Somkuti G A, Babel F J
J Bacteriol. 1968 Apr;95(4):1407-14. doi: 10.1128/jb.95.4.1407-1414.1968.
The protease produced by Mucor pusillus was recovered from a wheat bran medium by treatment with ammonium sulfate, ethyl alcohol, gel filtration and ion-exchange chromatography. The yield of the enzyme was 55%. The overall increase in the specific activity of the protease was 34-fold. The purified protease was most active at pH 3.8 and 5.6 against hemoglobin and casein, respectively. Optimal hydrolysis of casein was observed at 55 C. The enzyme was stable from pH 3.0 to 6.0. Enzyme inactivated by metal ions was reactivated by ethylenediaminetetraacetate and o-phenanthroline. Reducing agents and thiol poisons had no effect on the protease, suggesting that free sulfhydryl groups were not required for enzyme activity. Diisopropyl fluorophosphate did not inhibit the protease, indicating the probable absence of serine in the active center. The Michaelis-Menten constant for casein was 0.357%. Electrophoretic analysis of active protein recovered by ion-exchange chromatography showed that the protease preparation was homogeneous.
微小毛霉产生的蛋白酶通过硫酸铵处理、乙醇处理、凝胶过滤和离子交换色谱法从小麦麸皮培养基中回收。该酶的产率为55%。蛋白酶的比活性总体提高了34倍。纯化后的蛋白酶在pH 3.8时对血红蛋白活性最高，在pH 5.6时对酪蛋白活性最高。在55℃观察到酪蛋白的最佳水解。该酶在pH 3.0至6.0范围内稳定。被金属离子失活的酶可被乙二胺四乙酸和邻菲罗啉重新激活。还原剂和硫醇毒物对该蛋白酶无影响，这表明酶活性不需要游离巯基。二异丙基氟磷酸不抑制该蛋白酶，表明活性中心可能不存在丝氨酸。酪蛋白的米氏常数为0.357%。对通过离子交换色谱回收的活性蛋白进行电泳分析表明，蛋白酶制剂是纯的。
