Overexpression And Application Of The Alkaline Protease From Bacillus Alcalophilus

Since Alkaline protease has many advantages,including the high reactivity,no industrial pollution and wide adaptability,it is widely used in pharmaceutical,food,cosmetics,detergents,textile,leather,and other industries.In order to improve the activity of alkaline protease and study its application in preparing casein peptide phosphate,the work carried out in this study was as follows:DNA fragment encoding the alkaline protease gene?apr?was amplified from B.alcalophilus TCCC11006 by PCR.The gene obtained was then inserted into the expression vector pHY-WZX.The recombinant plasmid constructed was transformed into the protease-deficient strain Bacillus licheniformis HI 15,generating the genetically engineered bacterium B.licheniformis HI 15/pHY-WZX-apr.As analyzed by SDS-PAGE,the alkaline protease expressed by the recombinant bacterium showed as a major protein band of 35 KDa.Besides,after culturing for 56 h in basical fermentation medium,the activity of protease reached its maximum of 4910 U/mL,which was 2.79 times than starting strain.Compared with the enzymatic properties of commercially available 1398 neutral protease and alkaline protease,the stability ofthe recombinant alkaline protease were better under the high temperature and pH.Mn²⁺,Mg²⁺ and Ca²⁺could enhance its activity.Only a small amount of metal ions inhibited the activity of alkaline protease.The optimal meadium compositions were determined to be 50 g/L lactose,40 g/L soybean cake powder,3 g/L ammonium chloride and 15 mmol/L phosphate by orthogonal experiment.Besides,Through a single-factorial experiment,the optimal culture conditions in shakeflasks were determined as follows:size of the inoculum,time of inoculum,culturing temperature and fermentation time were 4%?v/v?,16 h,34? and 56 h,respectively,the activity of protease reached 6963 U/mL,which was 1.42 times higher than its unoptimized.Three different proteases were separately used to hydrolyze casein,and the degree of hydrolysis of recombinant alkaline protease was highest.The optimum hydrolyzing time,temperature,pH and the amount of the enzyme were found to be 2 h,50?,9.5 and 4000 U/g substrate through the single-factorial and orthogonal experiments.Under the optimal hydrolysis conditions,the yield and N/P mole ratio of the casein peptide phosphate?CPP?prepared were 14.8%and 8.47,respectively.Knowledge gained here lays a solid foundation for the applications ofalkaline protease in the manufacturing of functional food.