Construction Of Bacillus Subtilis Neutral Protease Producing Strain And Optimization Of Fermentation Medium

As a microbial enzyme, neutral protease A has a wide range of applications in food, animal husbandry, leather processing and pharmaceutical industry. Bacillus subtilis(B. subtilis) can express and secrete extracellular neutral protease A(NprE). The wild-type B. subtilis expresses and secrets limited production of neutral protease A due to the limitations of inherent regulation mechanism of gene expression in the cell, so it can not meet the need of practical application. In this study, gene manipulation techniques were adopted to constructe recombinant B. subtilis which can overexpress and secret neutral protease A. In addition, this study also optimizations the fermentation medium of the neutral protease A which is made of recombinant B. subtilis.The coding sequence and terminator sequence of gene nprE which codes neutral protease A in B. subtilis were connected with artificial expression cassette PAE to construct the recombined PAE-nprE gene which can express the neutral protease A constitutively. PAE-nprE gene was inserted into plasmid pHP13 which is a kind of the E. coli-B. subtilis shuttle vector to construct a plasmid named pHP13N that can express neutral protease A.The plasmid pHP13N was transformed into B. subtilis DB104 and its derivative strains BDY2, BDY3, BDY4 and BDY5 while it was also transformed into B. subtilis 168 and its derivative strain BN. Skim milk medium was used to screen and compare preliminarily the ability of expressing and secreting protease among the transformants. The results shows that large hydrolysis circle could be screened among transformants of B. subtilis DB104 and its derivative strains on the skim milk medium while it was not screened in teansformants of B. subtilis 168 and its derivative strains. This phenomenon indicated that neutral protease A can be expressed and secreted more efficiently in B. subtilis DB104 and its derivatives.As the the test strain, B. subtilis BDY2/pHP13N was used in the test of optimizate fermentation medium of neutral protease A. According to the resutle of single-factor tests, defined fermentation medium was composed of glucose, corn syrup powder, baking flour, phosphate, MgSO4, ZnSO4, CaCl2 and Tween-80. Corn syrup powder and Tween-80 were very significant factors while glucose?MgSO4 and ZnSO4 were significant factors in the fermentation of neutral protease A by Plackett-Burman experimental. The optimum fermentation medium consisted of glucose 24 g/L, corn steep powder 18 g/L, roasted soy flour 20 g/L, buffer of potassium phosphate 0.06 mol/L, ZnSO4 0.2 g/L, CaCl2 0.2 g/L and Tween-80 1 g/L determined by Slope-climbing test and response surface optimization though central composite design. Strain B. subtilis BDY2/pHP13N was fermented in shake using the optimum fermentation medium. After fermenting for 72 h at 37 ?, the activity of neutral protease A in the fermentation broth reached 520 U/mL which was improved by 3.8 times compared with the activity before optimization.