Purification and characterization of a novel keto ester reductase from the green alga, <Emphasis Type="Italic">Chlorella sorokiniana</Emphasis>: comparison of enzymological properties with other microbial keto ester reductases |
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Authors: | Kohji Ishihara Rieko Iwai Momoko Yoshida Mika Morishita Hitomi Yamaguchi Nobuyoshi Nakajima |
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Institution: | (1) Graduate School of Science, Okayama University of Science, 1-1 Ridai-cho, Kita-ku, Okayama 700-0005, Japan;(2) Department of Life Science, Okayama University of Science, 1-1 Ridai-cho, Kita-ku, Okayama 700-0005, Japan;(3) Graduate School of Biological Sciences, Nara Institute of Sciences and Technology, 8916-5 Takayama, Ikoma Nara, 630-0192, Japan;(4) Research and Development Center, Nagase & Co., Ltd., 2-2-3 Nishi-ku, Kobe Hyogo, 651-2241, Japan;(5) Graduate School of Health and Welfare Science, Okayama Prefectural University, 111 Kuboki, Soja Okayama, 719-1197, Japan |
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Abstract: | A novel keto ester reductase (Chlorella sorokiniana keto ester reductase, CSKER) from Chlorella sorokiniana SAG 211-8k cells was purified. The CSKER had a monomeric structure based on gel filtration chromatography (37 kDa) and SDS–polyacrylamide
gel electrophoresis (34 kDa). The purified CSKER showed a high reducing activity with β-keto esters, in particular, ethyl 4-chloro-3-oxobutanoate and ethyl 2-chloro-3-oxobutanoate. However, the purified enzyme
did not show any reducing activity with α-keto esters and 2-chlorobenzoylformamide (aromatic α-keto amide). The CSKER catalyzed
the reduction of ethyl 4-chloro-3-oxobutanoate, ethyl 3-oxobutanoate, and methyl 3-oxobutanoate to the corresponding (R)-, (S)-, and (S)-hydroxy ester, respectively, with high enantioselectivity (>99% e.e.), respectively. Furthermore, the reduction of ethyl
2-methyl-3-oxobutanoate by CSKER exclusively yielded the corresponding syn-(2R, 3S)-hydroxy ester. The purified CSKER was inactive with NADH, used instead of NADPH. None of the keto ester-reducing enzymes
already isolated from other microorganisms was identical to the CSKER. These results suggested that CSKER is a novel keto
ester reductase that has not yet been reported. |
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