Molecular cloning of subtilisin J gene from bacillus stearothermophilus and its protein engineering

Abstract

The structural gene for subtilisin J from Bacillus stearothermophilus NCIMB10278 was cloned in Bacillus subtilis using pZ124 as a vector, and its nucleotide sequence was deterimined. The nucleotide sequence revealed only one large open reading frame, composed of 1,143 base pairs and 381 amino acid residues. A Shine-Dalgarno sequence was found 8 bp upstream from the translation start codon (GTG). The deduced amino acid sequence revealed an N-terminal signal peptide and pro-peptide of 106 residues followed by the mature protein composed of 275 residues. The productivity of subtilisin J in the culture broth of the Bacillus subtilis was about 46-fold higher than that of the Bacillus stearothermophilus. The amino acid sequence deduced from the extracellular alkaline protease subtilisin J is highly homologous to that of subtilisin E and it shows 69\% homology with subtilisin Carlsberg, 89\% with subtilisin BPN`` and 70\% with subtilisin DY. Some properties of the subtilisin J with had been purified from the Bacillus subtilis were examined. The subtilisin J has alkaline pH characteristics and a molecular weight of 27,500. All enzymatic properties of the subtilisin J including thermostabilities were similar to those of subtilisin E and subtilisin Amylosacchariticus. Eight mutations were introduced to study the molecular basis of thermostability of subtilisin J and to analyze the structure-function relationship in the subtilisin J. Among eight mutant proteins, Gln-195 and Asp-49 mutant showed activity as wild-type subtilisin. Gln-195 mutant enzyme was expressed in Bacillus subtilis and was purified from the culture supernatant. When the mutant enzyme was expressed at 37$^\circ$C in the presence of 2 mM $CaCl_2$, the pattern of enzyme production was quite different from that of wild-type. The purified Gln-195 mutant enzyme was analyzed with respect to optimal temperature, optimal pH, and heat stability. The mutation was found to decrease the heat stability but not cataly...