Chiral amino acid derivatives can be synthesised by the biocatalytic conversion of substituted hydantoins using microbial enzymes or resting cells: a hydantoinase performs the ring-opening cleavage of the hydantoin to produce an N-carbamylamino acid and N-carbamylamidohydrolase then converts this intermediate to the amino acid, ammonia and CO 2. The hydantoinases from four locally isolated bacterial strains are currently being characterised in terms of conditions for optimal enzyme activity assay, and to demonstrate the effects of pH, temperature, metal ions, protease inhibitors, surfactants, and anti-oxidants on hydantoinase activity in crude extracts. Typically, pH 8, 50°C, and 0.3 mM Cu 2+ were found to be optimal. Disruption of cells using a detergent or membrane freeze-fracture resulted in increased activities, suggesting 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 % conversion of hydantoins to N-carbamylamino acids from 66% to 2%. Comparisons 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 whole cells and cell extracts, but amidohydrolase activity only in cell extracts, while RU-OR showed higher amidohydrolase activity than hydantoinase activity.