Establishment Of Transformation Method For Bacillus Subtilis And Construction Of PGLV Expression Vector For High Efficiency Expression

Escherichia coli expression system, although mature and easy operation it is, its expression product is easy to form inclusion bodies, production efficiency is low.In addition, Escherichia coli is a potential pathogenic bacteria, such as lipopolysaccha-ride endotoxin release of pyrogen in the growth process, that has been restricted for expression of pharmaceutical and food production.Bacillus subtilis, as a expression system has no pathogenicity, does not produce endotoxin, strict growth under aerobic conditions, easy to express the protein secreted into the culture medium. In addition, the fermentation technology of bacillus has been very mature, such as ?-amylase, protease and other commercial enzymes.The study was on the construction of Bacillus subtilis expression system on amylase gene, starting from the selection of strong promoter induced by maltose,named Pglv-pHCMC04, and drew a low salinity environment starvation for defective strain transformation of Bacillus subtilis by optimizing the conditions of efficient transformation methods, the S method,which transformation efficiency is up to 1.2 × 104 CFU/g DNA. To learn more about the expression system, we took from small and big gene, signal peptide gene on experiments, to connect with red fluorescent protein, Bacillus licheniformis in high temperature amylase gene with and without signal peptide, Streptomyces griseus amylase gene with signal peptide as reporter genes to research the characteristics, it can be concluded that the expression of Bacillus subtilis in intracellular is possible, but there are certain limitations on the gene size or other aspects.Extracellular secretion system needs the signal peptide of the same genus to complete. Then optimize the fermentation conditions of high tern perature amylase gene with Bacillus licheniformis signal peptide, from optimum maltose adding amount, optimum temperature, induction time, and optimum inoculation quantity of several aspects, to optimize the fermentation conditions of single factor, the optimum fermentation conditions were obtained:with inoculated 2% to OD600 reached 0.55 and added 1.5% maltose at the temperature of 35?, the enzyme activity reached the highest to 2.53 (U/mL).Finally, the mutation maltose promoter catabolite repression element ere sequence has changed from GC to AT by reverse PCR technology, experiment and based on the above optimization,Redetermin ation of enzyme activity, enzyme production that high-speed ahead of time, and the enzyme activity was increased to nearly five times, reached 12.2 (U/mL).