Keizou Yamamoto

Purification and characterization of the nitrilase from Alcaligenes faecalis ATCC 8750 responsible for enantioselective hydrolysis of mandelonitrile

Abstract 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 460,000±30,000 from HPLC gel filtration. The enzyme preferentially hydrolyzed substituted aliphatic nitriles, 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°C. 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.

Characterization and Cloning of an Enantioselective Amidase from Comamonas acidovorans KPO-2771-4

Abstract The characteristics and amino acid sequence of an enantiomer-selective amidase active on R-(−)-2-(3′-benzoyl phenyl)propionamide [R-(−)-ketoprofen amide] purified previously from Comamonas acidovorans KPO-2771-4 were studied. On gel filtration, this amidase appeared to be a monomer with a molecular mass of 55 kDa. It had maximal activity at 35°C and at pH values from 8.5 to 10.0. Except for Cu2+, Zn2+, Pb2+, and p-chloromercuribenzoate, it was not affected by chelating reagents, carbonyl reagents, reductants, most metal ions, or thiol reagents. The amidase had hydrolyzing activity against a broad range of aliphatic, aromatic, and amino acid amides, evidence that it is a wide-spectrum amidase. Oligonucleotide probes designed from limited peptide sequence information were used to clone the corresponding gene. The nucleotide-determined sequence indicated that the amidase consists of 473 amino acids (Mw 50,464 Da). Significant homologies were found at the amino acid level between the R-enantiomer-selective amidase of C. acidovorans KPO-2771-4 and the S-enantiomer-selective enzymes from Rhodococcus sp., Brevibacterium sp., and Pseudomonas sp. Only 39% homology was conserved in the consensus region of these amidases.

Efficient conversion of dinitrile to mononitrile-monocarboxylic acid by Corynebacterium sp. C5 cells during tranexamic acid synthesis

Use of peptone and yeast extract as the nitrogen source together with glucose as the carbon source was effective in the culture of Corynebacterium sp. C5 cells that convert trans -1,4-dicyanocyclohexane ( t -DCC) to trans -4-cyanocyclohexane-1-carboxylic acid ( t -MCC). The ratio of nitrile hydratase to amidase that converted t -DCC to t -MCC was 3 : 1, but the stability of the nitrile hydratase was one-tenth that of the amidase. Potassium phosphate buffer (pH 8.5) stabilized the two enzymes and produced maximum activity. High yields of t -MCC (99.4%) were produced from a high concentration of t -DCC (40%) in a resting cell system. t -DCC crystals disappeared, with the solution becoming clear as the reaction proceeded. The tranexamic acid synthesized from the t -MCC produced by the resting cells was of very high quality; 99.97% trans -isomer.

Production of S-(+)-ibuprofen with high optical purity from a nitrile compound by cells immobilized on cellulose porous beads

Abstract The production of S-(+)-ibuprofen (S-IP) from a nitrile compound by immobilized cells was investigated. Cells immobilized within both κ-carrageenan and Ca-alginate showed a lower reaction conversion and gave a product of lower optical purity than free, intact cells. However the reaction of cells immobilized in cellulose porous beads (CPBs) with pore sizes of 10–30 μm and an average diameter of 200 μm resulted in a yield and optical purity as high as those given by intact cells. Even when the CPBs were used repeatedly up to 12 times, an S-IP optical purity of more than 99% was maintained, but the reaction conversion decreased from 48% to 32%.