Purification and Characterization of the Nitrilase from Alcaligenes faecalis ATCC 8750 Responsible for Enantioselective Hydrolysis of Mandelonitrile

概要

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A nitrilase that converts racemic mandelonitrile to R-(-)-mandelic acid was purified to apparent homogeneity from a cell extract of Alcaligenes faecalis ATCC 8750. The molecular weight of this enzyme was estimated to be 32,000±2,000 from SDS-PAGE and that of the native enzyme 460,0000±30,000 from HPLC gel filtration. The enzyme preferentially hydrolyzed substituted aliphatic nitrilles, in particular benzyl cyanide and its p-substituted compounds, but hydrolyzed aromatic nitriles only with difficulty. The amino-terminal amino acids were sequenced and their sequences compared with those of other nitrilases. The purified enzyme had a pH optimum of 7.5 and an optimum temperature range of 40 to 45℃. The enzyme was inhibited by various thiol reagents. It hydrolyzed racemic mandelonitrile, producing optically pure R-(-)-mandelic acid and ammonia without the concomitant production of mandelamide, evidence that this nitrilase is highly enantioselective for R-mandelonitrile.
公益社団法人日本生物工学会の論文
1992-06-25