Efficient Overexpression Of Recombinant Pullulanase In Bacillus Subtilis

Pullulanase?EC 3.2.1.41?is a starch debranching enzyme that specifically hydrolyzes?-1,6 glucosidic linkages in polysaccharides such as pullulan and amylopectin,and it is an indispensable enzyme in the starch processing industry.Bacillus subtilis is a recognized safety strain?GRAS?and capable of secreting biologically active proteins directly to the medium.In this study,a mutant strain of Bacillus subtilis ATCC 6051?10 which is capable of expressing exogenous proteins with high efficiency was used as the host to the overexpression of the pullulanase gene pul in Bacillus subtilis.By optimizing the promoters and the fermentation conditions,extracellular activity of pullulanase was enhanced.The construction and optimization of the recombinant B.subtilis not only offered valuable strains for getting rid of the dependence on import of pullulanase in China,but would also provide advantageous options for efficient production of recombinant enzymes from B.subtilis.Focusing on the enhancement of pullulanase production,the main research contents are listed as follows:?1?Screening of natural promoters.Nineteen promoters were selected to study the effect on the expression of pullulanase pul in Bacillus subtilis from species of genus Bacillus including Bacillus subtilis,Bacillus licheniformis,Bacillus amyloliquefaciens and Bacillus megaterium.Six strong promoters,which were P43,PspovG,Psig W,Ppgk,PsodA and PamyL,were selected for further study.?2?Selection of pullulanase Genes.The pullulanase gene pul and its mutant pul324 from Bacillus naganoensis and the pullulanase gene pulA from Bacillus acidopullulyticus were selected and expressed using strong promoters P43 and PspovG,respectively.The activity of pullulanase pul mediated by promoter PspovG was the highest among the tested pullulanases,which was up to 389.85 U/mL at 72 h in shake flask fermentation.?3?Strong promoters P43,PspovG,PsigW and PamyL were selected to construct the tandem promoters,and the effect of the tandem promoters on the expression of pullulanase pul in B.subtilis was evaluated.The enzymatic activity showed that the strain BE307?PamyL-PspovG?displayed the highest expression level among all these strains and reached 620.80U/mL at 72 h,Compared to the strain BE105?PspovG-pul?,the pullulanase activity increased by 59.24%.?4?By studying the enzymatic properties of pullulanase,the optimum reaction temperature and pH for the enzyme activity was found to be 57.5?and 5.0,respectively.Furthermore,the pullulanase exhibited a stable activity under the p H from 5.5 to 6.5 and the temperature from45 to 55?,and it is suitable for storage at-20?.Adding low concentrations of metal ions such as Mg²⁺,Zn²⁺and Ca²⁺can increase the activity of pullulanase,but as the concentration increases,the promoting effect gradually turns into inhibition.The higher the concentration of metal ion is,the more significant the inhibition is.?5?The fermentation of recombinant strain BE307 was optimized.The preliminary optimized fermentation medium were as follows:xylose 2%,yeast extract 2%,corn starch 4%,cotton-seed-cake power 5%,Tween-80 2.0%,the initial concentration of kanamycin sulfate was20?g/mL and its feed concentration was 5?g/m L.The optimized fermentation conditions were as follows:the initial pH of the medium was 7.0,the fermentation temperature was 37°C,and the inoculum volume was 5%.According to the needs of industrial fermentation,2%xylose and 2%yeast extract were replaced by 2%sucrose and 5%corn syrup dry powder,respectively.As a result,the highest activity of pullulanase reached to 1352.61 U/m L in 500 mL baffled shake flask at 72 h,increased by 117.88%after fermentation optimization.