Purification, Characterization and Partial Amino Acid Sequences of a Novel Cephalosporin-C Deacetylase from Bacillus subtilis

概要

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Cephalosporin-C deacetylase [EC 3.1.1.41] was purified electrophoretically to homogeneity from the newly isolated Bacillus subtilis SHS 0133 (FERM BP-2755). The enzyme was purified about 27-fold with a yeild of 9 % and a specific activity of 187.4 U/mg protein. The native enzyme (molecular weight, 280,000) was composed of eight identical subunits with apparent molecular weights of 35,000. The cephalosporin-C deacetylase was stable up to 60℃ for 30min at pH 7.0. The enzyme exhibited Michaelis-Menten kinetics with the substrates cephalosporin C, 7-aminocephalosporanic acid (7-ACA) and p-nitrophenyl acetate; the K_m values were 24.0,7.9 and 1.0mM, respectively. One of the reaction products from 7-ACA, deacetyl-7-ACA, was a weak noncompetitive inhibitor and other product, acetate, was a weak competitive inhibitor; the K_i values were 171 and 290mM, respectively. However, these weak product inhibitors did not prevent the completion of the deacetylation of 7-ACA. The pI value of the enzyme was determined to be 5.3 using isoelectric focusing. The observed data indicate that the enzyme is different from known cephalosporin-C deacetylases. In addition, amino acid sequencing of the N-terminus and Achromobacter proteinase I-digested peptides yielded no sequences with similarities to other known proteins by a computer search.
公益社団法人日本生物工学会の論文
1994-01-25