Enantioselective hydrolysis of diethyl 3-hydroxyglutarate to ethyl (S)-3-hydroxyglutarate by immobilized Candida antarctica lipase B

Article abstract of DOI:10.1016/j.molcatb.2010.03.009

Optically pure ethyl (S)-3-hydroxyglutarate [(S)-3-EHG] is used as a key precursor for synthesis of a variety of pharmaceutically important compounds. In this work, we established an efficient procedure for enantioselectively hydrolyzing diethyl 3-hydroxyglutarate (3-DHG) to optically active (S)-3-EHG employing immobilized Candida antarctica lipase B (Novozym 435). Under the optimized conditions: pH 7.0, agitation speed 200 rpm, temperature 40 °C, 3-DHG concentration 0.15 mol L^-1, and enzyme loading 7 g L ^-1, (S)-3-EHG was prepared in above 95% ee value and 98.5% yield, and the reaction was free from external mass transfer and intra-particle diffusion limitations and kinetically controlled. The inhibitions of substrate (3-DHG) and product (3-EHG) were excluded because both displayed no decline in activity at elevated concentrations within the given ranges. In addition, ethanol, a byproduct of the reaction, inhibited lipase B following an uncompetitive inhibition pattern. The kinetic constants were obtained through non-linear regression, with values of V_max 1.29 mmol min^-1 g ^-1, K_m 0.06 mol L^-1, and K_i 0.37 mol L^-1, respectively.

Full text of DOI:10.1016/j.molcatb.2010.03.009

Journal of Molecular Catalysis B: Enzymatic 66 (2010) 90–94
Contents lists available at ScienceDirect
Journal of Molecular Catalysis B: Enzymatic
journal homepage: www.elsevier.com/locate/molcatb
Enantioselective hydrolysis of diethyl 3-hydroxyglutarate to ethyl
(S)-3-hydroxyglutarate by immobilized Candida antarctica lipase B
Hua-Ping Dong, Ya-Jun Wang, Yu-Guo Zheng∗
Institute of Bioengineering, Zhejiang University of Technology, Hangzhou, Zhejiang Province 310014, PR China
a r t i c l e i n f o
a b s t r a c t
Article history:
Optically pure ethyl (S)-3-hydroxyglutarate [(S)-3-EHG] is used as a key precursor for synthesis of a variety
of pharmaceutically important compounds. In this work, we established an efficient procedure for enan-
tioselectively hydrolyzing diethyl 3-hydroxyglutarate (3-DHG) to optically active (S)-3-EHG employing
immobilized Candida antarctica lipase B (Novozym 435). Under the optimized conditions: pH 7.0, agitation
speed 200 rpm, temperature 40 ◦C, 3-DHG concentration 0.15 mol L−1, and enzyme loading 7 g L−1, (S)-3-
EHG was prepared in above 95% ee value and 98.5% yield, and the reaction was free from external mass
transfer and intra-particle diffusion limitations and kinetically controlled. The inhibitions of substrate
(3-DHG) and product (3-EHG) were excluded because both displayed no decline in activity at elevated
concentrations within the given ranges. In addition, ethanol, a byproduct of the reaction, inhibited lipase
B following an uncompetitive inhibition pattern. The kinetic constants were obtained through non-linear
regression, with values of Vmax 1.29 mmol min−1 g−1, Km 0.06 mol L−1, and Ki 0.37 mol L−1, respectively.
© 2010 Elsevier B.V. All rights reserved.
Received 31 October 2009
Received in revised form 20 March 2010
Accepted 20 March 2010
Available online 27 March 2010
Keywords:
Ethyl (S)-3-hydroxyglutarate
Enantioselective hydrolysis
Uncompetitive inhibition
Enantioselectivity
Kinetic constants
1. Introduction
Optically active ethyl 3-hydroxyglutarate [3-EHG] is an attrac-
tive building block for synthesis of a variety of pharmaceutically
important compounds, such as statins, pimaricin and l-carnitine
[1–6]. Ethyl (S)-3-hydroxyglutarate [(S)-3-EHG] is a precursor
to various enantiopure intermediates for chiral drugs including
cholesterol-lowering drugs like Lipitor®, and l-carnitine which is
a required vitamin analogue for mammalian metabolism and is
applied in the active treatment of systemic and myopathic defi-
ciencies [7–9]. The enantiopure compounds with R-configuration
such as ethyl (R)-3-hydroxyglutarate (ee > 98%) and 4-cyano-
(R)-3-hydroxybutanoate are synthesized by enzymatic catalysis
[3,5,10,11]. By contrast, few reports concerning preparation of (S)-
3-EHG through enantioselective hydrolysis of prochiral diethyl
3-hydroxyglutarate (3-DHG) are available [1,2]. Gopalan et al. pre-
pared (S)-3-EHG with the yield and ee value of 38% and 69%,
using Arthrobacter sp. (ATCC 19140). Thereafter, Jacobsen et al.
provided a promising synthetic routine featured by improved
ee value of 91% and yield of 80% employing immobilized C.
antarctica lipase B (CALB, Novozym 435); and they also pre-
pared 4-cyano-(S)-3-hydroxybutanoate in ee value of 98% from
4-carbamoyl-(S)-3-hydroxybutanoate [12]. The problem in exist-
Scheme 1. Enantioselective hydrolysis of 3-DHG to (S)-3-EHG catalyzed by
Novozym 435.
ing (S)-3-EHG biosynthesis is that enantiopurities and the yields
of these methods are not satisfactory, which failed to meet the
requirement for enantiopure compound, and leaded to no com-
mercial (S)-3-EHG available. In addition, there are no reports by far
about the studies on the reaction kinetics and enzymatic mecha-
nisms.
Candida antarctica lipase B exhibits excellent thermal stabil-
ity, activity and stereospecificity [13,14], and is widely applied in
organic synthesis for esterification and hydrolysis [13], displaying
critical industrial importance. Its enzymatic performance can be
further enhanced to various extents through immobilization onto a
multitude of support materials, including hydrophilic or hydropho-
bic polymers [15–20].
In the present work, we attempted to establish an efficient
bioconversion of 3-DHG to enantiopure (S)-3-EHG (Scheme 1) by
commercial CALB (Novozym 435), and study the reaction kinetics in
detail. Moreover, the operating parameters including reaction tem-
perature, pH, and mass transfer limitations, as well as inhibitory
effects of substrate and products on commercial CALB were exam-
ined.
∗
Corresponding author. Tel.: +86 571 88320379; fax: +86 571 88320630.
E-mail address: zhengyg@zjut.edu.cn (Y.-G. Zheng).
1381-1177/$ – see front matter © 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.molcatb.2010.03.009

H.-P. Dong et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 90–94
91
2. Experimental
2.1. Enzyme and chemicals
Novozym 435 (CALB, fraction B of lipase from C. antarctica
immobilized on macroporous polyacrylate resin, diameter varying
between 0.3 mm and 0.9 mm) was supplied by Novozymes, Den-
mark. 3-DHG was purchased from Sigma. Other chemicals were of
analytical grade. (S)-3-EHG (ee ≥ 95%) was prepared in our labora-
tory.
2.2. Hydrolytic reaction
The hydrolytic reaction was carried out in a 50 ml erlenmeyer
flask in a shaking water bath tank at 40 ◦C and 200 rpm. 1.5 mmol
of 3-DHG was dissolved into 10 ml sodium phosphate buffer
(30 mmol L−1, pH 7.0), and then 0.07 g Novozym 435 was added
to initialize the reaction. During the reaction process for 30 min,
pH value of reaction media was kept at 7.0 by titrating with a 1 N
NaOH using a pH-Stat technique unless stated otherwise. At regu-
lar intervals, 0.5 ml aliquot sample was withdrawn to detect 3-DHG
and (S)-3-EHG.
Fig. 1. Time evolution of total conversion (᭹) and ee value of (S)-3-EHG (ꢀ) at 3-
DHG concentration of 0.15 mol L−1. Operating conditions: 40 ◦C, pH 7.0, 200 rpm,
and 0.07 g CALB.
2.6. ee Value of (S)-3-EHG determination
Otherwise, the enzyme was filtered off at the end of the reac-
tion, and the filtrate was subjected to ethyl acetate extraction after
pH adjusted to 2 with 2 N HCl. The extract phase was dehydrated
by anhydrous Na2SO4, and ethyl acetate was removed by reduced
distillation to obtain the product.
Determination of the ee value of (S)-3-EHG was based on a
normal phase HPLC separation of (R)- and (S)-3-EHG after con-
verting 3-EHG into two diastereomers by a derivative reaction
with chiral amine [21]. The normal phase HPLC conditions were as
follows: the mobile phase was petroleum ether–dichlormethane
(1:10), and flow rate was set at 1.5 ml min−1. The sample (20 ␮l)
was loaded onto a silica gel column (Kromasil; 250 mm × 4.6 mm,
5 ␮m) and visualized under 254 nm UV light. The retention times
of the derivatives of (R)-3-EHG (D-1) and (S)-3-EHG (D-2) were
3.85 min and 4.15 min, respectively. The ee values were obtained
by the ratio of peak areas of D-1 (A1) and D-2 (A2) as follows: ee
(%) = (A2 − A1)/(A2 + A1)×100.
2.3. Determination of the product
The chemical structure of the product was determined by 1H
(500 MHz) and 13C NMR (100 MHz) recorded with a NMR spectrom-
eter (Bruker Avace III), using deuteriochloroform as the solvent.
The stereoconfiguration of the product was determined by mea-
surement of specific rotation of the product (Rudolf AutoPol IV,
USA). The chemical purity (≥98%) was determined by HPLC, and ee
value of the product was measured by normal phase HPLC after a
derivative reaction with chiral amine [21].
3. Results and discussion
The product was elucidated to be enantiopure (S)-3-EHG
(ee ≥ 95%) based on the following experimental data. 1H NMR
(ppm): ı 1.28 (3H, t, J = 7.0 MHz), 2.59 (4H, dd), 4.18 (2H, q,
J = 7.0 MHz), 4.49 (1H, m), 13C NMR (ppm): ı 13.87, 40.32, 40.52,
60.83, 64.53, 171.82, 175.66. The specific rotation ([˛]2D0) of +1.90
(c 11.5, in acetone) confirmed S configuration [1], and ee value was
calculated to be ≥95%.
3.1. 3-EHG synthesis procedure
3-DHG conversion profile was firstly investigated under con-
trolled and uncontrolled pH conditions at 40 ◦C, catalyzed by
Novozym 435. In the uncontrolled pH case, pH of reaction solution
dropped sharply from 7.20 to 3.70, attributing to the accumulation
of 3-EHG. Accordingly, the conversion of 3-DHG declined to 70%
partly because of the inhibitory effect of acidified environments on
Novozym 435. In contrast with the uncontrolled pH case, the con-
version was elevated to be 98.5% at “regulated pH” of 7.0, showing
that the pH control during the reaction was advantageous to keep
high enzymatic activity. As shown in Fig. 1, the reaction rate was
fast in the initial 5 min, possessing a linear correlation with reac-
tion time; and it decreased with reduced substrate concentration.
The ee value of (S)-3-EHG remained invariant at high value dur-
ing the reaction process, and the conversion of the monoacids to
diacids was also not observed, demonstrating that Novozym 435
possesses excellent regioselectivity and enantioselectivity.
2.4. Determination of specific initial rate
Specific initial rate was defined as mmol of 3-EHG produced per
minute and per gram of enzyme, and determined from the time
course of 3-EHG formation using a second order polynomial fitting
by regression analysis of 3-EHG concentration and determining the
initial slope of the tangent to the curve.
2.5. High performance liquid chromatography (HPLC) analysis
The content of the substrate and product was determined
and quantified by HPLC using
a hypersil ODS 2 column
3.2. Influence of mass transfer limitation on hydrolytic reaction
(4.6 mm × 250 mm, 5 ␮m) at room temperature. The mobile phase
consisted of acetonitrile and 0.5% (v/v) aqueous triethylamine solu-
tion at ratio of 1:4 (v/v). The flow rate was set at 1 ml min−1; and the
injection volume of each sample was 10 ␮l. By using a UV-detector
(Spd-10 A Vp plus, Shimadzu), 3-DHG was monitored at 220 nm
with retention time of 5.50 min; 3-EHG was detected at 260 nm
with retention time of 4.10 min.
Due to two phases containing aqueous bulk and the solid bound
enzyme in the reaction media, the influence of mass transfer in the
external bulk and intra-particle should be properly assessed. The
effect of the external mass transfer limitation could be solved by
increasing the agitation speed. As shown in Fig. 2, the initial rate
increased with agitation speed up to 200 rpm. No dependence of

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H.-P. Dong et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 90–94
Fig. 3. Effect of pH on specific initial rate (ꢀ) and ee value (ꢁ) of (S)-3-EHG. Oper-
ating conditions: 40 ◦C, 200 rpm, 0.07 g CALB, 0.15 mol L−1 3-DHG, and pH value of
reaction media was kept at the initial value. Sodium acetate buffer, 4.5–5.5; sodium
phosphate buffer, 6.0–8.0; Tris–HCl buffer, 7.5–8.9.
Fig. 2. Effect of agitation speed on specific initial rate. Operating conditions: 40 ◦C,
pH 7.0, 0.07 g CALB, and 0.15 mol L−1 3-DHG.
initial rate on agitation speed over 200 rpm was observed, suggest-
ing that no external diffusion limitation was present at that speed.
Therefore, the agitation speed was set at 200 rpm to remove the
external mass transfer limitation.
Intra-particle diffusion resistance was confirmed by calculating
Thiele’s modulus, ˚, which is given as [22]:
to different extents beyond this range. On the other hand, the opti-
mum pH for the enantioselectivity was located at between 7.0 and
7.5; besides, the declining trend in ee values outside the optimal pH
range was strikingly more pronounced than that of yields, suggest-
ing that the enantioselectivity was more vulnerable to pH changes.
Due to the best activity and enantioselectivity of Novozym 435 at
pH 7.0, the hydrolytic reaction was suitably to be performed in the
neutral media.
ꢀ
ꢁ
2
v
VP
˚ =
(1)
DEC AP
where v is the reaction rate (mmol s−1g−1), DE is the effective
diffusivity coefficient (cm2 s−1), C is the substrate concentration
(mol L−1), VP is Novozym 435 particle volume (cm3), and AP is the
surface area of the Novozym 435 particle (cm2). Since the enzyme
particle is spherical, Thiele’s modulus can be expressed as:
3.4. Effect of temperature
The thermal stability of biocatalysts is always taken as one of
the most important criteria for industrial applications. Novozym
435 is regarded as being rather resistant to heat, so the exam-
ined temperature range was broadened from 20 to 70 ◦C in the
present work. The maximum values of the activity and enantios-
electivity were commonly observed at 40 ◦C (Fig. 4). The enzymatic
activity improved with temperatures from 20 ◦C to 40 ◦C, and the
enzyme was still very active at 50 ◦C. However, the higher enan-
tioselectivity was maintained at temperatures below 40 ◦C, and it
decreased sharply at temperatures exceeding 40 ◦C, demonstrating
that the excessive high temperature resulted in the conformational
change of the active site, which determines the enantioselectivity
of Novozym 435. In addition, within the temperature range from
ꢀ ꢁ
2
v
DEC
R
˚ =
(2)
3
and DE expression can be given as:
ε
DE = DS
ꢀ
(3)
where ε is the porosity of the particle, ꢀ is the tortuosity factor of the
particle, and DS is the substrate diffusivity in the reaction medium
(cm2 s−1). DS could be estimated to be 5.89 × 10−5 cm2 s−1 from the
Siebel equation [23]. In addition, the values of ε and ꢀ were 0.5 and
6, respectively, so the value of DE was calculated to be 4.91 × 10−6
from Eq. (3). By substituting the appropriate values into Eq. (2), with
v= 0.022 mmol s−1 g−1 (maximum reaction rate), C = 0.15 mol L−1
,
R = 0.03 cm, and DE = 4.91 × 10−6 cm2 s−1, ˚ was calculated to be
0.003. Such a small value for Thiele’s modulus indicated that the
reaction was kinetically controlled and the intra-particle diffusion
could be neglected, which may be attributed to the small particle
size and comparatively large pore size of Novozym 435.
3.3. Effect of pH
Enzymes are catalysts with a high dependence on pH. Generally,
they have an optimal working pH, and higher or lower values can
lead to partial inactivation. Extreme pH can disrupt enzyme ter-
tiary structure and result in catalytic activity loss. Different buffer
solutions with pH varying from 4.5 to 9.0 were chosen, and the pH
value for each treatment was kept constant during the reaction pro-
cess using a pH-Stat technique. As shown in Fig. 3, Novozym 435
showed higher activity in the range from pH 6.0 to 8.0, and declined
Fig. 4. Effect of temperature on specific initial rate (ꢀ) and ee value (ꢁ) of (S)-3-EHG.
Operating conditions: 200 rpm, pH 7.0, 0.07 g CALB, and 0.15 mol L−1 3-DHG.

H.-P. Dong et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 90–94
93
Fig. 5. Effect of 3-DHG concentration on specific initial rate ( ) and ee value (
of (S)-3-EHG. Operating conditions: 40 ◦C, 200 rpm, pH 7.0, and 0.07 g CALB.
)
Fig. 6. Effect of initial ethanol concentration on specific initial rate. Operating con-
ditions: 40 ◦C, 200 rpm, pH 7.0, 0.07 g CALB, and 0.15 mol L−1 3-DHG.
of byproduct ethanol on Novozym 435 at concentrations above
0.08 mol L−1 was shown in Fig. 6.
20 ◦C to 40 ◦C, the hydrolytic reaction catalyzed by Novozym 435
obeyed Arrhenius law, and the activation energy, Ea, was calculated
to be 16.63 kJ mol−1
.
3.7. Kinetics and mechanism of reaction
Generally, hydrolytic reactions performed in an aqueous solu-
tion conform to uni-bi kinetic mechanisms, because water is
present in large quantities and the variation in its concentration
is negligible. In the case of ethanol inhibition, the dissociation con-
stant Ki should be included in the kinetic model. Therefore, the
equations for different inhibitions are described below:
Competitive inhibition,
3.5. Determination of substrate inhibition
It was observed in Fig. 5 that the reaction rate increased with
the elevated amounts of 3-DHG up to 0.40 mol L−1, suggesting that
more 3-DHG molecules were available to bind the active sites of
the enzyme as the amount of 3-DHG increasing, and 3-DHG had no
inhibitory effect on enzyme activity. On the contrary, ee value of (S)-
3-EHG decreased with 3-DHG concentration exceeding 0.2 mol L−1
.
Vmax[S]
Km(1 + [I]/Ki) + [S]
v =
(4)
(5)
(6)
This was consistent with the finding of Jacobsen et al. that ee
value of (S)-3-EHG dropped from 90% to 87% as 3-DHG increased
from 0.30 mol L−1 to above 1 mol L−1 [1]. Such negative effect on
enantioselectivity was probably resulted from the strengthened
hydrophobic interaction between excessive 3-DHG and Novozym
435 [17], which leaded to the conformational change in the active
site of Novozym 435 [24]. Therefore, lowering 3-DHG concentration
was advantageous to raise ee value of (S)-3-EHG, and the similar
dependence of enantioselectivity of biocatalyst on the substrate
concentration was also found in the nitrilase-catalyzed hydrolytic
reaction [25].
Noncompetitive inhibition,
Vmax[S]
v =
Km(1 + [I]/Ki) + [S](1 + [I]/Ki)
Uncompetitive inhibition,
Vmax[S]
v =
Km + [S](1 + [I]/Ki)
where v is the apparent initial reaction rate, Vmax is the apparent
maximum reaction rate, [S] is the substrate (3-DHG) concentration,
[I] is the initial ethanol concentration, Km is the Michaelis–Menten
constant, and Ki is the dissociation constant of ethanol binding to
lipase–substrate complex.
3.6. Determination of product inhibition
Enzymes do not alter reaction equilibrium because they do
not affect thermodynamics; increasing the product concentration
always speeds up reverse reaction, yielding apparent product inhi-
bition. 3-EHG and ethanol were produced in the hydrolysis of
3-DHG. Addition of 3-EHG into the solution before reaction would
cause pH drop, which was proved to result in decreases in the
activity and enantioselectivity of Novozym 435. To exclude pH fluc-
tuation and effect thereof, pH of reaction media were maintained
at 7.0 with 1 N NaOH. Under this condition, the conversions (>98%)
and ee values (≥95%) almost remained invariant as the initial 3-
EHG concentration increased up to 0.2 mol L−1, indicating it was
the acidified conditions caused by 3-EHG accumulation, not 3-EHG
itself, that inhibited Novozym 435.
In order to validate the kinetic mechanism, the inhibition plot
was generated by measuring the initial reaction rate at vary-
ing 3-DHG concentrations at each fixed concentration of ethanol
(0–0.4 mol L−1). As shown in Fig. 7, the Lineweaver–Burk plots of
velocities versus 3-DHG concentrations at different fixed ethanol
concentrations yielded a set of lines with the same slope, indi-
cating a typical uncompetitive inhibition. This result was further
confirmed by non-linear fitting of the data to the uncompetitive
model (Eq. (6)). In other words, ethanol could not bind to the
free lipase, but bind to the bound forms of enzyme and 3-DHG,
forming a non-productive end complex. The maximum reaction
velocity (Vmax), Michaelis constant (Km) and inhibition constant
(Ki) were calculated to be 1.29 mmol min−1 g−1, 0.06 mol L−1 and
0.37 mol L−1 through non-linear regression analysis. The obtained
parameters were used to simulate initial rate versus concentration
of 3-DHG at 0.2 mol L−1 ethanol. Fig. 8 showed that the experimen-
tal model fitted the data very well.
However, 60% of enzymatic activity was lost when nearly pure
alcohol (containing 3% water as the substrate) was used as the
reaction media, suggesting that the activity was not totally lost
as Novozym 435 was exposed to the pure alcohol because of its
good tolerance to the organic phase. The details about inhibition

94
H.-P. Dong et al. / Journal of Molecular Catalysis B: Enzymatic 66 (2010) 90–94
40 ◦C, substrate concentration 0.15 mol L−1, and the enzyme load-
ing 7 g L−1, (S)-3-EHG with ee value above 95% was obtained by
Novozym 435 catalysis, and the conversion reached above 98%.
Both values were much improved in comparison with the previous
study.
In addition, the absence of internal diffusion limitation in the
immobilized particle in this hydrolytic reaction was confirmed.
It was also explained that 3-DHG and 3-EHG had no inhibi-
tion on Novozym 435. However, ethanol with concentrations
above 0.08 mol L−1 inhibited Novozym 435 following the uncom-
petitive inhibition pattern. The kinetic constants were obtained
subsequently through non-linear regression analysis, and the fit-
ted parameters are: Vmax 1.29 mmol min−1 g−1, Km 0.06 mol L−1, Ki
0.37 mol L−1. From the established kinetic equation, the simulated
data of initial rate coincided with the experimental data.
Acknowledgments
This work was supported by the Major Basic Research Devel-
opment Program of China (no. 2007CB714306) and the Natural
Scientific Foundation of Zhejiang (no. Z4090612).
Fig. 7. Lineweaver–Burk plot of reciprocal initial rates versus reciprocal 3-DHG con-
centration at different ethanol concentrations. (ꢀ) 0 mol L−1, (ꢁ) 0.1 mol L−1, (ꢂ)
0.2 mol L−1, (᭹) 0.4 mol L−1. Operating conditions: 40 ◦C, 200 rpm, pH 7.0, and 0.07 g
CALB.
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Fig. 8. Comparison of simulated (ꢀ) and experimental initial rates (ꢁ) versus con-
centration of 3-DHG. Operating conditions: 40 ◦C, 200 rpm, pH 7.0, 0.07 g CALB, and
0.2 mol L−1 ethanol.
4. Conclusion
The detailed investigation of reaction kinetics and enzymatic
mechanisms in enantioselective hydrolysis of 3-DHG was firstly
conducted in this study, in addition to optimization of reac-
tion conditions, especially the agitation speed, pH, temperature
and concentration of 3-DHG, which were key factors influenc-
ing the yield and ee value of (S)-3-EHG. Under the optimized
reaction conditions: pH 7.0, agitation speed 200 rpm, temperature