MOLECULAR-CLONING AND NUCLEOTIDE-SEQUENCE OF THE 90K SERINE-PROTEASE GENE, HSPK, FROM BACILLUS-SUBTILIS (NATTO) NO-16

We previously reported purification and characterization of a 90k seri ne protease with pI 3.9 from Bacillus subtilis (natto) No. 16 [Kato et al. 1992 Biosci Biotechnol Biochem 56:1166]. The enzyme showed differ ent and unique substrate specificity towards the oxidized B-chain of i nsulin from those of well-known bacterial serine proteases from Bacill us subtilisins. The structural gene, hspK, for the 90k serine protease was cloned and sequenced. The cloned DNA fragment contained a single open reading frame of 4302, bp coding a protein of 1433 amino acid res idues. The deduced amino acid sequence of the 90k-protease indicated t he presence of a typical signal sequence of the first 30 amino acids r egion and that there was a pro-sequence of 164 amino acid residues aft er the signal sequence. The mature region of the 90k-protease started from position 195 of amino acid residue, and the following peptide con sisted of 1239 amino acid residues with a molecular weight of 133k. It might be a precursor protein of the 90k-protease, and the C-terminal region of 43k might be degraded to a mature protein from the precursor protein. The catalytic triad was thought to consist of Asp33, His81, and Ser259 from comparison of the amino acid sequence of the 90k-prote ase with those of the other bacterial serine proteases. The high-molec ular-weight serine protease, the 90k-protease, may be an ancient form of bacterial serine proteases.