7-Aminocephalosporanic acid production. Purification and properties of 7.BETA.-(4-carboxybutanamido)-cephalosporanic acid acylase produced by mutants derived from Pseudomonas.

Abstract

7β-(4-Carboxybutanamido)cephalosporanic acid acylase (penicillin amidohydrolase, EC 3.5.1.11) was crystallized from cell-free extracts of a mutant derived from Pseudomonas SY-77-1. Purification of the enzyme was performed by a procedure involving ammonium sulfate fractionation and column chromatographies on DEAE-Sephadex, TEAE-cellulose and Sephadex G-200. The crystalline enzyme was homogeneous on polyacrylamide gel disc electrophoresis. The molecular weight of the acylase was estimated to be 1.3×105 by gel filtration. The enzyme was fully active at pH above 6.5 and was highly stable at a pH range of 6.0 to 8.0 and below 38°C . The Michaelis-Menten constant (Km) and Vmax for 7β-(4-carboxybutanamido)cephalosporanic acid were 0.16 mM and 4.91 μmol/min/mg-protein, respectively. It was also indicated that this enzyme-protein occupied 2.3 % of the dry-cell weight.