Highly enantioselective conversion of racemic 1-phenyl-1,2-ethanediol by stereoinversion involving a novel cofactor-dependent oxidoreduction system of Candida parapsilosis CCTCC M203011

Article abstract of DOI:10.1021/op0341519

An economical and convenient biocatalytic process was developed for the preparation of (S)-1-phenyl-1,2-ethanediol (PED), which is a valuable chiral building block for pharmaceuticals and liquid crystals, by stereoselective microbial conversion from the corresponding racemate. As a result of screening bacteria, yeasts, and molds, the enantioselective conversion of racemic PED by Candida parapsilosis CCTCC M203011 was found to be the most efficient process to produce (S)-PED with high optical purity of 98% ee and yield of 92%. By detecting the intermediate produced in the reaction by GC-MS, it was suggested that (S)-enantiomer was produced from the intermediate identified as β-hydroxyacetophenone by asymmetric reduction after stereoselective oxidation of (R)-enantiomer to β-hydroxyacetophenone. After investigating the cofactor requirement and stereospecificity of the reaction catalyzed by the cell-free extract from C. parapsilosis CCTCC M203011, it was found that the stereoselective conversion involved the oxidation of (R)-PED to the intermediate with NADP⁺ as the cofactor and the reduction reaction that formed the product used NADH as the cofactor, which was catalyzed by a novel cofactor-dependent oxidoreduction system. The NADP⁺-dependent (R)-specific alcohol dehydrogenase involved in stereoinversion was purified from C. parapsilosis CCTCC M203011, which has a relative molecular mass of 45kD.

Full text of DOI:10.1021/op0341519

Organic Process Research & Development 2004, 8, 246−251
Highly Enantioselective Conversion of Racemic 1-Phenyl-1,2-ethanediol by
Stereoinversion Involving a Novel Cofactor-Dependent Oxidoreduction System of
Candida parapsilosis CCTCC M203011
Yao Nie, Yan Xu,* and Xiao Qing Mu
Key Laboratory of Industrial Biotechnology of Ministry of Education and School of Biotechnology, Southern Yangtze
UniVersity, Wuxi 214036, P. R. China
Abstract:
oxidation catalyzed by glycerol dehydrogenase (GDH),4 and
microbial stereoinversion.5 Among various methods of
producing optically active PED, microbial stereoinversion
with a whole cell system can be used to avoid coenzyme
addition or regeneration system and obtain products with high
optical purity and yield.6-10 Various microorganisms, includ-
ing Candida parapsilosis, C. maltosa, C. boidinii, Loddero-
myces elongisporus, Pichia boVis, Nocardia fusca, Arthro-
bacter Viscosus, Lactobacillus kefir, etc., have been found
to possess the same ability of preparing optically active
alcohols from the racemates.11-14 However, the stereoselec-
tive reactions catalyzed by alcohol dehydrogenases from
these strains were different not only in the efficiency of
enantioselective conversion, such as ee value and yield of
products, but also in the coenzyme requirement and the
reaction mechanism. Among these microorganisms, C.
parapsilosis IFO0708 has been reported to prepare optically
active 1,2-diols from the corresponding racemates through
stereoinversion.5 In this procedure, (S)-PED with the optical
purity of 100% ee and the yield of 100% was produced from
the corresponding racemate with the substrate concentration
of 5 g/L, and the stereoinversion involved the oxidation of
(R)-diol to 2-keto-1-alcohol by an NAD+-linked (R)-specific
alcohol dehydrogenase and the reduction of 2-keto-1-alcohol
to (S)-diol by an NADPH-linked (S)-specific reductase.
Although (S)-PED with high optical purity and yield was
prepared by this means, it has not been considered to be
suitable for large-scale production and industrial application
due to the low concentration of substrate in the reaction. A
highly enantioselective and productive conversion system for
racemic PED is essential to prepare optically pure enantiomer
for potential large-scale application.
An economical and convenient biocatalytic process was devel-
oped for the preparation of (S)-1-phenyl-1,2-ethanediol (PED),
which is a valuable chiral building block for pharmaceuticals
and liquid crystals, by stereoselective microbial conversion from
the corresponding racemate. As a result of screening bacteria,
yeasts, and molds, the enantioselective conversion of racemic
PED by Candida parapsilosis CCTCC M203011 was found to
be the most efficient process to produce (S)-PED with high
optical purity of 98% ee and yield of 92%. By detecting the
intermediate produced in the reaction by GC-MS, it was
suggested that (S)-enantiomer was produced from the inter-
mediate identified as â-hydroxyacetophenone by asymmetric
reduction after stereoselective oxidation of (R)-enantiomer to
â-hydroxyacetophenone. After investigating the cofactor re-
quirement and stereospecificity of the reaction catalyzed by the
cell-free extract from C. parapsilosis CCTCC M203011, it was
found that the stereoselective conversion involved the oxidation
of (R)-PED to the intermediate with NADP+ as the cofactor
and the reduction reaction that formed the product used NADH
as the cofactor, which was catalyzed by a novel cofactor-
dependent oxidoreduction system. The NADP+-dependent (R)-
specific alcohol dehydrogenase involved in stereoinversion was
purified from C. parapsilosis CCTCC M203011, which has a
relative molecular mass of 45kD.
Introduction
Optically active 1-phenyl-1,2-ethanediol (PED) is a valu-
able and versatile chiral building block for the synthesis of
pharmaceuticals, agrochemicals, pheromones, and liquid
crystals, etc. Of the racemate, (S)-enantiomer can further be
used as precursor for the production of chiral biphosphines
and chiral initiator for stereoselective polymerization.1
Several bio-methods for preparation of optically active
PED and other 1,2-diols have been developed, including of
stereospecific dihydroxylation of styrene catalyzed by naph-
thalene dioxygenase (NDO),2 optical resolution of racemic
PED by lipase-catalyzed transesterification,3 enantioselective
Using whole cells for preparative conversions, the coen-
zyme requirement of the enzyme involved in the reaction
(4) Liese, A.; Karutz, M.; Kamphuis, J.; Wandrey, C.; Kragl, U. Biotechnol.
Bioeng. 1996, 51, 544.
(5) Hasegawa, J.; Ogura, M.; Tsuda, S.; Maemoto, S.; Kutsuki, H.; Ohashi, T.
Agric. Biol. Chem. 1990, 54, 1819.
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B. Nature 2001, 409, 258.
(7) Carnell, A. J. AdV. Biochem. Eng. Biotechnol. 1999, 63, 57.
(8) Matsuyama, A.; Yamamoto, H.; Kawada, N.; Kobayashi, Y. J. Mol. Catal.
B: Enzym. 2001, 11, 513.
* Author for correspondence.Telephone: +86-510-5864735. Fax: +86-510-
5864112. E-mail: yxu@sytu.edu.cn (Y. Xu).
(1) Iwasaki, F.; Maki, T.; Nakashima, W.; Onomura, O.; Matsumura, Y. Org.
Lett. 1999, 1, 969.
(2) Lee, K.; Gibson, D. T. J. Bacteriol. 1996, 178, 3353.
(3) Bosetti, A.; Bianchi, D.; Cesti, P.; Golini, P.; Spezia, S. J. Chem. Soc.,
Perkin Trans. 1992, 2395.
(9) Panke, S.; Wubbolts, M. G. Curr. Opin. Biotechnol. 2002, 13, 111.
(10) Chadha, A.; Baskar, B. Tetrahedron: Asymmetry 2002, 13, 1461.
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Catal. B: Enzym. 1998, 5, 69.
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10.1021/op0341519 CCC: $27.50 © 2004 American Chemical Society
Published on Web 02/18/2004

Figure 1. Screening of microorganisms producing optically active PED from the racemate. (× ) yeasts; (0) molds; (O) bacteria.
and the regeneration of the coenzyme are important for the
conversions by cofactor-dependent enzymes. Various enan-
tioselective oxidoreductases, such as alcohol dehydrogenase,
aryl-alcohol dehydrogenase, carbonyl reductase, phenylac-
etaldehyde reductase, etc., have been found to require
cofactors specially to catalyze the asymmetric reaction.15-18
Most of these oxidoreductases are highly selective for their
cofactor, the specific nature of the enzyme and its cofactor
determines its stereoselectivity, and the coenzyme specificity
could even be different from the similar reactions catalyzed
by the enzymes from the strains of the same species.
Therefore, the discovery of enzymes of novel cofactor
requirement can provide valuable information on the diversity
of microbial enzymes, especially related enzymes from
microorganisms of the same species. Furthermore, the
information on novel cofactor dependence of enzymes can
be useful for the alteration of the coenzyme requirement by
the approach of site-directed mutagenesis to change or extend
the coenzyme specificity of enzymes and make the enzymes
more available.19,20
in preparation of optically active PED from the racemate
(Figure 1).
Among microorganisms found to have the ability of
converting racemic PED to optically pure product, some
strains, such as C. parapsilosis, C. boidinii, Saccharomyces
cereVisiae, BreVibacterium protophormiae, B. flaVum, Bacil-
lus polymyxa and B. sphaericus, showed better conversion
yield. Among them, only the (S)-isomer was obtained, and
no microorganisms were found to produce (R)-PED from
the racemate by stereoselective conversion. Furthermore,
most of the molds did not show the capability of producing
optically pure PED, and several bacteria produced (S)-PED
with high optical purity but in the yields of below 50%, and
only a couple of yeasts converted the racemate to (S)-isomer
with high optical purity and yield of 98% ee and 92%,
respectively. These results suggested that the production of
(S)-PED from the racemate was through stereoselective
degradation by some bacteria, and some yeasts were able to
convert (R)-PED to the (S)-enantiomer through stereoinver-
sion.
The main objectives of this study were to develop an
efficient procedure to obtain optically active PED from the
corresponding racemate with high optical purity and yield,
to analyze the reaction mechanism, and to investigate the
cofactor requirement and stereospecificity of the suitable
catalyst in the asymmetric reaction.
From the results of screening, the species of C. parap-
silosis generally showed notable ability of producing (S)-
PED from the racemate. The strains of C. parapsilosis from
different sources were then compared in catalyzing stereo-
selective conversion in detail (Table 1). Although most of
C. parapsilosis was found to have the capability to convert
(RS)-PED to the (S)-isomer, the efficiency of stereoinversion
by the strains from various sources were different with the
span of ee values from 1 to 98%. These facts indicate that
the species of C. parapsilosis from various sources could
possess different physiological character and have distinctions
on the gene encoding the oxidoreductases,21 so that the
efficiency of stereoselective conversion presented different
levels by C. parapsilosis from various sources. Among the
strains screened, C. parapsilosis CCTCC M203011 was the
best one selected for further research as an efficient catalyst,
by which (S)-PED with high optical purity of 98% ee was
Results and Discussion
Screening of Microorganisms Producing (S)-PED from
the Racemate. More than 100 strains, including bacteria,
yeasts, and molds, were tested for screening suitable strains
(15) Xie, S. X.; Ogawa, J.; Shimizu, S. Biosci. Biotechnol. Biochem. 1999, 63,
1721.
(16) Zheng, C. S.; Phillips, R. S. J. Chem. Soc., Perkin Trans. 1992, 1083.
(17) Bastos, F. M.; Franca, T. K.; Machado, G. D. C.; Pinto, G. F.; Oestreicher,
E. G.; Paiva, L. M. C. J. Mol. Catal. B: Enzym. 2002, 19, 459.
(18) Hidalgo, A. G. D.; Akond, M. A.; Kita, K.; Kataoka, M.; Shimizu, S. Biosci.
Biotechnol. Biochem. 2001, 65, 2785.
(19) Corbier, C.; Clermont, S.; Billard, C. P.; Skarzynski, T.; Branlant, C.;
Wonacott, A.; Branlant, G. Biochemistry 1990, 29, 7101.
(20) Seelbach, K.; Riebel, B.; Hummel, W.; Kula, M.; Tishkov, V. I.; Egorov,
A. M.; Wandrey, C.; Kragl, U. Tetrahedron Lett. 1996, 37, 1377.
(21) Yamamoto, H.; Kawada, N.; Matsuyama, A.; Kobayashi, Y. Biosci.
Biotechnol. Biochem. 1999, 63, 1051.
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247

Figure 2. Analysis of reaction intermediate by GC-MS. (A) Analysis of the intermediate by GC. (B) Mass spectrum of the
intermediate and the structure of â-hydroxyacetophenone.
Table 1. Stereoselective conversion of racemic PED
catalyzed by C. parapsilosis from different sources
9.85 min), a possible intermediate which appeared at the
retention time of 9.00 min on GC was formed during the
reaction (Figure 2A). The mass spectrum of this material
exhibited a molecular ion (M+) at m/e 136.1, and its fragment
pattern well explained the structure of â-hydroxyacetophe-
none (Figure 2B).
Reaction Route of the Stereoselective Conversion.
During the asymmetric conversion catalyzed by C. parap-
silosis CCTCC M203011, the amount of (R)-PED decreased
with time, while the amount of the opposite stereoisomer
increased in the same course, and the optical purity of (S)-
enantiomer reached the highest point after incubation for
48 h (Figure 3).
residual
PED
ee of
(S)-isomer
yield
(S)-PED
microorganisms
(%)(A) (%)(B)
(%)(C)a
Candida parapsilosis ATCC 10232
C. parapsilosis ATCC 22019
C. parapsilosis ATCC 7330
C. parapsilosis CECT 10211
C. parapsilosis CECT 10304
C. parapsilosis CECT 10434
C. parapsilosis CECT 10437
C. parapsilosis AS 2.491
80
79
81
82
79
83
79
81
78
82
90
93
96
62
62
76
1
75
81
88
60
72
47
73
98
8
65
64
71
41
69
75
74
65
67
61
78
92
52
C. parapsilosis AS 2.590
C. parapsilosis AS 2.1497
C. parapsilosis CICC 1627
C. parapsilosis CCTCC M203011
C. parapsilosis IFO 0708
a Note: (S)-PED yield (C) ) net yield of (S)-PED from (RS)-PED ) A ×
[1 - (100 - B)/200].
obtained in high yield of 92%. In our research, highly
enantioselective conversion of racemic PED was carried out
with a higher substrate concentration of 0.8% (w/v) and a
lower cell concentration of 5% (w/v). Although excessive
substrate concentration might inhibit the reaction and de-
crease the reaction efficiency, from the results of this work,
it still has the potentiality of optimizing the reaction
conditions to increase the substrate concentration to a high
level and enlarging the reaction to large-scale level for
industrial application.
Identification of the Intermediate in the Asymmetric
Reaction. While analyzing the reaction mixture with GC-
MS, it was found that in addition to PED (retention time of
Figure 3. Asymmetric reaction process catalyzed by C.
parapsilosis CCTCC M203011. (0) (R)-PED; (O) (S)-PED; (9)
optical purity.
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Scheme 1. Schematic representation of stereoinversion of (RS)-PED to (S)-PED catalyzed by C. parapsilosis CCTCC M203011
Table 2. Cofactor requirement and stereospecificity in the
oxidation of PED catalyzed by cell-free extract and purified
enzyme from C. parapsilosis CCTCC M203011, respectively
Table 3. Cofactor requirement in the reduction catalyzed by
cell-free extract from C. parapsilosis CCTCC M203011
activity (units/mg protein)
activity (units/mg protein)
â-hydroxy-
catalyst
cofactor
(R)-PED
(S)-PED
cofactor acetone 2-hexanone acetophenone acetophenone
cell-free extract
NAD+
0.016
0.106
0.005
0.065
NADH
NADPH 0.012
0.212
0.089
0.015
0.117
0.011
0.039
0.009
NADP+
(RS)-PED
parapsilosis CCTCC M203011, the enzyme catalyzing the
oxidation of PED was purified from the cell-free extract.
The relative molecular mass of the native enzyme was found
to be 45 kD by HPLC on a Protein KW-803 Shodex gel
filtration column. On the cofactor requirement of this purified
alcohol dehydrogenase in catalyzing PED oxidation, the
alcohol dehydrogenase catalyzed the oxidation of PED with
NADP+ but not NAD+ as the cofactor, indicating that the
enzyme is an NADP+-dependent alcohol dehydrogenase,
which coincided with the observation on the cofactor
requirement in the oxidation catalyzed by the cell-free extract
from C. parapsilosis CCTCC M203011. For its dissimilarity
with other alcohol dehydrogenases from C. parapsilosis
reported in cofactor requirement,5,22 this NADP+-dependent
alcohol dehydrogenase was proposed to be an alcohol
dehydrogenase with novel cofactor dependence. The result
that alcohol dehydrogenases from same species of C.
parapsilosis showed different cofactor requirements in
catalyzing oxidation could be explained by saying that these
strains of the same species were different on the gene that
encodes dehydrogenases, so that the three-dimensional
structures and the active-site binding cofactors of the
enzymes presented dissimilarity.
The cofactor requirements in the reduction of various
ketones catalyzed by the cell-free extract from C. parapsilosis
CCTCC M203011 were also investigated. As shown in Table
3, the activities of the cell-free extract with NADH as
cofactor were much higher than those with NADPH as the
coenzyme, suggesting that an NADH-dependent reductase
catalyzing asymmetric reduction was contained in the cell-
free extract from C. parapsilosis CCTCC M203011.
From these observations, the reaction mechanism of
stereoinversion of (RS)-PED to (S)-PED by C. parapsilosis
CCTCC M203011 can be explained as follows. (R)-PED in
the racemate is oxidized to the intermediate of â-hydroxy-
purified enzyme
a ND: not detected.
NAD+
NADP+
NDa
2.35
These results indicated that (S)-PED was produced from
the intermediate identified as â-hydroxyacetophenone by
asymmetric reduction after the oxidation of (RS)-PED to
â-hydroxyacetophenone (Scheme 1).In addition to the (S)-
isomer that remained in the substrate of the racemate and
that converted from the (S)-isomer, the product of (S)-PED
also included the part converted from the (R)-isomer.
Therefore, the yield of (S)-PED from the racemate was over
50% by C. parapsilosis CCTCC M203011. This reaction
route of stereoselective conversion from (RS)-PED to the
(S)-enantiomer catalyzed by C. parapsilosis CCTCC M203011
coincided with that of asymmetric oxidoreduction catalyzed
by C. parapsilosis IFO 0708,5 suggesting that the same
species as C. parapsilosis showed some similarities in
catalyzing asymmetric reactions of the racemates of chiral
alcohols to the optical pure enantiomers.
Cofactor Requirement and Stereospecificity. In the
reaction of oxidoreduction, oxidoreductases such as alcohol
dehydrogenase or carbonyl reductase require NAD(P)+ or
NAD(P)H as cofactors to catalyze the oxidoreduction. To
obtain the information on the reaction mechanism, the
cofactor requirement and stereospecificity in stereoselective
oxidation catalyzed by the cell-free extract and the purified
enzyme from C. parapsilosis CCTCC M203011 were
investigated (Table 2). In the presence of NADP+ but not
NAD+, the cell-free extract catalyzed the oxidation, sug-
gesting that the enzyme is an NADP+-dependent dehydro-
genase, and in the oxidation of PED, (R)-PED was oxidized
much faster than (S)-PED, which indicated that the cell-free
extract contained an (R)-specific alcohol dehydrogenase. To
confirm this NADP+-dependent dehydrogenase and clarify
the mechanism of the asymmetric reaction catalyzed by C.
(22) Yamamoto, H.; Matsuyama, A.; Kobayashi, Y.; Kawada, N. Biosci.
Biotechnol. Biochem. 1995, 59, 1769.
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249

Scheme 2. Stereoinversion of (R)-PED to (S)-PED catalyzed by C. parapsilosis CCTCC M203011
acetophenone by an NADP+-dependent (R)-specific dehy-
drogenase, and then â-hydroxyacetophenone is reduced to
(S)-PED by an NADH-dependent (S)-specific reductase
(Scheme 2). This mechanism was found to be different from
a previous report, in which the oxidation of 1,2-diols
catalyzed by the cell-free extract of C. parapsilosis required
NAD+ as cofactor and the reduction catalyzed by the cell-
free extract required NADPH as cofactor.5 For the cell-free
extract from C. parapsilosis CCTCC M203011 showing
some activity to (S)-isomer in a low level, this alcohol
dehydrogenase purified from the cell-free extract could also
catalyze the oxidation of a small quantity of (S)-PED to the
intermediate in this asymmetric reaction. It appears that the
(S)-specific reductase might play a more important role in
catalyzing this enantioselective oxidoreduction to achieve
such high optical purity of the product.
In this study, C. parapsilosis CCTCC M203011 presented
some distinctions with other strains of the same species in
catalyzing asymmetric conversion, especially on the ef-
ficiency of catalyzing stereoselective conversion and the
mechanism of asymmetric reaction. In addition, the enzyme
catalyzing oxidation was purified and was found to be an
alcohol dehydrogenase with novel cofactor specificity. To
take a good advantage of the enzyme and make the
mechanism more clear, further research on characterization
of the purified enzyme and purification of the other enzyme
catalyzing reduction is now under investigation.
cells were harvested by centrifugation at 4000 rpm for 20
min, washed twice with physiological saline, and then used
for reaction.
Asymmetric Reaction Conditions. The reaction mixture
in 2 mL comprised 0.1 M potassium phosphate buffer (pH
6.5), 5% (w/v) wet cells, and 0.8% (w/v) (RS)-PED. The
reactions were carried out at 30 °C for 48 h with shaking.
After reaction, the cells were removed by centrifugation at
4000 rpm for 20 min, and then PED was extracted with three
volumes of ethyl acetate by vigorous mixing. Finally, the
organic layer was analyzed by HPLC.
Analytical Methods. The optical purity of PED was
determined by HPLC using a Chiralcel OB-H column (4.6
mm × 25 cm; Daicel Chemical Ind., Ltd., Japan). Enanti-
omers were eluted with hexane and 2-propanol (9:1) at a
flow rate of 0.5 mL/min. The effluent was monitored at 215
nm, and the areas under each peak were integrated. The
enantiomeric excess (ee) was calculated from the peak areas
of the stereoisomers. The intermediate was identified by gas
chromatography-mass spectrometry (GC-MS) with a Finni-
gan Trace-MS mass spectrometer (Finnigan, U.S.A.) using
a column of OV1701 (30 m × 0.25 mm × 0.25 µm). The
initial temperature was 60 °C, followed by an increase of
temperature at a rate of 10 °C/min. The final temperature
was held at 240 °C for 15 min. The carrier gas was helium
(0.8 mL/min).
Enzyme Assay. The assay mixture in 220 µl for oxidation
comprised 200 mM potassium phosphate buffer (pH 7.5),
3 mM NAD(P)+, 0.1 M (RS)-PED, and an appropriate
amount of the enzyme. For reduction, NAD(P)+ and
(RS)-PED were replaced by NAD(P)H (1 mM) and ketones
(0.1 M), respectively. The reaction mixture was incubated
for 2 min without the enzyme at 30 °C, and then the reaction
was started by the addition of the catalyst. The increase or
decrease in the amount of the coenzyme was measured
spectrophotometrically at 340 nm. One unit of enzyme
activity was defined as the amount of enzyme catalyzing the
reduction/oxidation of 1 µmol of NAD(P)+/NAD(P)H per
min under the assay conditions.
Experimental Section
Microorganisms and Chemicals. All strains were ob-
tained from American Type Culture Collection (ATCC),
China Center for Type Culture Collection (CCTCC), China
Center of Industrial Culture Collection (CICC), Spanish Type
Culture Collection (CECT), Chinese Academy of Science
(AS), and Institute for Fermentation of Osaka (IFO). (R)-,
(S)-, and (R,S)-PED as standard samples were purchased from
the Sigma-Aldrich Chemical Co. All other chemicals used
in this work were of analytical grade and commercially
available.
Medium and Cultivation. Medium for yeasts contained
4% (w/v) glucose, 0.3% (w/v) yeast extract, and 10% (v/v)
mineral solution (pH 7.0). Medium for molds contained 2%
(w/v) glucose, 0.3% (w/v) yeast extract, 0.5% (w/v) meat
extract, 0.5% (w/v) peptone, and 5% (v/v) mineral solution
(pH 7.0). Medium for bacteria contained 0.5% (w/v) glucose,
0.2% (w/v) yeast extract, 1% (w/v) meat extract, 1% (w/v)
peptone, and 5% (v/v) mineral solution (pH 7.0). The mineral
solution consisted of 13% (NH4)2HPO4, 7% KH2PO4, 0.8%
MgSO4‚7H2O, and 0.1% NaCl. Microorganisms were cul-
tured in 50-mL shaking flasks containing 5 mL of medium
at the temperature of 30 °C. After incubation for 48 h, the
Enzyme Purification. All purification procedures were
done at the temperature of 4 °C, and 20 mM potassium
phosphate buffer (pH7.5) was used as the buffer unless
otherwise specified.
Preparation of the Cell-Free Extract. The collected cells
suspended in buffer were disrupted with a French pressure
cells at 20000 psi. The cell debris was removed by
centrifugation (10000 rpm × 60 min) at 4 °C, and the
supernatant was used as the cell-free extract.
Ethanol Precipitation. The cell-free extract was added to
50% (v/v) ethanol and stirred at 4 °C for 10 min. The
precipitate was collected by centrifugation and dissolved in
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buffer. The undissolved part was removed by centrifugation,
and the supernatant was used for the next step.
column (8 mm × 300 mm) at a flow rate of 0.72 mL/min
with 25mM phosphate buffer (pH 6.8).15
DEAE-Sephacel Chromatography. The supernatant was
put on a HiPrep 16/10 DEAE-Sephacel column and eluted
with a linear gradient of NaCl (0.0-0.5 M). The active
fractions were combined and dialyzed against the buffer.
Superdex G-75 Chromatography. The dialyzed solution
was put on a HiLoad 16/60 Superdex G-75 column, and the
enzyme was eluted with the same buffer. Then the active
fractions were collected.
Blue Sepharose Chromatography. The enzyme solution
was put on a Blue Sepharose column and eluted with the
buffer of NADP+ (0.5 mM). The active fractions were
collected and concentrated and then used for characterization.
Measurement of the Molecular Mass of the Enzyme.
The relative molecular mass of the enzyme was measured
by HPLC on a Protein KW-803 Shodex Gel Filtration
Acknowledgment
Financial support of Ministry of Education, P.R.China,
under the Excellent Young Teachers Program (EYTP), and
Department of Science and Technology of Jiangsu Province,
P. R. China, under Hi-technology Program are gratefully
acknowledged. We also thank the National Science Founda-
tion of China (NSFC) (No. 20376031) and the National Key
Basic Research and Development Program of China (973
Program) for their valuable support.
Received for review October 17, 2003.
OP0341519
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