Production of thiolsubtilisin YaB and its application to peptide-ligation

由嗜碱性芽孢杆菌YaB淀粉蛋白产生的碱性弹性酶是一种丝氨酸蛋白酶，具有较高的最适pH值，可有效水解弹性蛋白。该酶有55%的氨基酸序列与枯草菌素BPN同源，可称为枯草菌素YaB。在BPN的枯草菌素催化中心的Ser221被Cys取代导致蛋白酶活性丧失，但被取代的称为硫代枯草菌素的那一个仍然保持肽连接酶活性。因此，硫代枯草菌素作为一种新的生物技术工具备受关注。然而，到目前为止，重组枯草芽孢杆菌生产硫代枯草菌素是非常困难的。枯草菌素在体内作为具有前序列的前体合成。前序列是自动催化加工的，被称为活性构象的基本引导序列。本研究的目的是利用一种新的基因工程方法，在枯草芽孢杆菌中制备巯基枯草菌素YaB。我们成功地在枯草芽孢杆菌中获得了活性枯草素YaB，该枯草素YaB携带一个质粒，该质粒编码两个独立的枯草素YaB前原和前成熟多肽基因，但效率较低。然而，以类似的方式生产巯基枯草菌素YaB并不成功。接下来，我们的目标是通过枯草芽孢杆菌WB600菌株（6胞外蛋白酶阴性突变体）分泌原硫枯草菌素YaB，该菌株携带编码巯基枯草菌素YaB预成熟多肽的质粒。在western blotting中，我们观察到52个kDa种的分泌呈阳性。经证实，该蛋白被加工成一个成熟大小的物种（26kDa）。这一结果清楚地表明52 kDa的物种为前型，而26 kDa的物种为36 kDa。因此，观察到的52 kDa的分子质量表明了一种不寻常的前型结构。

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.