论文部分内容阅读
为了研究棉铃虫（Ｈｅｌｉｃｏｖｅｒｐａ ａｒｍｉｇｅｒａ） 半胱氨酸蛋白酶的底物专一性，进一步阐明该蛋白酶的性质和应用前景，从棉铃虫卵巢中纯化了该蛋白酶，用电泳方法检测了该蛋白酶对牛血红蛋白（Ｈｂ）、牛血清蛋白（ＢＳＡ）、卵黄磷蛋白（Ｖｎ）、明胶（ｇｅｌａｔｉｎ）等 ４种动物蛋白，及大豆、玉米、马铃薯、豇豆、向阳花种子及棉籽蛋白等 ６种植物蛋白的水解作用。结果显示该酶对４种动物蛋白及６种植物蛋白都有明显水解活性。用吸光度测定法测定了棉铃虫半胱氨酸蛋白酶对牛血红蛋白、牛血清蛋白、卵黄磷蛋白、明胶等４种底物的米氏常数（Ｋｍ） 值。结果显示棉铃虫半胱氨酸蛋白酶对牛血红蛋白和卵黄磷蛋白的水解产物的吸光度值较高，而对牛血清蛋白和明胶的水解产物的吸光度值较低。测得该蛋白酶对牛血红蛋白、牛血清蛋白、卵黄磷蛋白、明胶的Ｋｍ值分别为 １０、０．９１、０．０８μｍｏｌ／Ｌ和２５０ｍｇ／Ｌ，其中对卵黄磷蛋白的Ｋｍ值最小，说明棉铃虫半胱氨酸蛋白酶与卵黄磷蛋白的亲和力最大，即卵黄磷蛋白是棉铃虫半胱氨酸蛋白酶的最适底物。同时用电泳方法检测了该蛋白酶对粗提的棉铃虫卵黄磷蛋白的水解作用，结果显示该酶对其有明显水解活性，由此推测该酶可能参与棉铃虫
In order to study the substrate specificity of cystatin in Helicoverpa armigera and further elucidate the nature and application of the protease, the protease was purified from the ovary of the cotton bollworm and the electrophoresis method was used to detect the specificity of the protease for bovine hemoglobin Hb, BSA, Vn and gelatin, and the hydrolysis of six plant proteins including soybean, corn, potato, cowpea, sunflower seed and cottonseed protein effect. The results showed that the enzyme has obvious hydrolytic activity on four animal proteins and six plant proteins. The Michaelis-Menten constant (Km) values of four substrates of cotton bollworm cysteine protease against bovine hemoglobin, bovine serum albumin, egg yolk phosphoprotein and gelatin were determined by absorbance assay. The results showed that the absorbency value of cotton bollworm cysteine protease for the hydrolyzate of bovine hemoglobin and yolk phosphoprotein was higher, while the absorbance value of bovine serum albumin and gelatin hydrolyzate was lower. The Km values of the protease to bovine hemoglobin, bovine serum albumin, egg yolk protein and gelatin were respectively 10, 0.91, 0.08 μmol / L and 250 mg / L, of which the Km value of the protein was the smallest, The cysteine protease has the highest affinity with the protein of the egg yolk phosphoprotein, which is the optimum substrate for the cotton bollworm cysteine protease. At the same time, the hydrolytic activity of the protease against the crude protein of the egg white protein of Helicoverpa armigera was detected by electrophoresis. The results showed that the enzyme had significant hydrolytic activity. Therefore,