Pawar Vishakha A, Prajapati Anil S, Akhani Rekha C, Patel Darshan H, Subramanian R B
1P. G. Department Of Biosciences, Satellite Campus, Sardar Patel Maidaan, Bakrol-Vadtal Road, Sardar Patel University, P.O. Box No. 39, Vallabh Vidyanagar, Gujarat 388120 India.
2Department of Biochemistry, P. D. Patel Institute of Applied Sciences, Charotar University of Science and Technology, Changa, Anand, Gujarat India.
3 Biotech. 2018 Feb;8(2):107. doi: 10.1007/s13205-018-1130-5. Epub 2018 Feb 2.
A thermostable keratinase designated as KBALT was purified from RBDV1 from a poultry farm in Gujarat, India. The molecular weight of the native KBALT (nKBALT) purified using ammonium sulfate and ion exchange and gel permeation chromatography with a 40% yield and 80-fold purification was estimated to be ~ 43 kDa. The gene for KBALT was successfully cloned, sequenced and expressed in . Recombinant KBALT (rKBALT) when purified using a single step Ni-NTA His affinity chromatography achieved a yield of 38.20% and a 76.4-fold purification. Comparison of the deduced amino acid sequence of rKBALT with known proteases of species and inhibitory effect of PMSF suggest that rKBALT was a subtilisin-like serine protease. Both native and rKBALT exhibited higher activity at 85 °C and pH 8.0 in the presence of Mg, Mn, Zn, Ba and Fe metal ions. Interestingly, 70% of their activity was retained at temperatures ranging from 35 to > 95 °C. The keratinolytic activity of both nKBALT and rKBALT was enhanced in the presence of reducing agents. They exhibited broad substrate specificity towards various protein substrates. KBALT was determined for its kinetic properties by calculating its (0.61 mg/ml) and (1673 U/mg/min) values. These results suggest KBALT as a robust and promising contender for enzymatic processing of keratinous wastes in waste processing plants.
一种名为KBALT的耐热角蛋白酶是从印度古吉拉特邦一家家禽养殖场的RBDV1中纯化得到的。使用硫酸铵、离子交换和凝胶渗透色谱法纯化得到的天然KBALT（nKBALT），其分子量估计约为43 kDa，产率为40%，纯化倍数为80倍。成功克隆、测序了KBALT基因并在[具体宿主]中进行了表达。使用一步法镍-亚氨基二乙酸（Ni-NTA）组氨酸亲和色谱法纯化重组KBALT（rKBALT）时，产率达到38.20%，纯化倍数为76.4倍。将rKBALT推导的氨基酸序列与已知物种的蛋白酶进行比较以及苯甲基磺酰氟（PMSF）的抑制作用表明，rKBALT是一种枯草杆菌蛋白酶样丝氨酸蛋白酶。在镁、锰、锌、钡和铁金属离子存在的情况下，天然和重组的KBALT在85°C和pH 8.0时均表现出较高的活性。有趣的是，在35至>95°C的温度范围内，它们70%的活性得以保留。在还原剂存在的情况下，nKBALT和rKBALT的角蛋白分解活性均增强。它们对各种蛋白质底物表现出广泛的底物特异性。通过计算其Km（0.61 mg/ml）和Vmax（1673 U/mg/min）值来确定KBALT的动力学性质。这些结果表明，KBALT是垃圾处理厂中用于角蛋白废物酶处理的一种强大且有前景的备选酶。
