PURIFICATION AND PROPERTIES OF CEPHALOSPORIN-C DEACETYLASE FROM THE YEAST, RHODOTORULA-GLUTINIS 38B1, USEFUL FOR BIOCONVERSION OF 7-AMINOCEPHALOSPORANIC ACID-DERIVATIVES

A 7-aminocephalosporanic acid (7-ACA) deacetylating enzyme was purifie d to homogeneity from Rhodotorula glutinis 38B1, whose resting cells h ave been previously reported as useful for the conversion of 7-ACA der ivatives [Sakai et al., Appl. Environ. Microbiol., 62, 2667-2672, 1996 ]. The purified enzyme was a dimer comprised of identical subunits wit h a molecular mass of 82 kDa. The purified enzyme used cephalosporin C and several 7-ACA derivatives with low K-m and high k(cat) values as substrates, as well as some acetyl esters with relatively long-chain a lcohols. Based on this substrate specificity, the enzyme is classified as cephalosporin-C deacetylase (EC 3.1.1.41). The enzyme was most act ive at 35 degrees C and pH 5.5, and was inhibited by several serine en zyme inhibitors. The purified enzyme was glycosylated on the addition of an asparagine-linked ''hybrid type'' oligosaccharide, and most of t he enzyme activity was found in the purified cell wall fraction. The e nzyme localization and kinetic properties explain the high efficiency of 7-ACA deacetylation in a resting-cell reaction.