PURIFICATION, CHARACTERIZATION AND PARTIAL AMINO-ACID-SEQUENCES OF A NOVEL CEPHALOSPORIN-C DEACETYLASE FROM BACILLUS-SUBTILIS

Cephalosporin-C deacetylase [EC 3.1.1.41] was purified electrophoretic ally to homogeneity from the newly isolated Bacillus subtilis SHS 0133 (FERM BP-2755). The enzyme was purified about 27-fold with a yield 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 w ith apparent molecular weights of 35,000. The cephalosporin-C deacetyl ase was stable up to 60-degrees-C for 30 min at pH 7.0. The enzyme exh ibited 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 1.0 mM, respectively. One of the reaction products from 7-ACA, deacetyl-7-ACA, was a weak noncompetitive inhibit or and other product, acetate, was a weak competitive inhibitor; the K (i) values were 171 and 290 mM, respectively. However, these weak prod uct 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 isoel ectric focusing. The observed data indicate that the enzyme is differe nt from known cephalosporin-C deacetylases. In addition, amino acid se quencing of the N-terminus and Achromobacter proteinase 1-digested pep tides yielded no sequences with similarities to other known proteins b y a computer search.