Abstract
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°C was 7.5. The esterase had higher activity toward triglycerides with short-chain fatty acids than with long-chain ones. The activity of 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 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 : Km was 0.132 mM, and Vmax was 4.8 μmol·(mg protein)-1·min<-1>.
