Abstract

Extracellular protease production by a novel strain, Bacillus sp. EBTA6, has been optimized by using central composite design of response surface methodology and properties and industrial applications of crude enzyme have been investigated. Three independent variables (temperature, pH and yeast extract concentration) chosen in the experimental design were significant terms and reduced cubic model fit with the design at p < 0.0001 level. The recommended temperature, pH and yeast extract concentration were 30 °C, 8, and 15 g/L, respectively. Crude enzyme displayed activity over a wide pH and temperature ranges having the optimum at 50–60 °C and pH 8. It was quite stable at high pH values and at 50 °C. Amongst the metal ions (Mg⁺, Cu²⁺, Ca²⁺, Zn²⁺, K²⁺, and Sn²⁺), Ca²⁺ enhanced the activity and the others either decreased or did not change it. The enzyme activity was reduced by phenyl-methyl-sulfonyl fluoride (PMSF), and ethylene diamine tetra acetic acid (EDTA). The results revealed that the protease was serine alkaline type. Tween 20 and Tween 80 did not inhibit the enzyme, however, sodium dodecyl sulfate (SDS), reduced it by 39%. It completely removed blood stain in 20 min and coagulated milk in the presence of CaCl₂.

摘要

一种新菌株芽孢杆菌的胞外蛋白酶生产。EBTA6，已经通过使用响应面方法和粗酶的性质和工业应用的中心复合设计进行了优化。实验设计中选择的三个自变量（温度、pH 和酵母提取物浓度）是显着项，并且在p  < 0.0001 水平上与设计拟合的简化三次模型。推荐的温度、pH 值和酵母提取物浓度分别为 30 °C、8 和 15 g/L。粗酶在很宽的 pH 值和温度范围内表现出活性，最佳温度为 50–60 °C 和 pH 8。它在高 pH 值和 50 °C 下非常稳定。其中金属离子（Mg ⁺、Cu ²⁺、Ca ²⁺、Zn ²⁺、K ²⁺和Sn ²⁺ )、Ca ²⁺增强活性，其他则降低或没有改变。苯甲基磺酰氟 (PMSF) 和乙二胺四乙酸 (EDTA) 会降低酶活性。结果表明该蛋白酶为丝氨酸碱性型。吐温 20 和吐温 80 不抑制该酶，但是，十二烷基硫酸钠 (SDS) 将其降低了 39%。它在20分钟内完全去除血渍，并在CaCl ₂存在下凝固牛奶。