干酪乳杆菌蛋白酶纯化与动力学性质

Purification and Kinetics Characteristics of Proteinase from Lactobacillus casei 6033

干酪乳杆菌(Lactobacilluscasei)6033经增菌培养、超声波破碎、冷冻离心后获得粗酶液。粗酶液经硫酸铵分级沉淀后,分别进行阴离子交换层析、疏水层析、分子筛层析,最终获得1种纯蛋白酶。该酶分子量为61 2kD,可被苯甲基磺酰氟(PMSF)和乙二胺四乙酸(EDTA)强烈抑制,Co2+和Ca2+能增强该酶活力,Fe3+、Zn2+和Cu2+对其活性则有抑制作用。该酶在37℃时,最大酶活力出现在pH值7 0附近,pH4 0时相对酶活力下降到39 0%,而pH9 0时相对酶活力仅维持在8 0%左右。pH7 0时该酶的最适温度为37℃左右,20℃时相对酶活力下降到54 0%,而60℃时相对酶活力仅为34 0%。以酪蛋白作为底物时测得该酶Km值为1 5mg/ml、Vmax为32 0。

Ammonium sulfate and three successive chromatography i.e.ion exchange,hydrophobic,and molecular sieve were employed to isolate and purify a kind of proteinase from crude extracts of Lactobacillus casei.Results showed the proteinase had a molecular weight of 61.2 kD and was inhibited by PMSF and EDTA significantly.Co^(+) and Ca^(+) could enhance its activity,while Fe^(3+),Zn^(2+) and Cu^(2+) were on the contrary.At 37℃,the activity reached maximum at pH 6.0-7.0,declined to 39.0% at pH 4.0 and only maintained at the level of 8.0% at pH 9.0.At pH7.0,the activity reached maximum at about 37℃(the optimal temperature),declined to 54.0% at 20℃ and was only 34.0% at 60℃.With casein as substrate,the K_m and V_(max) value of the proteinase were 1.5 mg/ml and 32.0,respectively.