ISSN:
1476-5535
Schlagwort(e):
p-nitrobenzyl carboxy esterase
;
biocatalyst
;
beta-lactam antibioticp-nitrobenzyl ester
;
Bacillus subtilis
;
deesterification
;
carboxyl protection
Quelle:
Springer Online Journal Archives 1860-2000
Thema:
Biologie
,
Werkstoffwissenschaften, Fertigungsverfahren, Fertigung
Notizen:
Abstract 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 purifiedp-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 α-naphthyl acetate and catalyzed deblocking (ester hydrolysis) of beta-lactam antibiotic PNB esters such as cephalexin-PNB and loracarbef-PNB. TheN-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 diethylp-nitrophenyl phosphate; one mole of diisopropyl fluorophosphate titration was required per mole of PNB carboxy-esterase for complete inhibition. When the [3H]-diisopropyl fluorophosphate-treated biocatalyst was digested with Lys C and the resulting peptides separated by HPLC, a single [3H]-labeled peptide was obtained; its amino acid sequence is reported. Inhibition of the PNB carboxy esterase by diethyl pyrocarbonate suggests that a histidinyl residue (or residues) is (are) also involved in the catalytic site of the esterase.
Materialart:
Digitale Medien
URL:
http://dx.doi.org/10.1007/BF01570007
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