Construction And High Efficiency Expression A Recombinant Beta-Amylase-Trehalose Synthase In Escherichia Coli

Trehalose is a nonreducing disaccharide of glucose, its unique properties make it has excellent protection function. Trehalose has high water-holding activities, which maintain the fluidity of membranes under dry conditions. It also stabilizes enzymes, foods, cosmetics, and pharmaceuticals at high temperatures. In addition, trehalose can peotect proteins against denaturation because of desiccation and freezing, it’s an essential component for maintaining cell viability. Due to its desirable physical and chemical characteristiscs, commercial production of trehalose is anticipated.However, trehalose has a high price because the complex method of production. So, in order to reduce the production cost and find a simplification production, in this paper taking the beta-amylase gene of Bacillus cereus and trehalose synthase gene of Pseudomonas putida as starting point, and successfully constructed a DNA fragment that contains two genes encoding beta-amylase gene and trehalose synthase gene, this gene was expressed in Escherichia coli.In the first place, beta-amylase and trehalose synthase gene were clone from Bacillus cereus and Pseudomonas putide genomic DNA, respectively. And successfully constructed the recombinant plasmid pET-28a-ba and pET-28a-ts, the recombinant beta-amylase and trehalose synthase were expressed in Escherichia coli. The 2% of starch solution was catalysed by recombinant beta-amylase after 12h at pH6.47 and 45 ℃,the concentration of maltose in the reaction liquid was 15.71g/L, the enzyme activity of beta-amylase was 12.11U/ml and the yield of maltose was 78.5%.15% of maltose was catalysed by recombinant trehalose synthase after 12h at pH7.4 and 45℃, the concentration of trehalose was 57.38g/L, the enzyme activity of trehalose synthase was 42.13U/ml and the yield of trehalose was 38.9%.In this paper, the pepides of LGSRSAEL, (GGGGS)2 and (EAAAK)3 were used as linkers and chimeric genes were constructed with two fused direction. Six correct chimeric genes were constructed by ligase. All the six chimeric genes were successfully expressed in Escherichia coli BL21(DE3). In the SDS-PAGE profiles of the fusion protein molecular weight was about 120kDa. To express the recombinant enzymes, the transformed Escherichia coli BL21(DE3) was inoculated into Luria-Bertani(LB) medium, but only three fusion enzymes (BAP3TS,TSP2BA and TSP3BA)exhibited both beta-amylase and trehalose synthase.In the study, LB medium and M9-ZJ medium for fusion enzymes were studied and compared. The conclusion had been made form the research that the more favorable medium for transformed Escherichia coli BL21(DE3) to express the fusion enzyme. On this base, the medium composition were investigated to find the best culture medium in which the transformed strains express more efficient recombinant fusion enzyme. The transformed strains was cultured in optimization of culture medium, and all the six fusion enzymes were successfully exhibited both beta-amylase and trehalose synthase activities. The fusion enzyme BAP3TS have the highest activity among six fusion enzymes.6% soluble starch as the substrate was catalyzed by fusion enzyme BAP3TS at pH7.0 and 45℃ for 6h. The concentration of trehalose in the reaction solution was 25.36g/L, the enzymt activity of BAP3TS was 37.24U/ml and the productivity of trehalose was 42.27%. The yield of trehalose was 11 times of LB medium, and compared with the mixed enzyme, trehalose production increased by 2.5 times.