文献类型： 外文期刊

作者： You, Shuai ¹ ; Tu, Tao ¹ ; Zhang, Lujia; Wang, Yuan ¹ ; Huang, Huoqing ¹ ; Ma, Rui ¹ ; Shi, Pengjun ¹ ; Bai, Yingguo ¹ ;

作者机构： 1.Chinese Acad Agr Sci, Feed Res Inst, Key Lab Feed Biotechnol, Minist Agr, 12 Zhongguancun South St, Beijing 100081, Peoples R China

2.Chinese Acad Agr Sci, Feed Res Inst, Key Lab Feed Biotechnol, Minist Agr, 12 Zhongguancun Sou

关键词： Talaromyces leycettanus JCM12802;Endo-beta-1,3-1,4-glucanase;Thermostability improvement;High specific activity;Charge-charge interaction

期刊名称：BIOTECHNOLOGY FOR BIOFUELS （ 影响因子：6.04； 五年影响因子：6.485 ）

ISSN： 1754-6834

年卷期： 2016 年 9 卷

页码：

收录情况： SCI

摘要： Background: beta-Glucanase is one of the most extensively used biocatalysts in biofuel, food and animal feed industries. However, the poor thermostability and low catalytic efficiency of most reported beta-glucanases limit their applications. Currently, two strategies are used to overcome these bottlenecks, i.e., mining for novel enzymes from extremophiles and engineering existing enzymes. Results: A novel endo-beta-1,3-1,4-glucanase of GH16 (Tlglu16A) from the thermophilic fungus Talaromyces leycettanus JCM12802 was produced in Pichia pastoris and characterized. For potential industrial applications, recombinant TlGlu16A exhibits favorable enzymatic properties over most reported glucanases, i.e., remarkable stability over a wide pH range from 1.0 to 10.0 and superior activity on glucan substrates (up to 15,197 U/mg). The only weakness of TlGlu16A is the thermolability at 65 degrees C and higher. To improve the thermostability, the enzyme thermal stability system was then used to engineer TlGlu16A through optimization of residual charge-charge interactions. Eleven mutants were constructed and compared to the wild-type TlGlu16A. Four mutants, H58D, E134R, D235G and D296K, showed longer half-life time at 80 degrees C (31, 7, 25, 22 vs. 0.5 min), and two mutants, D235G and D296K, had greater specific activities (158.2 and 122.2 %, respectively) and catalytic efficiencies (k(cat)/K-m, 170 and 114 %, respectively). Conclusions: The engineered TlGlu16A has great application potentials from the perspectives of enzyme yield and properties. Its thermostability and activity were apparently improved in the engineered enzymes through charge optimization. This study spans the genetic, functional and structural fields, and provides a combination of gene mining and protein engineering approaches for the systematic improvement of enzyme performance.