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 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: Km was 0.132 mM, and Vmax was 4.8 μmol• (mg protein)-1 • min-1.
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Amaki et al. (1992) studied this question.
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