Cloning, Expression And Activities Of Recombinant Maltogenic α-amylase And Glutamyl Endopeptidase

Biotechnology have more influence on the production and use of enzyme, especially with the genetic engineering and protein technology becoming more and more mature. People also don’t need to depend on natural enzymes. We can clone various kinds of natural enzymes to make them highly effective express in biological systems,improve enzyme activities, and even create new enzymes through genetic engineering. This research cloned two enzymes from different sources and studied their activities.Maltogenic a-amylase can hydrolyze starch or related polysaccharide, generating maltose mainly. Maltogenic a-amylase can be used in starch to delay the aging speed, maintain flexibility, make maltose syrup and so on. Novamyl imported from abroad is too expensive, and domestic researches have just started. In this study, we optimize the codon of maltogenic a-amylase from Bacillus stearothermophilus and successfully expressed in E.coli. On the other hand, we also cloned it into Bacillus subtilis. Eventually, the activity of the enzyme is improved by optimizing.Glutamyl endopeptidase belongs to the serine protease family, it can specifically hydrolyzed the peptide bond formed by the alpha-carboxyl of glutamic acid or aspartic acid,so that the polypeptide molecule or protein is cleaved into small peptides. Glutamyl endopeptidase enzyme can be used in multi-functional peptide preparation, peptide spectrum analysis, glycated hemoglobin as well as food and other industries. Glutamyl endopeptidase isolated abroad are most pathogens, limiting their applications. In this study, we cloned the Glutamyl endopeptidase of Bacillus licheniformis, and succeeded in increasing the amount of soluble expression in E. coli.Our study included three parts as follows:1. The cloning expression and activities of maltogenic a-amylase in E. coliaccording to the codon bias of bacillus subtilis,we optimized the gene of maltogenic a-amylase from Bacillus stearothermophilus C599. Optimized gene was cloned into E. coli plasmid vector pET-32a (+) to construct thioredoxin fusion expression vector pET32a (+)-M. The recombinant plasmid was transformed into E. coli BL21(DE3).We induced the recombination strain pET32a (+)-M/E. coli BL21(DE3) with IPTG, the strain expressed the thioredoxin fusion protein as inclusion bodies whose molecular weight was about90kD. We did not get the active protein by reducing the inclusion bodies or refolding experiments. Ultrasound sonicated supernatant as a crude enzyme solution, the enzyme activity measured by DNS was1.56U/m L.2. The cloning..expression activities and optimization of Maltogenic α-amylase in B. bacillusAccording to the maltogenic a-amylase gene, we construct a Bacillus expression vector pWB980-M. It was transformed to Bacillus subtilis1A474which had no maltogenic a-amylase activity.36h fermentation supernatant as a crude enzyme solution, the enzyme activity was2.53U/mL. then the engineered strain pWB980-M/1A474was used to design the best fermentation conditions. The parameters included medium components, initial pH, oxygen, inoculum size, carbon sources, nitrogen sources, and secretion accelerator. The best fermentation conditions were as follows:12%soybean solution,1%Trypton, PBS (pH7.0),0.01%SDS,20%of the amount of bottling, inoculation amount of8%. After24hour’s fermentation in37℃,the maximum activity reached14.33U/mL.3. The cloning,expression and activities of Glutamyl endopeptidase in E.coliThe Glutamyl endopeptidase gene was amplified by PCR with genomic DNA of Bacillus licheniformis ATCC14580as template, and expressed in Escherichia coli BL21(DE3) by yielding hybrid plasmid pET28a-GE. Induced with IPTG, the E.Coli BL21harboring pET28a-GE successfully expressed the as inclusion bodies of which molecular weight was about25kD. we explore how to improve the solubility, the results showed that the chaperone could improve the solubility of recombinant Glutamyl endopeptidase, while thioredoxin and low temperature was futile. The Enzymatic Properties analysis showed that the enzyme optimum reaction temperature was42℃. The optimum pH was8.0, and the metal ions Mn2+could preferably activate the enzyme activity.To sum up, in this study we successfully realize the heterologous expression of two proteases by means of genetic engineering,which lays a foundation for our research group to continue to study these two enzymes,and provide references for the development and application of these two enzymes.