Expression Of CotA Protein From Bacillus Subtilis By Pichia Pastoris And Recombinant Protein Characterization Study

The study amplified CotA gene from Bacillus subtilis strain LS02accroding to the conservative sequence of functional gene and successfully inserted it into pMD18-T vector. Recombinant CotA-T vector was transformed to E.coli JM109. Sequencing result corroborates the CotA gene contains1524bp and encodes513amino acids; the GC content is48.18%. The DNA sequence has been submitted to the Genbanck, and the accession number is GU972587. Bioinformatis softwares were used to test the genetic relationship and basic information of CotA. The results indicate that CotA belongs to the bacterial laccase group, and is closest to the laccase of subtilis genera, the predicted molecular mass is58.14kDa; the sequence doesn't contain signal peptide and has5N-glycosylation and2O-glycosylation sites.Recombinant CotA-pPICαB vector was constructed and transformed into Pichia pastoris strain SMD1168H in the form of linearized state by electroporation. Positive transformants were detected at plate level by BMM plate with0.5mM ABTS. SDS-PAGE result demonstrates the recombinant CotA (r-CotA) has been secreted to the media and the target band shows a molecular mass of approximate62kDa. Besides, the background protein content is much lower than prokaryotic expression system, which makes it easy to purify the protein in subsequent process. Research was carried out to test the ferment biomass of recombinant P. pastoris strain, enzymatic expression level and media pH. It turns out that adding alanine obviously facilitate the production of r-CotA. At the same time alanine can maintain the pH of media and efficiently improve the stability of r-CotA in cultivation process.Enzyme characterization of r-CotA was tested and compared with that of spore suspension. The best catalytic pH of r-CotA with ABTS, SGZ and DMP is3.4-4.2,6.2and8.6. R-CotA was proved to possess notable pH stability under a wide range of pH. The optimum biocatalytic temperate of r-CotA is80℃and70℃as react with ABTS and SGZ, which is higher than that of wild spore suspension. In addition, r-CotA has shown better thermostable stability than other reported prokaryotic r-CotA till now. Mg2+has weak promoption for r-CotA, while other metal ions demonstrate inhibitions to the enzyme in defferent level. R-CotA exhibits conspicuous tolerance towards organic solvent. Nevertheless, it shows less resistant to enzyme inhibitors.