Purification and Gene Cloning of an Enantioselective Thioesterification Enzyme from Brevibacterium ketoglutamicum KU1073, a Deracemization Bacterium of 2-(4-Chlorophenoxy)propanoic Acid

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概要

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• We succeeded in the purification and gene cloning of a new enzyme, α-methyl carboxylic acid deracemizing enzyme 1 (MCAD1) from Brevibacterium ketoglutamicum KU1073, which catalyzes the (S)-enantioselective thioesterification reaction of 2-(4-chlorophenoxy)propanoic acid (CPPA). The cloned gene of MCAD1 contained an ORF of 1,623 bp, encoding a polypeptide of 540 amino acids. In combination with cofactors ATP, coenzyme A (CoASH), and Mg2+, MCAD1 demonstrated perfect enantioselectivity toward CPPA. The optimal pH and temperature for reaction were found to be 7.25 and 30 °C. Under these conditions, the Km and kcat values for (S)-CPPA were 0.92±0.17 mM and 0.28±0.026 s−1 respectively. The results for substrate specificity revealed that MCAD1 had highest activity toward fatty acid tails with a medium chain-length (C8). This result indicates that MCAD1 should be classified into a family of medium-chain acyl-CoA synthetase. This novel activity has never been reported for this family.

• 社団法人 日本農芸化学会の論文

著者

• TAKEO Masahiro

  Graduate School of Engineering, University of Hyogo

• NEGORO Seiji

  Graduate School of Engineering, University of Hyogo

• Ohta Hiromichi

  Department Of Chemistry Faculty Of Science And Technology Keio University

• Takeo Masahiro

  兵庫県立大学 工学研究科物質系工学専攻

• KATO Dai-ichiro

  Graduate School of Engineering, University of Hyogo

• YOSHIDA Hiromitsu

  Graduate School of Engineering, University of Hyogo

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