PURIFICATION AND PROPERTIES OF A P-NITROBENZYL ESTERASE FROM BACILLUS-SUBTILIS

A procedure for purifying to homogeneity a microbially produced biocat alyst useful for deblocking intermediates in the manufacture of beta-l actam antibiotics is reported, in aqueous solution the purified p-nitr obenzyl (PNB) carboxy-esterase was soluble, monomeric (molecular weigh t: 54 000 by SDS-PAGE or by gel filtration) and exhibited an acidic pl , 4.1, The PNB carboxy-esterase catalyzed rapid ester hydrolysis for s imple organic esters such as PNB-acetate, benzyl acetate and alpha-nap hthyl acetate and catalyzed deblocking (ester hydrolysis) of beta-lact am antibiotic PNB esters such as cephalexin-PNB and loracarbef-PNB. Th e N-terminal amino acid sequence and the amino acid composition are re ported. A serine residue is involved in ester hydrolysis: the PNB carb oxy esterase was inhibited by phenylmethylsulfonyl fluoride and diethy l p-nitrophenyl phosphate; one mole of diisopropyl fluorophosphate tit ration was required per mole of PNB carboxy-esterase for complete inhi bition, When the [H-3]-diisopropyl fluorophosphate-treated biocatalyst was digested with Lys C and the resulting peptides separated by HPLC, a single [H-3]labeled peptide was obtained; its amino acid sequence i s reported, Inhibition of the PNS carboxy esterase by diethyl pyrocarb onate suggests that a histidinyl residue (or residues) is (are) also i nvolved in the catalytic site of the esterase.