Abbott_1975_Appl.Microbiol_30_413

An esterase that deacetylates cephalosporins was recovered from the supernatant of a Bacillus subtilis culture. It was partially purified by ammonium sulfate fractionation and ultrafiltration. The enzyme had a temperature optimum between 40 and 50 C and a pH optimum of 7.0. The molecular weight was estimated by gel filtration to be 190,000. The enzyme was very stable and retained greater than 80% of its activity after storage in solution at 25 C for 1 month. The esterase exhibited Michaelis-Menton kinetics with the substrates 7-aminocephalosporanic acid (7-ACA) and 7-(thiophene-2-acetamido)cephalosporanic acid (cephalothin); the K(m) values were 2.8 X 10(-3) and 8.3 X 10(-3) M, respectively. The products of 7-ACA deacetylation were weak competitive inhibitors, and a K(i) value of 5.0 X 10(-2) M was determined for acetate and of 3.6 X 10-2 M for deacetyl-7-ACA. Weak product inhibition did not prevent the deacetylation reaction from going to completion. A 5-mg/ml solution of partially purified esterase completely hydrolyzed (greater than 99.5%) a 24-mg/ml solution of 7-ACA in 3 h. Because of the kinetic properties and excellent stability, this enzyme may be useful in an immobilized form to prepare large quantities of deacetylated cephalosporin derivatives.