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

A procedure for purifying to homogeneity a microbially produced biocatalyst useful for deblocking intermediates in the manufacture of beta-lactam antibiotics is reported, in aqueous solution the purified p-nitrobenzyl (PNB) carboxy-esterase was soluble, monomeric (molecular weight: 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 simple organic esters such as PNB-acetate, benzyl acetate and alpha-naphthyl acetate and catalyzed deblocking (ester hydrolysis) of beta-lactam antibiotic PNB esters such as cephalexin-PNB and loracarbef-PNB. The N-terminal amino acid sequence and the amino acid composition are reported. A serine residue is involved in ester hydrolysis: the PNB carboxy esterase was inhibited by phenylmethylsulfonyl fluoride and diethyl p-nitrophenyl phosphate; one mole of diisopropyl fluorophosphate titration was required per mole of PNB carboxy-esterase for complete inhibition, 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 is reported, Inhibition of the PNS carboxy esterase by diethyl pyrocarbonate suggests that a histidinyl residue (or residues) is (are) also involved in the catalytic site of the esterase.