Purification and properties of carboxylesterase B of Escherichia coli.

Carboxylesterase B produced by Escherichia coli was purified 1,350-fold with a recovery of 12% by successive gel filtrations, DEAE-trisacryl, phenyl-Sepharose chromatography and preparative electrophoresis. The purified enzyme was found to be homogeneous, as judged by a single precipitation line in Ouchterlony double diffusion in an experiment with homologous antiserum. The apparent molecular weight determined by gel filtration and the isoelectric point determined by electrofocusing were 57,000 and 4.6, respectively. Using acetate, propionate and butyrate esters of 1-naphtol, it was observed that elongation of the acyl carbon chain resulted in a progressive increase in velocity of ester hydrolysis. The apparent Km for 1-naphtyl acetate was found to be 0.25 mM. The enzyme was maximally active at pH 7.4 and was found to be unstable below pH 5. Hydrolytic activity was preserved after heat treatment for 30 min at 60 degrees C, but was abolished by heating for 10 min at 70 degrees C. The enzyme was strongly inhibited by low concentrations of di-isopropyl fluorophosphate. This suggested that a serine residue is required for catalytic activity. Esterase was unaffected by tosyl-L-lysin chloromethylketone, iodoacetamide, 4-hydroxy-mercuribenzoate and EDTA. Using antiserum against purified carboxylesterase B of E. coli, significant immunological cross-reactions were observed between this antigen and carboxylesterase B produced by Shigella flexneri, S. boydii and S. sonnei.