Production of thiolsubtilisin YaB and its application to peptide-ligation

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Alkaline elastase produced by alkalophilic Bacillus YaB starin is a serine-protease having high optimum pH and effectively hydolyzes elastin, a sclero-protein. The enzyme has a 55% amino acid sequence homology to subtilisin BPN, and can be called as subtilisin YaB.The substitution of Ser221 at the catalytic center of subtilsin BPN, to Cys causes loss of protease activity but the substituted one, called as thiolsubtilisin, still retains peptide-ligase activity. So the thiolsubtilisin is greatly interested as a new tool for biotechnology. The production of thiolsubtilisin by a recombinant Bacillus subtilis is, however, highly difficult so far. Subtilisins are synthesized in vivo as precursors having pro-sequences. The pro-sequences are auto-catalytically processed and known as essential guide sequences for active conformation. The aim of this study is to try to produce thiolsubtilisin YaB in Bacillus subtilis by a new genetic engineering method. We succeeded to produce active subtilsin YaB in Bacillus subtilis which carried a plasmid encoding two separate genes for pre-pro and pre-mature polypeptides of subtilsin YaB,though the efficiency is low. The production of thiolsubtilisin YaB in a similar fashion is, however, not successful. In the next place, we aimed to secrete pro-thiolsubtilisin YaB by Bacillus subtilis WB600 strain(6 extracellular proteases-negative mutant) carrying a plasmid encoding pre-pro-mature polypeptide of thiolsubtilisin YaB.We observed a secreted 52 kDa species positive in the western blotting. The proteins was proved to be processed to a mature size species (26kDa). This result clearly shows that the 52 kDa species is the pro-form and that the 26 kDa pro-form is 36 kDa. The observed molecular mass of 52 kDa, therefore, suggests an unusual structure of the pro-form.