Shankar Shiv, Laxman Ryali Seeta
Division of Biochemical Sciences, National Chemical Laboratory, Pune, 411 008, India,
Appl Biochem Biotechnol. 2015 Jan;175(1):589-602. doi: 10.1007/s12010-014-1314-3. Epub 2014 Oct 22.
This study illustrates the biophysicochemical properties of an alkaline protease, BAP (Beauveria sp. alkaline protease) from Beauveria sp. MTCC 5184. This protease exhibited maximum activity at 50 °C, pH 9.0, and stability in a broad pH range, in the presence of organic solvents, denaturants, as well as detergents. Wash performance studies revealed that BAP was able to remove blood clots/stains from blood-soaked cloth. Peptide mass fingerprinting results demonstrated partial homology of BAP with subtilisin-like proteinase. BAP showed catalytic activity against natural as well as synthetic substrates. Active site characterization of BAP confirmed the involvement of serine, tryptophan, and aspartic acid in catalytic activity. Detailed kinetic and thermodynamic studies of BAP demonstrated that the activation energy (Ea) for casein hydrolysis was 82.55 kJ/M, the specificity constant (Kcat/K m), and the values of ∆G (change in Gibbs free energy) decreased with increase in temperature, whereas ∆H (change in enthalapy) and ∆S (change in entropy) were constant. The results of the present study indicate that BAP has potential for applications as detergent additive, in peptide synthesis, and in basic research.
本研究阐述了来自球孢白僵菌MTCC 5184的碱性蛋白酶BAP（球孢白僵菌碱性蛋白酶）的生物物理化学性质。该蛋白酶在50°C、pH 9.0时表现出最大活性，并且在有机溶剂、变性剂以及洗涤剂存在的情况下，在较宽的pH范围内具有稳定性。洗涤性能研究表明，BAP能够从浸血的布料上去除血凝块/污渍。肽质量指纹图谱结果证明BAP与枯草杆菌蛋白酶样蛋白酶存在部分同源性。BAP对天然底物和合成底物均表现出催化活性。BAP的活性位点表征证实丝氨酸、色氨酸和天冬氨酸参与了催化活性。对BAP进行的详细动力学和热力学研究表明，酪蛋白水解的活化能（Ea）为82.55 kJ/M，特异性常数（Kcat/Km）以及吉布斯自由能变化值（∆G）随温度升高而降低，而焓变（∆H）和熵变（∆S）保持恒定。本研究结果表明，BAP在作为洗涤剂添加剂、肽合成以及基础研究方面具有应用潜力。
