A CYSTEINE-DEPENDENT SERINE-PROTEASE ASSOCIATED WITH THE DORMANT SPORES OF BACILLUS-CEREUS - PURIFICATION OF THE PROTEIN AND CLONING OF THECORRESPONDING GENE

Subtilisin-like serine protease, which is associated with the dormant spores of Bacillus cereus, was solubilized by washing the spores with 2 M KCl and purified to homogeneity by carbobenzoxy-D-phenylalanine-li ganded affinity column chromatography and hydrophobic interaction colu mn chromatography. Enzyme activity was completely inhibited by reagent s for sulfhydryl groups such as HgCl2 as well as by conventional subti lisin inhibitors, suggesting the enzyme to be cysteine-dependent. The enzyme retained activity in 5 M urea at 4 degrees C for at least 2 mon ths, and the specific activity was 50 times that of subtilisin BPN' wh en measured for a common chromogenic substrate, carbobenzoxy-glycl-gly cyl-L-leucine p-nitroanilide. The gene encoding this protease was clon ed in Escherichia coli, and its nucleotide sequence was analyzed. The deduced amino acid sequence suggested that the protease is produced as a precursor comprising three portions; a signal sequence (28 amino ac id residues), a prosequence (80 amino acid residues) and a mature enzy me (289 amino acid residues), The mature region of the enzyme had high similarity with a thermitase from Thermoactinomyces vulgaris (72% ide ntity) and a thermostable alkaline protease from Thermoactinomyces sp. E79 (66% identity), which have the N-terminal sequence showing scarce ly noticeable similarity with corresponding stretches of subtilisins a nd mercuric ion-sensitive free cysteine in the equivalent position of the primary structure.