摘要
用硫酸铵分部盐析及离子交换层析技术从米黑毛霉半固体培养物的浸提液中提纯了天冬氨酸蛋白酶,酶活力收率为19.5％,,比活力达3080SU/mg蛋白,提纯8.5倍。用聚丙烯酰胺凝胶电泳、SDS-凝胶电泳、等电聚焦、双向免疫扩散及免疫电泳等方法鉴定该酶均一。本文还报道了关于该酶生化性质、动力学性质及化学修饰的研究结果。该酶以天冬氨酸为其活性的必需基团,系一典型的天冬氨酸蛋白酶。
Purification and basic biochemical properties of the aspartate proteinase from M.miehei were studied. The enzyme was purified up to 8.5-fold with specific activity 3080 SU/mg of protein and 19.5% recovery from crude water-extract by means of ammonium sulphate fractionation, DEAE-Sephadex and CM-Cellulose chromatography. The purified aspartate proteinase was homogeneous as judged by polyacrylamide gel electrophoresis, discontinuous SDS-polyacrylamide gel electrophoresis, thin layer polyacrylamide gel isoelectric focusing, immunodiffusion and immunoelectrophoresis. Some basic biochemical properties of the enzyme were examined. Optimal pH was-3.6, optimal temperature was 60℃, pI was 4.2. Km and Vmax for hemoglobin were 5.6×to pH 5 at 30℃. Buffered enzyme exhibited more heat-resistant than that dissolved in water. The molecular weight was estimated to be about 40,500 daltons by SDS pol yacrylamide gel electropharesis. Its total carbohydrate content was about 5.2%. The relationship between catalytic activity and various residues of the aspartate proteinase was studied by selective chemical modification. The results showed that the Trp, Arg, His, Tyr residues, SH groups and metal ion had no relationship to the activity. But the enzyme activity was almost lost with the modification of aspartyl residues by carboxyl-group-specific reagent EDC and aspartate proteinase-specific reagents EPNP and DAN. In addition, aspartate proteinase-apecific inhibitor Pepsta-tin exhibited obvious inhibition. The evidence obtained from both chemical modification and inhibition implied that the enzyme was a typical aspartate proteinase with the aspartic acid as essential residues for the enzyme activity.
关键词
米黑毛霉
天冬氨酸
蛋白酶
纯化
M .miehei Purification of aspartate proteinase Selective chemical modification.
