PRODUCTION OF ENANTIOMERICALLY PURE AMINO-ACIDS - CHARACTERIZATION OFSOUTH-AFRICAN HYDANTOINASES AND HYDANTOINASE-PRODUCING BACTERIA

Chiral amino acid derivatives can be synthesised by the biocatalytic c onversion of substituted hydantoins using microbial enzymes or resting cells: a hydantoinase performs the ring-opening cleavage of the hydan toin to produce an N-carbamylamino acid and N-carbamylamidohydrolase t hen converts this intermediate to the amino acid, ammonia and CO2. The hydantoinases from four locally isolated bacterial strains are curren tly being characterised in terms of conditions for optimal enzyme acti vity assay, and to demonstrate the effects of pH, temperature, metal i ons, protease inhibitors, surfactants, and anti-oxidants on hydantoina se activity in crude extracts. Typically, pH 8, 50 degrees C, and 0.3 mM Cu2+ were found to be optimal. Disruption of cells using a detergen t or membrane freeze-fracture resulted in increased activities, sugges ting that the hydantoinase enzymes may be membrane bound. It was also found that three Pseudomonas strains exhibited higher activities than the Agrobacterium strain, in terms of hydantoin conversion, with % con version of hydantoins to N-carbamylamino acids from 66% to 2%. Compari sons of hydantoinase and amidohydrolase activity in resting cells and in cell extracts also show marked differences in activity profile for different strains, e.g., strain RU-KM1 exhibited hydantoinase activity in whale cells and cell extracts, but amidohydrolase activity only in cell extracts, while RU-OR showed higher amidohydrolase activity than hydantoinase activity. (C) 1998 Elsevier Science B.V. All rights rese rved.