
Chance BiBi mechanism, which does not form central ternary
complexes. The simplest method, the study of product inhibition
caused by the first product of the reduction reaction compared to
the second substrate of the carbonyl reductase, was carried out to
analyze the kinetic significance of the ternary complexes (Table 3).
Although most parameters shown in Table 1 were similar in
fitting both the rate Eqs. (4) and (5), the estimate of Kip of the
sequential order BiBi mechanism was much larger than that of
the Theorell-Chance BiBi mechanism, and the asymptotic standard
error of the estimate of Kii of the sequential order BiBi mecha-
nism was higher than the parameter estimate of Ki. Consequently
[I]/Kii in Eq. (4) could be discarded. Then the linear mixed-type
inhibition model (Eq. (4)) of the sequential order BiBi mechanism
was transformed into the linear competitive inhibition model (Eq.
(5)) of Theorell-Chance mechanism. It proved that the reduction
reaction of stereoinversion process follows Theorell-Chance BiBi
mechanism which is a special case of the ordered BiBi mechanism
where the central complex (E·NADPH·Ketone → E·NADP+·S-PED)
becomes kinetically insignificant and does not contribute to the
rate of reduction reaction.
3.4. Kinetic parameters of key enzymes catalyzed stereoinversion
It was also very important to find the restrictive step of a sequen-
tial two-step reaction by comparing the kinetic parameters such
as the maximum initial reaction rate and Michaelis constant. The
kinetic parameters of CPADH and CPCR were estimated at their
optimal conditions and listed in Table 2. According to the maxi-
mum initial reaction rate of reduction which was almost 10 times
higher than the oxidation reaction, it was suggested that the oxida-
tion step was the restrictive step in the whole process. Temperature
along with pH value, the main factors affecting the initial reac-
tion rate, were another key factors to improve the efficiency of
stereoinversion.
Fig. 2. Kinetic studies of reduction reaction catalyzed by CPCR. (a) Effect of ketone
on the initial velocity of the reduction reaction of ketone with NADPH as the vari-
able substrate. The concentration of ketone were: (ꢀ) 0.2 mM, (ꢁ) 0.3 mM, (×)
0.5 mM, and (ꢂ) 20 mM. (b) Slope replot of trendline in Fig. 2a versus 1/[␤-Hydroxy-
hypnone]. (c) Intercept replot of trendline in Fig. 2a versus 1/[␤-hydroxy-hypnone].
with varied concentrations of NADPH in the absence of products
(Fig. 2). From Eqs. (1) and (2), the parameters of the initial rate were
obtained (Table 2). The good fit of these data to Eq. (1), as well as the
fact that the secondary plots derived from it, such as the gradients
and ordinate intercepts versus the reciprocal of the fixed substrate
concentration were also linear, strongly suggests that the reaction
was most probably of the sequential type involving ternary central
complexes. Therefore, these patterns excluded the ping-pong type
of mechanism involving more than one stable form of the enzyme.
3.5. Effects of temperature on activities of key enzymes
Temperature affects the enzymatic reaction rate significantly.
The effects of temperature on CPADH and CPCR activities are shown
in Fig. 3. These indicated that both enzymes closely followed the

X.Q. Mu et al. / Process Biochemistry 46 (2011) 233–239
237
Fig. 4. Effects of temperature on the efficiency of stereoinversion reaction with
whole cells as catalysts with 15 g/l PED ((ꢂ) yield value; (᭹) e.e. value).
pared with the reduction reaction. The optimization studies of
reaction temperature for whole cells (Fig. 4) indicate that the effi-
ciency of stereoinversion would reach the maximum at the optimal
temperature of oxidation reaction.
3.6. Effects of pH on the thermodynamic properties of key
enzymes
Enzymes are catalysts with a high dependence on the pH. Higher
or lower pH levels lead to partial or complete inactivation and
different enzymes have their own optimal pH values. It is also
important to observe the kinetic and thermodynamics properties
of both CPADH and CPCR at different pH levels to get more infor-
mation in order to understand the stereoinversion process when
resting cell used as biocatalyst. Some important kinetic and ther-
modynamics parameters of the two enzymes at different pH values
were determined. The corresponding values of Vm, Km, Kcat, Kcat/Km
are listed in Table 5. The results indicate that the affinity and cat-
alytic efficiency of CPADH and CPCR were greatest at pH 9 and 4.5,
respectively. According to the optimization studies of reaction con-
dition for whole cells, it was proved that the optimal pH value
of stereoinversion reaction was 6.5–7.0 (Fig. 5), and the kinetic
parameters including Vm, Km, Kcat, Kcat/Km were quite lower at the
neutral condition than that at their optimal pH value. The optimal
pH values inconsistent of CPADH and CPCR decrease the efficiency
of stereoinversion.
Fig. 3. Effect of temperature to stereoinversion of key enzyme. (a) Arrhenius plots
for the estimation of the thermodynamic parameters of CPCR and CPADH. (b) Effects
of temperature on half-life time of CPCR and CPADH. (ꢃ) for CPCR, (ꢀ) for CPADH.
Arrhenius model where the enzyme activities increased with tem-
perature up to a maximum value. A semi-log plot versus the
reciprocal of the absolute temperature showed that the threshold
temperature of CPADH and CPCR were 35 ◦C and 45 ◦C, respec-
tively (Fig. 3). The activation energy (Ea) was calculated from the
slope by plotting of ln vversus 1/T below the T threshold, which
was 34.3 kJ mol−1 and 9.2 kJ mol−1, respectively. The energies of
the reversible unfolding of the enzyme were estimated from the
plots above the threshold in Fig. 3(a), which were 82.3 kJ mol−1
and 60.5 kJ mol−1. It is also important to observe the effect of tem-
perature on the stability of these two enzymes by thermodynamic
parameter determination. The data show that these two enzymes
followed the first-order rate model (data not shown). Fig. 3(b) indi-
cates that the half time of CPADH and CPCR decreased rapidly with
increasing temperature. It shows that CPADH is more stable at tem-
perature below 30 ◦C than CPCR. The values of t1/2 for CPADH and
CPCR are quite similar when the temperature is above 40 ◦C.
The thermodynamic activation parameters of irreversible
denaturation of CPADH and CPCR were estimated at temperatures
from 20 ◦C to 50 ◦C according to the formula by Eyring [27] and
are listed in Table 4. All these thermodynamic parameters, espe-
cially the positive entropy value of the oxidation reaction, indicate
that the oxidation reaction is thermodynamically unfavorable com-
3.7. Elimination of substrate inhibition to improve the efficiency
of stereoinversion
According to Section 3.1, the substrate inhibition was the
great barrier to improve the efficiency of stereoinversion reac-
tion. Extractive biocatalytic methods of two-phase system, such
as aqueous/organic two-phase system, aqueous two-phase system
and liquid–solid two-phase system, were the most common ways
to eliminate the inhibition of organic substrate at higher concentra-
tion. As described in Table 6, several different two-phase systems
were compared. Aqueous two-phase system and liquid–solid two-
phase system with resin could improve the substrate concentration
Table 4
Thermodynamic activation parameters of the irreversible denaturation of CPADH and CPCR.
Reaction
Enzyme
ꢀEd (kJ mol−1
)
ꢀG (kJ mol−1
)
ꢀH (kJ mol−1
)
ꢀS (J mol−1 K−1
)
Oxidation
Reduction
CPADH
CPCR
21.16
70.61
80.89–89.98
80.44–83.00
108.01
70.24
85.38
−36.34

238
X.Q. Mu et al. / Process Biochemistry 46 (2011) 233–239
Table 5
Comparison of estimates of kinetic and thermodynamics parameters of oxidation and reduction reaction catalyzed by CPADH and CPCR at different pH values.
pH
CPADH
CPCR
Vm
Km
(mmol)
ꢀE
(kJ mol−1
Kcat
(s−1
Kcat/Km
Vm
Km
(mmol)
ꢀE
(kJ mol−1
Kcat
(s−1
Kcat/Km
(␮mol min−1 mg−1
)
)
)
(mmol−1 s−1
)
(␮mol min−1 mg−1
)
)
)
(mmol−1 s−1
)
4
4.5
5
6
7
8
8.5
9
10
–
–
–
–
–
–
–
–
–
–
86.9
107.5
95.2
60.0
58.2
33.6
29.0
25.8
–
3.02
2.77
2.95
3.72
3.96
4.59
4.83
5.41
–
72.05
70.61
71.25
79.18
81.35
89.97
92.33
94.66
–
43.58
53.91
47.74
30.09
29.19
16.85
14.54
12.94
–
14.43
19.46
16.18
8.09
7.37
3.67
3.01
2.39
–
1.1
1.3
2.0
3.2
8.5
10.7
8.8
12.4
11.2
7.9
7.2
5.6
5.0
5.5
36.69
34.56
31.86
26.25
23.15
21.16
22.56
0.66
0.78
1.20
1.92
5.10
6.42
5.28
0.05
0.07
0.15
0.27
0.91
1.28
0.96
Table 6
Improvement of the efficiency of stereoinversion by elimination of substrate inhibition.
Different reaction system
e.e. at 30 g/l PED
Yield at 30 g/l PED
e.e. at 50 g/l PED
Yield at 50 g/l PED
Mono aqueous phase
Aqueous–organic
Aqueous–organic
Liquid–solid
35.5%
14.3%
99.1%
99.4%
67.7%
57.2%
90%
10.3%
9. 2%
76.2%
91.6%
55.8%
54.5%
81.4%
82.5%
94.2%
tive Research Team in University IRT0532 and Jiangsu Development
Strategies of Science and Technology (BG2007008), for their finan-
cial support.
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Acknowledgments
We would like to thank the National Science Foundation of China
(NSFC) (no. 20776060) and the National Key Basic Research and
Development Program of China (973 program), the Ministry of Edu-
cation, the Hi-Tech Research and Development Program of China
(2007AA02Z226), the Program for Changjiang Scholars and Innova-
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