State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, 130 Meilong Road, Shanghai 200237, People's Republic of China.
Appl Biochem Biotechnol. 2011 Dec;165(7-8):1473-84. doi: 10.1007/s12010-011-9368-y. Epub 2011 Oct 12.
As one critical enzyme in deconstructing complicated cellulose matrix, endoglucanase (EG) is needed to exhibit high activity and thermostability under severe industrial conditions. Driven by this purpose, EGtf1 (Q08166) and EGtf2 (Q7X2N2), with relatively high specific activities, were selected out of 43 putative EG genes from SWISS-PROT database. These distinguished EGs were successfully overexpressed in Escherichia coli and purified by one-step affinity chromatography. The maximal activity was shown at approximate pH 5.0 and 50 °C. It is worth noting that EGtf1 and EGtf2 displayed outstanding thermostability with a half-life of up to 1,386 h at 50 °C, which is almost 100-fold higher than other reported EGs. Furthermore, the presence of various metal ions (1 mM) or organic solvents (50%, v/v) did not cause significant effect on the activities of EGtf1 and EGtf2 and even showed 2.1- and 2.7-fold enhancement in the case of dodecanol. All these features, especially the excellent thermostability of EGtf1 and EGtf2, enable them to become a good candidate for further protein engineering to realize the ultimate practical application in biomass industry.
作为分解复杂纤维素基质的关键酶之一，内切葡聚糖酶（EG）需要在苛刻的工业条件下表现出高活性和热稳定性。受此目的驱动，从 SWISS-PROT 数据库中选择了 43 个假定的 EG 基因中的 EGtf1（Q08166）和 EGtf2（Q7X2N2），它们具有相对较高的比活性。这些有区别的 EG 在大肠杆菌中成功过表达，并通过一步亲和层析进行纯化。最大活性出现在约 pH5.0 和 50°C。值得注意的是，EGtf1 和 EGtf2 表现出出色的热稳定性，在 50°C 下半衰期长达 1,386 h，比其他报道的 EG 高近 100 倍。此外，各种金属离子（1 mM）或有机溶剂（50%，v/v）的存在对 EGtf1 和 EGtf2 的活性没有显著影响，在十二醇的情况下甚至表现出 2.1-和 2.7 倍的增强。所有这些特性，特别是 EGtf1 和 EGtf2 的优异热稳定性，使它们成为进一步进行蛋白质工程的良好候选者，以实现生物质工业的最终实际应用。
