Characterization of hydantoinase from Pseudomonas fluorescens strain DSM 84.

The hydantoinase (EC 3.5.2.2) from Pseudomonas fluorescens strain DSM 84 was purified either by hydrophobic interaction chromatography on phenyl-Sepharose or by salting out chromatography on Sepharose 4B, gel filtration on Sephacryl S-400, and preparative electrophoresis. Molecular weight values of 230,000 and 60,000 for the native enzyme and each of the four subunits were estimated for the hydantoin hydrolysing activity. The hydantoinase was stable at temperatures up to 40 degrees C but showed an optimal activity at 55 degrees C. The enzyme was markedly inhibited by copper, para-hydroxymercuribenzoate, 8-hydroxyquinoline, and 2,2'-dipyridyl but not by zinc, and poorly by EDTA and o-phenanthroline. The hydantoin-hydrolyzing activity could be reactivated by ferrous ions. Dihydrouracil was the most readily hydrolyzed substrate. The dihydropyrimidinase produced by strain DSM 84 could also hydrolyze 5-substituted hydantions such as isopropylhydantoin (valine derivative) continuously for 10 days in a membrane reactor at a conversion rate of 30%. The only identified end product was N-carbamyl-D-valine.