High-performance Expression of Recombinant Pullulanase in Bacillus Subtilis
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Abstract:
Pullulanase can specifically hydrolyze amylopectin to get amylose, which is important in Starch industry applications. In this study, the pullulanase gene pul was cloned from the Bacillus naganoensis ATCC 53909 genome and inserted into the E.coli-Bacillus subtilis shuttle vector pBE to construct the expression vector pBE-pul. Based on these, seventeen strong promoters from Bacillus subtilis, Bacillus licheniformis and Bacillus amyloliquefaciens were respectively cloned into the expression vector pBE-pul and transformed into Bacillus subtilis ATCC6051∆10. Finally, Seventeen recombinant strains containing different strong promoters were constructed, the secretory expression of pullulanase was achieved and the activity of pullulanase was measured. The results indicated that the activity of pullulanase mediated by promoter P43 and PspovG was significantly higher than that mediated by the other promoters and the activity of pullulanase mediated by promoter PspovG was higher than that mediated by the promoter P43.Meanwhile, the pullulanase gene mutant pul324 which deleted N-terminal 108 amino acids was investigated, which was mediated by promoter PspovG. Through comparing seventeen promoters and two pullulanase genes, a recombinant strain that can efficiently overexpress pullulanase was constructed successfully. The highest pullulanase activity reached 389.85 U/mL, significantly higher than that reported in the literature.
