Shivanand Pooja, Jayaraman Gurunathan
School of Biosciences and Technology, VIT University, Vellore 632014, India.
Indian J Biochem Biophys. 2011 Apr;48(2):95-100.
A halotolerant bacterium Bacillus acquimaris VITP4 was used for the production of extracellular protease. Fractional precipitation using ammonium chloride was used to obtain the enzyme. The protease exhibited optimum activity at pH 8.0 and 40 degrees C and retained 50% of its optimal proteolytic activity even in the presence of 4 M NaCl, suggesting that it is halotolerant. The molecular mass of protease, as revealed by SDS-PAGE was found to be 34 kDa and the homogeneity of the enzyme was confirmed by gelatin zymography and reverse-phase HPLC. Upon purification, the specific activity of th enzyme increased from 533 U/mg to 1719 U/mg. Protease inhibitors like phenyl methane sulphonyl fluoride and 2-mercaptoethanol did not affect the activity of the enzyme, but EDTA inhibited the activity, indicating the requirement of metal ions for activity. Cu2, Ni2+ and Mn2+ enhanced the enzyme activity, but Zn2+, Hg2+ and Fe2+ decreased the activity, while Mg2+, Ca2+ and K+ had no effect on the enzyme activity. The protease was quite stable in the presence of cationic (CTAB), anionic (SDS) and neutral detergents (Triton X-100 and Tween-20) and exhibited antimicrobial activity against selected bacterial and fungal strains. The stability characteristics and broad spectrum antimicrobial activity indicated the potential use of this protease in industrial applications.
一种耐盐细菌海栖芽孢杆菌VITP4被用于生产胞外蛋白酶。使用氯化铵进行分级沉淀以获得该酶。该蛋白酶在pH 8.0和40℃时表现出最佳活性，即使在存在4 M NaCl的情况下仍保留其最佳蛋白水解活性的50%，这表明它具有耐盐性。通过SDS-PAGE显示，蛋白酶的分子量为34 kDa，并且通过明胶酶谱法和反相HPLC确认了该酶的同质性。纯化后，该酶的比活性从533 U/mg增加到1719 U/mg。蛋白酶抑制剂如苯甲基磺酰氟和2-巯基乙醇不影响该酶的活性，但EDTA抑制其活性，表明该酶的活性需要金属离子。Cu2+、Ni2+和Mn2+增强了该酶的活性，但Zn2+、Hg2+和Fe2+降低了其活性，而Mg2+、Ca2+和K+对该酶的活性没有影响。该蛋白酶在阳离子（CTAB）、阴离子（SDS）和中性去污剂（Triton X-100和吐温-20）存在下相当稳定，并对选定的细菌和真菌菌株表现出抗菌活性。其稳定性特征和广谱抗菌活性表明该蛋白酶在工业应用中具有潜在用途。
