Comparison of seven microbial d-hydantoinases

Abstract

d-Hydantoinase (E.C.3.5.2.2) from Pseudomonas fluorescens (strains 1.2 and 1.9), Pseudomonas putida (strains 2.2, 2.5 and DSM 84), Serratia liquefaciens (strain 1.15) and Corynebacterium pseudodiphtheriticum (strain 14.10) were compared according to their stability to different purification procedures. With exception to C. pseudodiphtheriticum where between 75 and 85% of the enzyme was recovered after purification, yields less than 30% were observed for all other strains. These low recovery levels were not due to the removal of essential ions or to the absence of substrate during chromatography. Purification yields could not be improved by the addition of detergents and of a cryoprotectant to prevent aggregation and/or to increase hydantoinase stability. Hydantoinases from all the strains examined except C. pseudodiphtheriticum, were stable up to 37°C and at pH 5. The C. pseudodiphtheriticum enzyme was stable at temperatures up to 57°C and at pH values above 8. All hydantoinases exhibited maximum activity at temperatures between 45 and 55°C and at pH 8.0 or above. Native and SDS-PAGE gave M r estimates ranging from 160 000 to 235 000 and 60 000 ± 4000 for the native enzymes and their subunits, respectively.