PURIFICATION AND PROPERTIES OF A THERMOSTABLE ESTERASE OF BACILLUS-STEAROTHERMOPHILUS PRODUCED BY RECOMBINANT BACILLUS-BREVIS

A thermostable carboxyl esterase of Bacillus stearothermophilus excreted by recombinant B. brevis was purified to homogeneity using column chromatographies on DEAE Bio-Gel A, Sephacryl S-200HR, and Mono Q. The purified enzyme had a molecular weight of 29,000. The optimum pH at 37-degrees-C was 7.5. The esterase had higher activity toward triglycerides with short-chain fatty acids than with long-chain ones. The activity of the esterase was completely or significantly inhibited by diisopropyl fluorophosphate, phenylmethylsulfonyl fluoride, iodoacetamide, and p-chloromercuribenzoic acid, while it was slightly stimulated in the presence of 2-mercaptoethanol. This observation suggests that the enzyme is a serine enzyme having a sulfhydryl group for expressing the enzyme activity. The activation energy for inactivation of the enzyme was estimated to be 131 kcal.mol-1. The kinetic parameters of this enzyme with p-nitrophenyl butyrate were measured: K(m) was 0.132 mM, and V(max) was 4.8-mu-mol.(mg protein)-1.min-1.