Jain Kavish Kumar, Kumar Sandeep, Bhardwaj Kailash N, Kuhad Ramesh Chander
Lignocellulose Biotechnology Laboratory, Department of Microbiology, University of Delhi South Campus, Benito Juarez Road, New Delhi, 110021, India.
Department of Microbiology, Maharshi Dayanand University, Rohtak, Haryana, 124001, India.
Mol Biotechnol. 2018 Oct;60(10):736-748. doi: 10.1007/s12033-018-0106-3.
Thermostable cellulases offer several advantages like higher rates of substrate hydrolysis, lowered risk of contamination, and increased flexibility with respect to process design. In the present study, a thermostable native endoglucanase nEG (EC 3.2.1.4) was purified and characterized from T. aurantiacus RCKK. Further, it was cloned in P. pastoris X-33 and processed for over expression. Expression of recombinant endoglucanase (rEG) of molecular size ~ 33 kDa was confirmed by SDS-PAGE and western blotting followed by in gel activity determination by zymogram analysis. Similar to nEG, the purified rEG was characterized to harbor high thermostability while retaining 50% of its initial activity even after 6- and 10-h incubation at 80 and 70 °C, respectively, and exhibited considerable stability in pH range 3.0-7.0. CD spectroscopy revealed more than 20% β-sheets in protein structure consistently when incubated upto 85 °C as a speculated reason for protein high thermostability. Interestingly, both nEG and rEG were found tolerant up to 10% of the presence of 1-ethyl-3-methylimidazolium acetate [C2mim][OAc]. Values of the catalytic constants K and V for rEG were recorded as 2.5 mg/ml and 303.4 µmol/mg/min, respectively. Thermostability, pH stability, and resistance to the presence of ionic liquid signify the potential applicability of present enzyme in cellulose hydrolysis and enzymatic deinking of recycled paper pulp.
耐热纤维素酶具有多种优势，如底物水解速率更高、污染风险降低以及在工艺设计方面具有更大的灵活性。在本研究中，从金黄嗜热栖热菌RCKK中纯化并表征了一种耐热天然内切葡聚糖酶nEG（EC 3.2.1.4）。此外，将其克隆到巴斯德毕赤酵母X-33中并进行过表达处理。通过SDS-PAGE和蛋白质免疫印迹法确认了分子大小约为33 kDa的重组内切葡聚糖酶（rEG）的表达，随后通过酶谱分析进行凝胶内活性测定。与nEG相似，纯化后的rEG具有高热稳定性，即使在80℃和70℃分别孵育6小时和10小时后仍保留其初始活性的50%，并且在pH值3.0 - 7.0范围内表现出相当的稳定性。圆二色光谱显示，在高达85℃孵育时，蛋白质结构中始终存在超过20%的β-折叠，推测这是蛋白质高热稳定性的原因。有趣的是，发现nEG和rEG在1-乙基-3-甲基咪唑醋酸盐[C2mim][OAc]存在高达10%的情况下仍具有耐受性。rEG的催化常数K和V值分别记录为2.5 mg/ml和303.4 μmol/mg/min。热稳定性、pH稳定性以及对离子液体存在的耐受性表明该酶在纤维素水解和再生纸浆酶法脱墨方面具有潜在的应用价值。
