Study On Expression Of An Alkaline Protease Gene From Bacillus Pumilus

Bacillus pumilus UN-31-C-42 come from strain BA(06), which is separated from life waste in ChengDu, after many times mutation. From the result of small-scale and enlarged experiments, it is indicated that the fermentation has high dehairing activity and low collagen-degradation activity. This result suggests that the protease produced by this strain might be ideal for leather industry. The protease DHAP has been purified, which can accomplish the whole dehairing process lonely.The AP gene has been cloned, which has complete coding region and partial promoter region. In order to validate whether the AP gene code the protease DHAP and the expression product of it can accomplish the whole dehairing process lonely, and construct the engineering bacteria of the AP gene , the study is performed on the expression of AP gene in B. subtilis WB600. The study includes four parts: the construction of the transform system of B. subtilis WB600, cloning of the strong promoter from bacillus, the construction of expression plasmid of AP gene and the expression of AP gene in B. subtilis WB600.In the first part, the following work was performed. The growth curves of B.pumilus UN-31-C-42 and B. subtilis WB600 were measured. The sensitivity of the two stains to the two kinds of antibiotic was determined.They were both sensitive to kanamycin. So, the kanr gene was chosen to be the marker of the transform system of them. B.pumilus UN-31-C-42 was sensitive to chloramphenicol, while B. subtilis WB600 could grow at 20 g / mL chloramphenicol. The kanr gene was cloned from the E.coli - B.subtilis shuttle vector pSUGV4, and the homology of nucleic acid between it and the one of pUB110 from Staphylococcus aureus is 100%. The competent-cell, protoplast and electroporation transformation methods were used to transform B. subtilis WB600 with the vector pSUGV4.With the competent-cell transformation methods, when the OD560 reach 1.0, the culture was transformed into MD media to cultivate, the high transformation efficiency 1.4 X 10/ g DNA was obtained. The effect of concentration of lysozyme and how long it works to the protoplast formation rate and regeneration rate were studied on B. subtilis WB600. When lysozyme concentration is 0.5mg/ml, and it works for 1 hour at 37 , the protoplast formation rate will be almost 100% and the transformation efficiency was 1.0 xio3 g DNA. Several factors affecting electroporation transformation efficiency were investigated. The parameters that resulted in high transformation efficiency were: OD560 0.8, electricity intensity 16KV/ cm, buffer SHMG , capacitance 25uF, resistance 200 , 60uL cell volume and the transformation efficiency was 2.4 x 102 gDNA.In the second part, the following study work was performed. The promoter fragments were obtained from B.pumilus UN31-C-42 and B.subtil is WB600 with the promoter-probe vector pSUPV4 in E.coli. There were 5 strong promoter fragments cloned from B.pumilus UN31-C-42, which were named Bp21 Bp38, Bp53 Bp57 and Bp70. There were 3 strong promoter fragments cloned from B.subtilis WB600, which were named Bs2, Bs4 and Bs8. These fragments were sequenced and analyzed respectively and they all contained some conserved sequences of prokaryotic gene promoters. The most efficient promoter fragment Bp53 from B.pumilus UN31-C-42 could promote the alkaline protease gene of B.pumilus expression not only in E.coli but also in B.subtilis cells. While, the most efficient Bs2 fragment from B.subtilisWB600 could promote the alkaline protease gene expression in E.coli and it also could promote the kanr gene expression in B.subtilis cells. The constitutive promoter P43 and the inducible promoter and signal peptide of the SacB gene were cloned from B.subtilis WB600, which were sequenced and analyzed. The nucleic acid of them has 100% homology to the sequence reported in Genbank. They could not promote the AP gene expression in B.subtilis WB600.In the third part, the following study work was performed. The coding region and the terminator of AP gene about 1.3kb and the Bp53 promoter fra...