A novel Pseudomonas putida strain with high levels of hydantoin-convertingactivity, producing L-amino acids

Optically pure chiral amino acids and their derivatives can be efficiently synthesised by the biocatalytic conversion of 5-substituted hydantoins in r eactions catalysed by stereo-selective microbial enzymes: initially a hydan toinase catalyses the cleavage of the hydantoin producing an N-carbamyl ami no acid. In certain bacteria where an N-carbamyl amino acid amidohydrolase (NCAAH) is present, the N-carbamyl amino acid intermediate is further conve rted to amino acid, ammonia and CO,. In this study we report on a novel Pse udomonas putida strain which exhibits high levels of hydantoin-converting a ctivity, yielding L-amino acid products including alanine, valine, and norl eucine, with bioconversion yields between 60% and 100%. The preferred subst rates are generally aliphatic, but not necessarily short chain, 5-alkylhyda ntoins. In characterizing the enzymes from this microorganism, we have foun d that the NCAAH has L-selectivity, while the hydantoinase is non-stereosel ective. In addition, resting cell reactions under varying conditions showed that the hydantoinase is highly active, and is not subject to substrate in hibition, or product inhibition by ammonia. The rate-limiting reaction appe ars to be the NCAAH-catalysed conversion of the intermediate. Metal-depende nce studies suggest that the hydantoinase is dependent on the presence of m agnesium and cobalt ions, and is strongly inhibited by the presence of copp er ions. The relative paucity of L-selective hydantoin-hydrolysing enzyme s ystems, together with the high level of hydantoinase activity and the unusu al substrate selectivity of this P. putida isolate, suggest that is has sig nificant potential in industrial applications. (C) 2001 Elsevier Science B. V. All rights reserved.