[Extracellular serine proteinase of Bacillus thuringiensis]

Pure extracellular serine proteinase has been isolated from a broth filtrate of Bacillus thuringiensis, strain 69-6R by fractionation with ammonium sulfate and affinity chromatography on Sepharose 4B derivatives containing p-(omega-aminomethyl)-phenylboronic acid and cyclopeptide bacillichin as ligands. The enzyme is completely inactivated by phenylmethylsolfonyl fluoride, a specific reagent for serine proteinases, has the molecular weight of 29 000 and pI of 8.4, reveals maximal activity and stability at pH 8.5 and is inactivated at pH values below 4 and above 10 and at temperatures above 60 degrees. The enzyme hydrolyzed azokasein, bovine serum albumin and synthetic chromogenic peptide substrates, e.g. benzyloxycarbonyl-L-alanyl-L-alanyl-L-leucyl p-nitroanilide and possesses the esterolytic activity. In terms of its physico-chemical characteristics, interaction with specific inhibitors and substrates, extracellular serine proteinase from Bacillus thuringiensis can be related to subtilisins. However, its amino acid composition-Lys16, His4, Arg8, Asx28, Thr16, Ser18, Glx29, Pro12, Gly32, Ala31, Val19, Met5, Ile12, Leu18, Tyr11, Phe10, Trp4 appears to be an intermediate between that of subtilisins and intracellular serine proteinases of Bacilli.