新酵素D-アミノペプチダーゼ : 構造, 機能, および有機合成への利用

概要

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A new enzyme named “D-Aminopeptidase” has been isolated and characterized from a soil bacterium Ochrobactrum anthropi, SCRC Cl-38. It showed strict D-stereospecificity toward substrates including low molecular weight D-amino acid amides, D-alanine N-alkylamides, and peptides with a D-alanine at the N-terminus. The gene for the enzyme was cloned in Escherichia coli and an expression plasmid constructed. The amount of the enzyme in the cell-free extract of an E. coli transformant was elevated up to 288, 000 units/liter culture, which is about 3, 600-fold over that of O. anthropi SCRC Cl-38. The deduced amino acid sequence of the enzyme showed that it is related to the “penicillin-recognizing enzymes”. Mutants of the enzyme were generated by site-specific mutagenesis. We propose that the enzyme is a new member of the “penicillin-recognizing enzymes”. The cells of E. coli transformant were used as a catalyst for the D-stereospecific hydrolysis of several racemic amino acid amides HCl. The concentration of D-alanine reached up to 220 g/liter from racemic alanine amide HCl. D-Amino acid N-alkylamides were stereoselectively synthesized in organic solvents from racemic amino acid esters by the use of the enzyme immobilized by urethane prepolymer PU-6. The enzyme was also active in synthesizing D-alanine oligopeptides in non-aqueous media.
社団法人有機合成化学協会の論文