| In this study, we obtained three producing pullulanase microorganisms:Bacillus licheniformis、Bacillus cereus、Bacillus subtilis. The gene encoding pullulanase were amplified by PCR using degenerate primers and specific primers. The obtained pullulanase gene were2133bp、2142bp、2157bp, respectively. The nucleotide sequences had the high similarity to reported pullulanase. These three pullulanase gene were inserted into the expression vector pET28a/pET32a/pET42a, and transformed into E.coli BL21. SDS-PAGE electrophoresis analysis, the pullulanase gene were successfully expressed in E.coli BL21, the protein molecular weight were about80kDa.The properties of pullulanse were studied. The pullulanase of Bacillus licheniformis in expression vector pET42a have the maximum activity of4.48U/ml, the optimum temperature of this enzyme was40℃, optimum pH value was7.0. The pullulanase of Bacillus cereus and Bacillus subtilis optimum temperature were40℃and30℃, respectively. the optimum pH were6.0.In this research paper, shuttle intracellular expression vector pBNSl and secretion expression vector pBNS2were constructed using pBE2a-based plasmid. Both vectors are based on the strong promoter p43and the ampicillin resistance gene expression cassette. To obtain the secretion expression vector pBNS2, an efficient Shine-Dalgarno (SD) sequence、the amyQ signal peptide and a multiple cloning site (BamHI,SaЛ,SacI,XhoI,Pstl,Sphl) were inserted into the downstream of p43promoter. To test the vectors, pBNSl-gfp and pBNS2-gfp were first constructed to expression green fluorescent protein (GFP) intra-and extracellular in Bacillus subtilis1A751, respectively. Fluorescence detection and SDS-PAGE electrophoresis analysis, green fluorescent protein were successfully expressed in E.coli BL21and Bacillus subtilis1A751, the expressed protein molecular weight were about30kDa, gfp gene of pBNSl plasmids achieved efficient secretion expression in Bacillus subtilis1A751. |
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