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Purification and characterization of a novel keto ester reductase from the green alga, Chlorella sorokiniana: comparison of enzymological properties with other microbial keto ester reductases

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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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Acknowledgments

This study was partially supported by the Ministry of Education, Culture, Sports, Science and Technology of Japan through a Financial Assistance Program for Social Collaborative Research (2006–2010). It was also supported by a grant from the UESCO Scientific Promotion Foundation (2007–2009), Japan. We thank the central research laboratory, Okayama University Medical School, Japan, for their help with the n-terminal region amino acid sequencing analysis of the purified enzyme.

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Authors and Affiliations

  1. Graduate School of Science, Okayama University of Science, 1-1 Ridai-cho, Kita-ku, Okayama, 700-0005, Japan

    Kohji Ishihara & Rieko Iwai

  2. Department of Life Science, Okayama University of Science, 1-1 Ridai-cho, Kita-ku, Okayama, 700-0005, Japan

    Mika Morishita

  3. Graduate School of Biological Sciences, Nara Institute of Sciences and Technology, 8916-5 Takayama, Ikoma, Nara, 630-0192, Japan

    Momoko Yoshida

  4. Research and Development Center, Nagase & Co., Ltd., 2-2-3 Nishi-ku, Kobe, Hyogo, 651-2241, Japan

    Hitomi Yamaguchi

  5. Graduate School of Health and Welfare Science, Okayama Prefectural University, 111 Kuboki, Soja, Okayama, 719-1197, Japan

    Nobuyoshi Nakajima

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  1. Kohji Ishihara
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  2. Rieko Iwai
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  3. Momoko Yoshida
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  4. Mika Morishita
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  5. Hitomi Yamaguchi
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  6. Nobuyoshi Nakajima
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Correspondence to Kohji Ishihara.

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Ishihara, K., Iwai, R., Yoshida, M. et al. Purification and characterization of a novel keto ester reductase from the green alga, Chlorella sorokiniana: comparison of enzymological properties with other microbial keto ester reductases. World J Microbiol Biotechnol 27, 17–24 (2011). https://doi.org/10.1007/s11274-010-0421-8

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