Chakrabarti S K, Matsumura N, Ranu R S
Laboratory of Plant Molecular Biology and Biotechnology, Department of Bioagricultural Sciences & Pest Management, Colorado State University, Fort Collins, CO 80523, USA.
Curr Microbiol. 2000 Apr;40(4):239-44. doi: 10.1007/s002849910048.
An extracellular alkaline serine protease has been purified from Aspergillus terreus (IJIRA 6.2). The purification procedure involved chromatography on DEAE-Sephadex A25, phosphocellulose, hydroxyapatite, casein-Sepharose, gel filtration on Sephacryl-S-300 and by glycerol density gradient centrifugation. The enzyme was further purified to apparent homogeneity through a combination of electrophoresis in polyacrylamide gel containing 0.1% sodium dodecyl sulfate (SDS) with or without protease substrate (gelatin) and subsequent regeneration of its activity in situ by removal of SDS. The active enzyme was visualized in a zymogram or on the basis of protease activity exhibited on an X-ray film. The protein in the unstained segment of the gel was electroeluted. The eluted protein with protease activity exhibited a molecular mass of 37,000-daltons on electrophoresis in SDS-polyacrylamide gel. A sedimentation coefficient of 3.2S was obtained by glycerol density gradient contrifugation. Maximum activity of protease was observed at pH 8.5 and at 37 degrees C. Purified protease was active between pH 5.5 and 9.5 and was found to be stable up to 60 degrees C. With Na-caseinate, the K(m) of the purified protease was found to be 0.055 mM. Antipain, phenylmethane sulfonyl fluoride, and chymostatin served as non-competitive inhibitors. Substrate specificity was determined by using a synthetic chromogenic peptide containing N-P-Tosyl-Gly-Pro-Arg-p-nitroanilide. Results showed that the protease cleaved the peptide on the -COOH end of arginine residue.
已从土曲霉（IJIRA 6.2）中纯化出一种细胞外碱性丝氨酸蛋白酶。纯化过程包括在DEAE - 葡聚糖A25、磷酸纤维素、羟基磷灰石、酪蛋白 - 琼脂糖上进行层析，在Sephacryl - S - 300上进行凝胶过滤以及甘油密度梯度离心。通过在含有0.1%十二烷基硫酸钠（SDS）且有或没有蛋白酶底物（明胶）的聚丙烯酰胺凝胶中进行电泳，并随后通过去除SDS原位恢复其活性，该酶进一步纯化至表观均一性。活性酶在酶谱中或基于在X射线胶片上显示的蛋白酶活性可视化。凝胶未染色部分的蛋白质进行电洗脱。在SDS - 聚丙烯酰胺凝胶电泳中，具有蛋白酶活性的洗脱蛋白显示分子量为37,000道尔顿。通过甘油密度梯度离心获得沉降系数为3.2S。在pH 8.5和37℃时观察到蛋白酶的最大活性。纯化的蛋白酶在pH 5.5至9.5之间有活性，并且在高达60℃时稳定。对于酪蛋白酸钠，纯化蛋白酶的K(m)为0.055 mM。抗蛋白酶、苯甲基磺酰氟和抑肽酶作为非竞争性抑制剂。通过使用含有N - P - 甲苯磺酰 - 甘氨酰 - 脯氨酰 - 精氨酸 - 对硝基苯胺的合成生色肽来确定底物特异性。结果表明，该蛋白酶在精氨酸残基的 - COOH末端切割该肽。
