Abstract

In this study, it is aimed to search for new proteases with novel properties from bacteria and to characterise the properties of the enzyme. The best protease producing microorganisms was isolated from soil and characterised by 16 s rDNA sequence analysis. A 33 kDa molecular weight bacillolycin metallo–serine protease from newly isolated Bacillus sp. BHC01 was purified 2.6–fold with a final 38,617.0 ± 1013.7 U/mg protein specific activity. Optimum reaction temperature and pH of the enzyme was found to be 50° C and pH 7.5, respectively. Fe²⁺, N⁺, Co²⁺ and Mg²⁺ ions were detected as activators, and Cu²⁺ metal ions was observed as inhibitors. The kinetic constants of the enzyme K_m and V_max is measured as 0.574 mg/mL, 338.1 mmol/ml.min, respectively. Because ethylenediaminetetraacetic acid (EDTA) and phenylmethylsulfonyl fluoride (PMSF), both inhibit the enzyme, it is concluded that this enzyme is a mixed catalytic type peptidase which has metals in active site and also has some serine residues in order to reach an active folding.

摘要

在这项研究中，旨在从细菌中寻找具有新特性的新蛋白酶并表征酶的特性。从土壤中分离出最好的产蛋白酶微生物，并通过16 s rDNA 序列分析进行表征。来自新分离的芽孢杆菌属的33 kDa 分子量杆菌肽金属丝氨酸蛋白酶。BHC01 纯化了 2.6 倍，最终的蛋白质比活性为 38,617.0 ± 1013.7 U/mg。发现酶的最佳反应温度和 pH 值分别为 50°C 和 pH 7.5。Fe ²⁺、N ⁺、Co ²⁺和 Mg ²⁺离子被检测为活化剂，Cu ²⁺观察到金属离子作为抑制剂。酶K _m和V _max的动力学常数分别测量为0.574 mg/mL、338.1 mmol/ml.min。由于乙二胺四乙酸 (EDTA) 和苯甲基磺酰氟 (PMSF) 均对该酶有抑制作用，因此可以得出结论，该酶是一种混合催化型肽酶，其活性位点具有金属，并且还具有一些丝氨酸残基以达到活性折叠。