碱性蛋白酶的异源表达及其在制备CPP中的应用

Heterologous Expression and Application of Gene Encoded Alkaline Protease in Preparation of CPP

碱性蛋白酶是现代工业酶制剂中最重要的酶类之一,在人类日常生活中扮演着非常重要的角色.本研究以嗜碱芽胞杆菌(B.alcalophilus)TCCC11006基因组为模板,从中扩增出碱性蛋白酶基因apr,并构建重组表达质粒p HYapr,将重组质粒电转入蛋白酶基因缺失的地衣芽胞杆菌(B.licheniformis)H115中,实现了碱性蛋白酶的分泌表达.重组菌在LB培养基中最高酶活力达到3,199,U/m L,是出发菌株酶活力的1.82倍,发酵周期缩短了8,h.然后通过单因素和正交实验对重组碱性蛋白酶水解酪蛋白的工艺参数进行优化,确定了最佳水解温度、p H和酶添加量分别为50,℃、9.5和4,000,U/g.在最佳水解条件下,通过钡-乙醇沉淀的方法制备生物活性肽——酪蛋白磷酸肽(CPP),其得率和氮磷物质的量比分别为14.8%,和8.47.这为碱性蛋白酶在功能性食品制造方面的应用奠定了良好的基础.

Alkaline protease is one of the most important enzymes in industrial enzymes.It plays a very important role in human daily life.Alkaline protease encoded gene(apr)was amplified from B.alcalophilus TCCC11006,genome.Then the recombined expression vector pHY-apr was successfully constructed and transformed into protease-deficient strain B.licheniformis H115.The alkaline protease gene was expressed in the recombined B.licheniformis H115.The highest alkaline protease activity was 3,199,U/mL in LB medium,which was 1.82,times of that of the starting strain.The fermentation cycle was decreased by 8,h.Then the hydrolyzing casein paramenters of recombined alkaline protease were optimized with single factor and orthogonal experiments.The optimum hydrolyzing time,temperature,pH and the amount of the enzyme were found to be 2,h,50,℃,9.5,and 4,000,U/g substrate.Under the optimal hydrolysis conditions,the yield and N/P mole ratio of the casein peptide phosphate(CPPs)prepared were 14.8%, and 8.47,respectively.Knowledge gained in the research can be a solid foundation for the application of alkaline protease in the manufacturing of functional food.