Enrichment and selection of hydantoinase-producing micro-organisms

Abstract

A screening method that allows rapid, efficient and reliable detection and selection of hydantoinase-positive micro-organisms was devised. Out of 76 strains selectively isolated from soil, 15 strains possessing hydantoin-hydrolysing activity were selected using a spot-test based on the direct detection in agar of N-carbamyl amino acids. The results obtained were confirmed by quantitative determination of hydantoinase activity in whole cells and crude extracts of positive strains. Inactivity and/or instability of some hydantoin-hydrolysing activity were observed upon preparation of a few crude extracts. Preliminary experiments indicated that some selected strains had an inducible hydantoinase activity. The reliability and the indicative value (presence of hydantoinase) of the spot-test were confirmed by incubating crude extracts in enzyme membrane reactors for up to 112 h. The six strains that were positive with the spot-test and had high hydantoinase activity continuously produced N-carbamylvaline. Following nitrite treatment of N-carbamylvaline, only D-valine could be detected as confirmed by three different methods (enzymic and chromatographic).