| Co-Investigator(Kenkyū-buntansha) |
MORIKAWA Tadashi Denka Co., Sogo Institute, Researcher, 総合研究所, 研究員
OGAWA Jun Grad. Sch. Agriculture, KYOTO UNIVERSITY, Assistant Professor, 農学研究科, 助手 (70281102)
SHIMIZU Sakayu Grad. Sch. Agriculture, KYOTO UNIVERSITY, Professor, 農学研究科, 教授 (70093250)
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| Research Abstract |
Threonine aldolase (TA) catalyzes reversible degradation of β-hydroxy-α-amino acids to glycine and corresponding aldehydes. We found and characterized several microbial TAs showing stereospecificity, and cloned genes encoding these enzymes. By using recombinant TAs, an enzymatic resolution process was established for the production of optically pure β-hydroxy-α-amino acids. In addition, the TA-catalyzed direct synthesis of β-hydroxy-α-amino acid from aldehyde and glycine was investigated.
Low-specificity L-TAs (LSLTAs) from Pseudomonas sp. NCIMB 10558, Saccharomyces cerevisiae and Escherichia coli cleave both L- and L-allo-Thr, while L-allo-TA (LATA) of Aeromonas jandaei almost specifically acts on the L-allo-isomer (L-allo-/L- ratio > 100) In spite of these different stereospecificities, LATA shows significant homology to LSLTAs in primary structure. Introduction of random point mutagenesis into the LATA gene by error-prone PCR was carried out in order to alter its stereospecificity toward Thr isomers. Among 800 mutants screened, strain F48 showed TA activity toward not only L-allo-Thr but also L-Thr (L-allo-/L- ratio 【approximately equal】 10). In the DNA sequence analysis of a variant F48, two substitutions (T at positions 765 and 876 to C and A, respectively) were found. The former substitution was silent with respect to amino acid sequence, while the latter changed Ser at the position 292 to Arg. From cell-free extract of the mutant strain F48, two TAs with different molecular mass were isolated. These two enzymes consisted of 4 or 8 identical subunits with molecular mass of 38,000, which showed same N-terminal amino acid sequence. Tetramer structure is the native state, and octamer might be formed by combination of tetramer. The tetramer enzyme showed higher activity than the octamer form.
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