Dual enzymatic dynamic kinetic resolution by Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase and Candida antarctica lipase B

Article abstract of DOI:10.1039/c6ra18895h

The immobilization of Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase (TeSADH) using sol-gel method enables its use to racemize enantiopure alcohols in organic media. Here, we report the racemization of enantiopure phenyl-ring-containing secondary alcohols using xerogel-immobilized W110A TeSADH in hexane rather than the aqueous medium required by the enzyme. We further showed that this racemization approach in organic solvent was compatible with Candida antarctica lipase B (CALB)-catalyzed kinetic resolution. This compatibility, therefore, allowed a dual enzymatic dynamic kinetic resolution of racemic alcohols using CALB-catalyzed kinetic resolution and W110A TeSADH-catalyzed racemization of phenyl-ring-containing alcohols.

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Dual enzymatic dynamic kinetic resolution by
Thermoanaerobacter ethanolicus secondary
alcohol dehydrogenase and Candida antarctica
lipase B†
Cite this: RSC Adv., 2016, 6, 96616
a
a
Ibrahim Karume, Musa M. Musa, Odey Bsharat,a Masateru Takahashi,b
*
Samir M. Hamdanb and Bassam El Alia
The immobilization of Thermoanaerobacter ethanolicus secondary alcohol dehydrogenase (TeSADH) using
sol–gel method enables its use to racemize enantiopure alcohols in organic media. Here, we report the
racemization of enantiopure phenyl-ring-containing secondary alcohols using xerogel-immobilized
W110A TeSADH in hexane rather than the aqueous medium required by the enzyme. We further showed
that this racemization approach in organic solvent was compatible with Candida antarctica lipase B
(CALB)-catalyzed kinetic resolution. This compatibility, therefore, allowed a dual enzymatic dynamic
kinetic resolution of racemic alcohols using CALB-catalyzed kinetic resolution and W110A TeSADH-
catalyzed racemization of phenyl-ring-containing alcohols.
Received 25th July 2016
Accepted 30th September 2016
DOI: 10.1039/c6ra18895h
www.rsc.org/advances
micro composites were also used as racemization catalysts for
secondary alcohols in a one-pot DKR with lipase-catalyzed KR.6
Introduction
Candida antarctica lipase B (CALB), known as Novozyme 435,
is the most popular lipase used in the KR-catalyzed trans-
esterication of alcohols.7 Lipases catalyze the enantioselective
transesterication with preference to R-congured alcohols in
KR, nevertheless W104A CALB mutant and proteases such as
subtilisin have shown selectivity for S-alcohols in KR of
secondary alcohols.8 The inexpensive and environmentally
benign nature of enzymes presents a preferable alternative to
challenges by organometallic catalysts. The scarcity of race-
mases due to the high stereoselectivity of the biosynthetic
pathways, however, leaves less hope for a full enzymatic DKR
approach. Alcohol dehydrogenases (ADHs), which catalyze the
enantioselective redox reactions of ketones and their corre-
sponding alcohols,9–11 have been successfully reported to
accomplish racemization of enantiopure alcohols.12 In this
front, we have shown that Thermoanaerobacter ethanolicus
secondary alcohol dehydrogenase (TeSADH) mutants can be
used to racemize enantiopure secondary alcohols.13,12a
TeSADH is a nicotinamide adenine dinucleotide phosphate
(NADP+)-dependent ADH that is well-known for its high thermal
stability and high tolerance to organic media.14 Xerogel-
encapsulated W110A TeSADH mutant has been successfully
employed in the asymmetric reduction of ketones in non-
aqueous media.15 Unlike the wild-type TeSADH, W110A
TeSADH reduces a variety of ketones with high conversions and
enantiomeric excess (ee).15 Although W110A TeSADH follows
Prelog's rule,16 the stereoselective reduction of prochiral
ketones by TeSADH may occur by delivering the hydride from
The vital role of optically active compounds in pharmaceuticals,
agrochemicals, and other synthetic chemicals unceasingly
demands cheap and environmentally benign methodologies to
obtain enantiomerically pure compounds. For example, drug
efficacy could be inuenced in cases where one enantiomer
outrages the activity of the opposite enantiomer.1 Deracemiza-
tion approaches that involve the formation of a single enan-
tiomer from its corresponding racemate, such as cyclic
deracemization, enantioconvergence, stereoinversion and
dynamic kinetic resolution (DKR), are ideal alternatives to
kinetic resolution (KR).2,3 DKR that couples a KR reaction with
an in situ racemization for the slow-reacting enantiomer in KR
has been the most advanced deracemization approach that
exceeds the 50% yield limit of KR and reaches up to a 100%
yield.3 Non-enzymatic systems, for example, chiral ferrocene–
ruthenium complexes, have been employed in DKR of racemic
alcohols.4,5 A signicantly popular approach for DKR of alcohols
relies on combining an enzymatic KR with a transition metal
complex based racemization.2b,3a Core–shell beta-silicalite-1
aDepartment of Chemistry, King Fahd University of Petroleum and Minerals, Dhahran,
31261, Saudi Arabia. E-mail: musam@kfupm.edu.sa; Fax: +966-13-860-4277; Tel:
+966-13-860-7343
bDivision of Biological and Environmental Sciences and Engineering, King Abdullah
University of Science and Technology, Thuwal 23955-6900, Saudi Arabia
† Electronic supplementary information (ESI) available: Gas chromatograms,
nuclear magnetic resonance spectra, and mass spectra are provided. See DOI:
10.1039/c6ra18895h
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Scheme 1 Racemization of enantiopure alcohols using W110A TeSADH. ADPR ¼ adenosine diphosphoribose, R1 is more sterically hindered than R2.
removal of water molecules required to maintain the enzyme's
activity. The efficiency of W110A TeSADH-catalyzed racemiza-
tion showed an improvement in various solvents with high
log P. Among the solvents tested, hexane and cyclohexane
showed the best racemization performance (Table 1, entries 6
and 7). We also tested the racemization of (S)-1a using xerogel-
immobilized W110A TeSADH in Tris–HCl buffer solution, and
noticed an improvement in racemization efficiency. This indi-
cates that using hydrophobic solvents like hexane restricts the
diffusion of the substrate through the xerogel. The xerogel
contains water that is enough to maintain the enzyme's active
conformer and perhaps too little to trigger CALB-hydrolysis of
ester products.15
The course of the racemization reaction of (S)-1a was moni-
tored with time using free and immobilized W110A TeSADH to
check the effect of the reaction medium and the enzyme
immobilization on the racemization efficiency. We monitored
the racemization of (S)-1a at various time intervals using free
W110A TeSADH in Tris–HCl buffer solution (pH 8.0, 50 mM)
and in biphasic system containing Tris–HCl buffer solution (pH
8.0, 50 mM)/hexane (1 : 1, v/v), and compared their perfor-
mances with that for xerogel-immobilized W110A TeSADH in
hexane (Fig. 1). Nearly complete racemization was observed
aer 20 h when the free enzyme was used in an aqueous
medium (from >99% to 5% ee). Moderate racemization effi-
ciency was noticed in a biphasic system containing Tris–HCl
buffer solution/hexane (1 : 1, v/v) and these conditions resulted
in a reduction in the ee of (S)-1a from >99% to 33% in 24 h. A
rapid racemization was observed in the rst 4 hours in Tris–HCl
(54% ee) and in the biphasic system (64% ee). Good enzyme
activity in both systems is attributed not only to the abundance
of water required to maintain enzyme's activity but also to the
free state of the enzyme, which facilitate the diffusion of the
substrate in and out of the active site of the enzyme.
Racemization of (S)-1a using xerogel-immobilized W110A
TeSADH containing 0.2 mg of the enzyme in hexane (1.0 mL)
resulted in poor racemization efficiency, however, higher
enzyme loading resulted in signicant improvement (81% vs.
63% ee using 0.2 mg and 0.6 mg of the enzyme, respectively,
Table 1). In a previously reported asymmetric reduction of
phenyl-ring-containing ketones using xerogel-immobilized
W110A TeSADH in hexane,17 isopropanol was used as a cosub-
strate and a cosolvent, which might have facilitated the diffu-
sion of the substrate to the active site, and thus resulting in high
conversions to alcohols. The low racemization performance of
the xerogel-immobilized W110A TeSADH in hexane when
compared to that of the free enzyme is attributed to the slow
diffusion rate of the substrate through the sol–gel matrix due to
Scheme 2 One-pot dual enzymatic DKR of secondary alcohols.
CALB: Candida antarctica lipase B, R1 has higher Cahn–Ingold–Prelog
priority that R2.
the undesired Si face producing the anti-Prelog alcohols,
a phenomenon known as “selectivity mistakes”, leading to
racemization (Scheme 1).12a Such selectivity mistakes may occur
because of the reverse t of substrates in the enzyme's active
site.
Herein, we successfully report a strategy and its imple-
mentation for a dual enzymatic DKR of secondary alcohols. This
approach relies on racemization of enantiopure secondary
alcohols using xerogel-immobilized W110A TeSADH in water-
immiscible organic solvents, which enabled its use with
CALB-catalyzed KR in one pot (Scheme 2).
Results and discussion
We started by immobilizing W110A TeSADH together with
NADP+ and NADPH using the sol–gel method as described
previously.15 The wet sol–gel (hydrogel) was then dried to
produce a xerogel. We selected W110A TeSADH because it
showed good performance in racemization of enantiopure
alcohols and high tolerance to organic solvents.12a,15 We then
conducted the racemization of (S)-1-phenyl-2-propanol [(S)-1a]
using xerogel-immobilized W110A TeSADH in different organic
solvents (Table 1). This substrate was selected because it was
reported as a substrate for W110A TeSADH17 and its counterpart
enantiomer as a substrate for CALB.18 Racemization of (S)-1a in
water-miscible organic solvents like methanol and dimethyl
sulfoxide (DMSO) showed no activity, which could be due to
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Table 1 Racemization of (S)-1a using xerogel-encapsulated W110A TeSADH in organic mediaa
% ee S enantiomerc
Entry
Solvent
log Pb
Before
aer
1
2
3
4
5
6
7
8
DMSO
Methanol
MTBE
Dichloromethane
Toluene
Cyclohexane
Hexane
ꢀ1.4
ꢀ0.7
0.9
1.3
2.7
3.2
3.8
3.8
4.3
4.4
4.8
—
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
>99
96
>99
94
84
81
Hexane
Heptane
63d
96
9
10
11
12
2,2,4-Trimethylpentane
Octane
93
92
59
Tris–HCle
a
Unless stated, reactions were performed at 50 ꢁC and 200 rpm for 24 h using (S)-1-phenyl-2-propanol (0.04 mmol), xerogel [W110A TeSADH (0.2
mg), NADPH (1.0 mg) and NADP+ (1.0 mg)] in a solvent (1.0 mL). b log P (partition coefficient) values based on octanol–water partition coefficient.19
c
The ee of the corresponding acetate ester derivative of the produced alcohol was determined by chiral GC. d 0.6 mg of W110A TeSADH was used
e
instead of 0.2 mg. Aqueous medium [Tris–HCl (pH 8.0, 50 mM)/acetonitrile (95 : 5 v/v)].
the absence of the cosubstrate. However, using immobilized might lead to a highly restricted t of this enantiomer in the
ADHs in non-aqueous media to perform racemization of active site (i.e., high preference to (S)-enantiomer with less
enantiopure alcohols is still interesting because of the versatile selectivity mistakes). Similar results were previously obtained
applications in such media.20
using various mutants of TeSADH in racemization of (R)-1c
Xerogel containing W110A TeSADH (0.6 mg) and hexane as using a free enzyme.13
a solvent were adopted as optimum conditions for racemization
of other enantiopure alcohols in non-aqueous media (Table 2).
Under these conditions, racemization of (R)-1a gave better
results than (S)-1a (Table 2, entries 1 and 2). Similar results were
obtained in racemization of (S)-4-phenyl-2-butanol [(S)-1b] and
(R)-4-phenyl-2-butanol [(R)-1b], however, with lower efficiencies
(Table 2, entries 3 and 4). This is attributed to the structure of
1b, which seems to properly t in the active site of W110A
TeSADH and thus increasing the substrates' stereospecicity of
the enzyme, and hence slowing racemization. TeSADH is re-
ported to have two pockets within the active site, which are
varying in size.21 The relatively small size of 1a might enable the
entire molecule to t in the large pocket with either direction
allowing more selectivity mistakes hence the enhanced race-
mization. The improved racemization efficiency of (R)-alcohols
compared to (S)-alcohols is attributed to the fact that W110A
TeSADH obeys Prelog's rule, and thus expected to produce (S)-
congured alcohols from the generated ketones whilst the (R)-
congured alcohol is formed by selectivity mistakes; similar
results were observed previously in W110A TeSADH-catalyzed
racemization of phenyl-ring-containing enantiopure alcohols
using free enzyme in an aqueous medium.12a Least racemiza-
tion efficiency was observed for (R)-1-phenyl-2-butanol [(R)-1c]
Fig. 1 Comparison of racemization of (S)-1a using free and immobi-
lized W110A TeSADH in aqueous, biphasic and organic media.
(98% to 90% ee), which is due to the extended chain length that
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Table 2 Xerogel-encapsulated W110A TeSADH-catalyzed racemization of enantiopure phenyl-ring-containing alcoholsa
% eeb
Entry
Substrate
R1
R2
before
aer
1
2
3
4
5
(S)-1a
(R)-1a
(S)-1b
(R)-1b
(R)-1c
C6H5CH2
C6H5CH2
C6H5CH2CH2
C6H5CH2CH2
C6H5CH2
CH3
CH3
CH3
CH3
>99(S)
>99(R)
>99(S)
>99(R)
98(R)
63(S)
48(R)
93(S)
80(R)
90(R)
CH3CH2
a
Unless stated, reactions were performed at 50 ꢁC at 200 rpm, for 24 h using the alcohol (0.04 mmol), xerogel-[W110A TeSADH (0.6 mg), NADPH (1.0
mg) and NADP+ (1.0 mg)] in hexane (1.0 mL). b The ee of the corresponding ester derivative of the produced alcohol was determined by chiral GC.
The ability of xerogel-immobilized W110A TeSADH to
Compatibility of a stereoselective KR reaction with an efficient
partially racemize enantiopure alcohols in hexane as the sole racemization method is an essential requirement for a successful
reaction medium encouraged us to attempt a one-pot dual DKR. We, therefore, proceeded to check compatibility of W110A
enzymatic DKR that combines a CALB-catalyzed KR and W110A TeSADH-catalyzed racemization and CALB-catalyzed KR in one-
TeSADH-catalyzed racemization. The rst requirement for pot. The presence of water in xerogel might retard the CALB-
a successful DKR is an efficient KR. We started by comparing catalyzed KR of alcohols. As previously reported,15 the xerogel
the selectivity of CALB-catalyzed KR of (rac)-1a using selected contains more than 50% by weight water, and thus the two
acyl donors in hexane (Table 3). Isopropenyl acetate showed catalyst systems (i.e., xerogel-immobilized W110A TeSADH and
high activity (48% conv.) with moderate selectivity (E ¼ 13–17). CALB) were positioned such that direct contact is avoided as
Similar results were obtained with vinyl acetate with lower explained in the experimental section (see Fig. S2 of ESI†).
selectivity. Methyl methoxy acetate showed excellent selectivity Deracemization of (rac)-1a via DKR using CALB and W110A
(E > 100) with low conversion (Table 3, entry 4). 2,2,2-Tri- TeSADH dual enzymatic approach resulted in 74% conversion to
uoroethyl butyrate gave moderate conversion and selectivity the corresponding (R)-acetate ester in 68% ee. The calculated total
(Table 3, entry 5).
composition of R enantiomers (i.e., (R)-1a and its corresponding
Table 3 Comparison of reactivity and selectivity of CALB-catalyzed kinetic resolution of (rac)-1a using selected acyl donorsa
Entry
Acyl donor
Conv. (%)
eepb (%)
E(R/S)d
1
2
3
4
5
Isopropenyl acetate
Isopropenyl acetate
Vinyl acetate
Methyl methoxy acetate
2,2,2-Triuoroethyl butyrate
26
48
31
5
81(R)
78(R)c
75(R)
>99(R)
87(R)
13
17
10
>100
17
15
a
Unless stated, reactions were performed using (rac)-1a (0.05 mmol), acyl donor (0.1 mmol) and CALB (1.0 mg) in hexane (1.0 mL) at 50 ꢁC at 50 rpm
b
c
d
for 3 h. The ee of the produced ester was determined by chiral GC. Reaction aer 6 h. E values were calculated according to eqn (1) in the
Experimental section.
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Fig. 2 GC chromatogram of products of DKR of (rac)-1a using CALB-catalyzed KR and W110A TeSADH-catalyzed racemization.
Table 4 W110A TeSADH-CALB one-pot DKR of selected racemic phenyl-ring-containing alcoholsa
Entry
Substrate
R1
R2
Conv. (%)
eeb (%)
Total Rc (%)
1
2
3
4
5
(rac)-1a
(rac)-1b
(rac)-1c
(rac)-1d
(rac)-1e
C6H5CH2
C6H5CH2CH2
C6H5CH2
C6H5CC
p-MeO-C6H4CH2CH2
CH3
CH3
CH3CH2
CH3
CH3
74
70
15
71
69
68(R)
66(R)
99(R)
63(R)
71(R)
62
58
52
58
59
a
Unless stated, reactions were performed using (rac)-1a–e (0.05 mmol), 2b (0.1 mmol), xerogel-immobilized enzyme [W110A (0.6 mg), NADP+ (1.0
mg) and NADPH (1 mg)], CALB (1 mg) in hexane (1.0 mL) at 50 ꢁC at 50 rpm for 24 h. b The ee of the ester product was determined by chiral GC.
c
Total R (%) ¼ R-alcohol + R-ester.
(R)-acetate ester) was 62%, which indicates that CALB-catalyzed functionalized silanes that might improve substrate diffusion
KR and W110A TeSADH are compatible (Fig. 2).
rate is needed; this might improve the racemization efficiency.
To our knowledge, this is the rst report that shows that
Among the tested substrates, best DKR results were obtained
with (rac)-1a (74% conv. & 68% ee, Table 4, entry 1); this is a dual enzymatic DKR of isolated alcohols is possible. Devel-
attributed to the good extent of racemization of its enantiopure oping new mutants of TeSADH is crucial for the improvement of
forms by W110A TeSADH when compared with other the racemization efficiency of enantiopure alcohols, and thus
substrates. Under the same conditions, (rac)-1b, (rac)-4-phenyl- improving the effectiveness of the reported dual DKR. Extensive
3-butyn-2-ol [(rac)-1d] and (rac)-4-(40-methoxyphenyl)-2-butanol effort is currently conducted in our laboratory to generate new
[(rac)-1e], gave similar results (69–71% conv. & 58–59% total mutants of TeSADH through protein engineering to improve the
R), Table 4, entries 2, 4 and 5. Lowest activity was observed with racemization efficiency of TeSADH.
(rac)-1c (15% conv. & 52% R) due to slow KR (Table 4, entry 3).
To obtain an efficient DKR of alcohols, the rate of the racemi-
zation of the slow reacting enantiomer in KR should be faster
than the rate of the esterication of the fast reacting alcohol.22
Conclusion
W110A TeSADH has shown activity in racemization of enan-
This is to ensure having a racemic alcohol over the course of the
reaction and thus minimizing the selectivity mistakes that
could be encountered in KR. This is apparently not the case for
our racemization approach using xerogel-immobilized W110A
TeSADH. Further investigation of various types of sol–gel with
tiopure alcohols in organic media contra to aqueous media
required by the enzyme. We encapsulated W110A TeSADH
using the sol–gel method and used the immobilized enzyme
in organic solvents to racemize enantiopure phenyl-ring-
containing alcohols. The use of W110A TeSADH-catalyzed
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racemization in pure hexane allowed the incorporation of this immobilized enzyme and coenzymes was le to dry in open air
racemization method with in situ CALB-catalyzed KR. The for 24 h forming the xerogel-encapsulated W110A TeSADH.
results obtained in this work indicate that the two enzymes are
compatible presenting an opportunity for one-pot dual enzy-
matic DKR. In a follow-up, we are currently conducting an
extensive protein engineering studies for TeSADH to improve its
racemization efficiency and investigating a variety of sol–gel
prepared using different silanes with hydrophobic moieties that
might facilitate substrate diffusion during racemization hence
improving this DKR approach.
General procedure for racemization of enantiopure alcohol
using xerogel-encapsulated W110 TeSADH
The enantiopure alcohol (0.05 mmol) and xerogel-encapsulated
enzyme containing W110A TeSADH (0.2–0.6 mg), NADP+ (1.0
mg) and NADPH (1.0 mg) in a solvent (1.0 mL) were placed in
a 1.5 mL Eppendorf tube. The reaction mixture was shaken at 200
rpm in a thermostat-controlled shaker at 50 ꢁC for 24 h. The
reaction mixture was extracted with diethyl ether (2 ꢂ 500 mL).
The combined organic layers were dried over Na2SO4 and
concentrated. For ee determination, the alcohols were derivat-
ized by treatment with acetic anhydride (one drop) and pyridine
(two drops) for 6 h prior to their analysis by chiral GC to deter-
mine the % conversion and % ee of the acetate ester derivatives.
Experimental
General
Compounds (R)-1a, (S)-1a, (R)-1b, (S)-1b, (rac)-1a, (rac)-1b,
NADPH, NADP+, CALB (Novozyme 435), and solvents (HPLC
grade) were used as purchased from Sigma-Aldrich; (R)-1c was
obtained by CALB-catalyzed kinetic resolution of (rac)-1c as re-
ported;23 (rac)-1c–e were synthesized by NaBH4 reduction of the
corresponding commercially available ketones, as reported.24
Capillary gas chromatography (GC) measurements were per-
formed on Agilent 7890A gas chromatograph equipped with
a ame ionization detector and an Agilent HP-Chiral-20B column
(30 m, inner diameter 0.32 mm, lm thickness 0.25 mm) using
helium as the carrier gas. The following temperature program
was used for the oven: 70 ꢁC (initial, hold time 10 min) to 180 ꢁC
(nal, hold time 20 min) at a rate of 5 ꢁC minꢀ1. The injector and
the detector temperature were 220 and 230 ꢁC, respectively. The
Procedures for racemization using free enzyme (aqueous and
biphasic)
The enantiopure alcohol (0.05 mmol) W110A TeSADH (0.4 mg),
NADP+ (1.0 mg) and NADPH (1.0 mg), were added to solvent (1.0
mL) in a round bottomed ask. The reaction in aqueous
medium was conducted in Tris–HCl buffer (50 mM, pH 8.0)/
acetonitrile (95 : 5 v/v) whereas the biphasic reaction in Tris–
HCl buffer (50 mM, pH 8.0)/hexane (50 : 50 v/v). The reaction
mixture was shaken at 200 rpm in a thermostat-controlled
shaker at 50 ꢁC for 24 h. The reaction mixture was extracted
with diethyl ether (2 ꢂ 500 mL). The combined organic layers
were dried over Na2SO4 and concentrated. For ee determina-
tion, the alcohols were derivatized by treatment with acetic
anhydride (one drop) and pyridine (two drops) for 6 h prior to
their analysis by chiral GC to determine the % conversion and
% ee of the acetate ester derivatives.
gas ow rates for air, H2, and He were 300, 30, and 15 mL minꢀ1
,
respectively. The split mode with a split ratio of 10 : 1 was used.
GC-MS analysis was performed using an instrument
combined with an Agilent 5975C inert mass selective detector
with a triple-Agilent GC 7890A axis detector. The same temper-
ature program and gas ow rates used for the GC were used for
the GC-MS. NMR spectra were recorded on a JOEL JNM-LA500
FT NMR at 500 MHz (1H) and at 125 MHz (13C) at room
temperature, using deuterated chloroform (CDCl3) peak as an
internal standard.
General procedure for KR of racemic alcohols
Racemic alcohol [(rac)-1a–e, (0.05 mmol)], acyl donor (0.1
mmol), CALB (1 mg), and hexane (1.0 mL) were placed in a 1.5
mL Eppendorf tube. The mixture was shaken at 50 rpm at 50 ꢁC
for 3–6 h (monitored by GC equipped with a chiral column). A
portion (50 mL) from the reaction was dried with Na2SO4 prior to
analysis. Retention times of the resultant peaks were compared
with standard samples of enantiopure alcohols and esters.
The E values were calculated according to the relationship:25
Â
À
ÁÃ
ln 1 ꢀ c 1 þ eep
Â
À
ÁÃ
E ¼
(1)
ln 1 ꢀ c 1 ꢀ eep
where c is percent conversion and eep is the ee of the product.
Gene expression and purication of W110A TeSADH
General procedure for dual enzymatic DKR of racemic
alcohols
W110A TeSADH was constructed and expressed in BL21(DE3)
cells and puried as described previously.12a,21b
Xerogel [containing W110A TeSADH (0.6 mg), NADPH (1.0 mg)
and NADP+ (1.0 mg)], CALB (1.0 mg), (rac)-1a–e (0.03 mmol) and
isopropenyl acetate (0.06 mmol) in hexane (1.0 mL) were placed
Preparation of xerogel-encapsulated W110A TeSADH
Tetramethoxysilane (2035 mL, 1 mol eq.) water (470 mL, 2 mol in the same Eppendorf tube ensuring no physical contact
eq.), and HCl (40 mL, 0.04 M) were vigorously shaken to form between the two enzyme systems as shown in Fig.ꢁS2 of ESI.†
a sol. The enzyme stock containing NADP+ (1.0 mg), NADPH (1.0 The reaction mixture was shaken at 50 rpm at 50 C for 24 h.
mg) and W110A TeSADH (0.2–0.6 mg) in Tris–HCl buffer solu- The reaction content including the lipase and the xerogel were
tion (400 mL, pH 8.0) was gently mixed with an equal volume of washed with ethyl acetate (2 ꢂ 500 mL). The combined organic
the sol for 5 seconds and le to stand for 24 h in a closed layers were dried over Na2SO4 and concentrated. The ee of the
Eppendorf tube to allow gel aging. The hydrogel containing the ester derivatives was determined by chiral GC.
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Paper
9 M. M. Musa and R. S. Phillips, Catal. Sci. Technol., 2011, 1,
1311–1323.
Conflict of interest
10 F. Hollmann, I. W. C. E. Arends and D. Holtmann, Green
Chem., 2011, 13, 2285–2314.
The authors declare no competing nancial interest.
11 Y. Ni and J.-H. Xu, Biotechnol. Adv., 2012, 30, 1279–1288.
12 (a) M. M. Musa, R. S. Phillips, M. Laivenieks, C. Vieille,
M. Takahashi and S. M. Hamdan, Org. Biomol. Chem.,
2013, 11, 2911–2915; (b) C. C. Gruber, B. M. Nestl, J. Gross,
P. Hildebrant, U. T. Bornscheuer, K. Faber and W. Kroutil,
Chem.–Eur. J., 2007, 13, 8271–8276.
13 M. M. Musa, J. M. Patel, C. M. Nealon, C. S. Kim,
R. S. Phillips and I. Karume, J. Mol. Catal. B: Enzym., 2015,
115, 155–159.
14 (a) D. Burdette and J. G. Zeikus, Biochem. J., 1994, 302, 163–
170; (b) D. Burdette, C. Vieille and J. G. Zeikus, Biochem. J.,
1996, 316, 115–122; (c) V. T. Pham and R. S. Phillips, J. Am.
Chem. Soc., 1990, 112, 3629–3632.
15 M. M. Musa, K. I. Ziegelmann-Fjeld, C. Vieille, J. G. Zeikus
and R. S. Phillips, Angew. Chem., Int. Ed., 2007, 46, 3091–
3094.
Acknowledgements
The authors acknowledge the support provided by King Abdu-
laziz City for Science and Technology (KACST) through the
Science and Technology Unit at King Fahd University of Petro-
leum and Minerals (KFUPM), for funding this work through
project No. 11-BIO1666-04, as part of the National Science,
Technology, and Innovation Plan as well as baseline research
funding offered to Prof. Hamdan through King Abdullah
University of Science and Technology. The authors thank Prof.
Claire Vieille, from the Department of Microbiology and
Molecular Genetics at Michigan State University, for providing
the plasmids of TeSADH.
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