Leucine Dehydrogenase of an Thermophilic Anaerobe, Clostridium thermoaceticum: Gene Cloning, Purification and Characterization(Microbiology & Fermentation Industry)

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概要

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• The leucine dehydrogenase (L-leucine: NAD^+ oxidoreductase, deaminating, EC 1.4.1.9) gene of Clostridium thermoaceticum was cloned and expressed in Escherichia coli C600 with a vector plasmid, pICD242, which was constructed from pBR322 and the leucine dehydrogenase gene derived from C. thermoaceticum. The enzyme overproduced in the clone was purified about 12 fold to homogeneity by heat treatment and another two steps with a yield of46%. The enzyme of E. coli-pICD242 was immunochemically identical with that of C. thermoaceticum. The enzyme has a molecular weight of about 350,000 and consists of six subunits identical in molecular weight (56,000). The enzyme is not inactivated by heat treatment: at pH 7.2 and 75℃ for 15min; at 55℃ and various pH's between 6.0 and 10.0 for 10 min. The enzyme catalyzes the oxidative deamination of branched-chain L-amino acids and the reductive amination of their 2-oxo analogues in the presence of NAD^+ and NADH, respectively. The pro-S hydrogen at C-4 of the dihydronicotin-amide ring of NADH is exclusively transferred to the substrate; the enzyme is B stereospecific. The enzymological properties are very similar to those of the Bacillus stearothermophilus enzyme [T. Ohshima, S. Nagata and K. Soda, Arch. Microbiol., 141, 407(1985)].

• 社団法人日本農芸化学会の論文
• 1987-12-23

著者

• Esaki Nobuyoshi

  Kyoto Univ.

• Esaki Nobuyoshi

  Nstitute For Chemical Research Kyoto University

• Esaki Nobuyoshi

  Laboratory Of Microbial Biochemistry Institute For Chemical Research Kyoto University

• TANAKA Hidehiko

  Laboratory of Microbiology, Institute for Chemical Research, Kyoto University

• SODA Kenji

  Laboratory of Microbiology, Institute for Chemical Research, Kyoto University

• Soda Kenji

  Laboratory Of Microbial Biochemistry Institute For Chemical Research Kyoto University

• NAGATA Shinji

  Laboratory of Bioorganic Chemistry, Department of Applied Biological Chemistry, Graduate School of A

• Esaki Nobuyoshi

  Institute For Chemical Resarch Kyoto University

• Soda K

  Department Of Biotechnology Kansai University

• Tanaka Hidehiko

  Laboratory Of Biochemistry Kyoto College Of Pharmacy

• Nagasaki S

  Department Of Bioresources Science Kochi University

• Nagata Shinji

  Laboratory Of Applied Microbiology Department Of Bioresources Science Kochi University

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