Improvement of the thermostability and catalytic efficiency of a highly active β-glucanase from Talaromyces leycettanus JCM12802 by optimizing residual charge-charge interactions.
BACKGROUND
β-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 β-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-β-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 °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 °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.
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing, 100081 People's Republic of China.
Shuai
S
You
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing, 100081 People's Republic of China.
Tao
T
Tu
State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai, 200237 People's Republic of China.
Lujia
L
Zhang
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing, 100081 People's Republic of China.
Yuan
Y
Wang
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing, 100081 People's Republic of China.
Huoqing
H
Huang
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing, 100081 People's Republic of China.
Rui
R
Ma
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing, 100081 People's Republic of China.
Pengjun
P
Shi
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing, 100081 People's Republic of China.
Yingguo
Y
Bai
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing, 100081 People's Republic of China.
Xiaoyun
X
Su
Institute of Animal Science and Veterinary Medicine, Hainan Academy of Agricultural Sciences, Haikou, 571100 People's Republic of China.
Zhemin
Z
Lin
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing, 100081 People's Republic of China.
Huiying
H
Luo
Key Laboratory for Feed Biotechnology of the Ministry of Agriculture, Feed Research Institute, Chinese Academy of Agricultural Sciences, No. 12 Zhongguancun South Street, Beijing, 100081 People's Republic of China.
Bin
B
Yao
Charge–charge interaction; Endo-β-1,3-1,4-glucanase; High specific activity; Talaromyces leycettanus JCM12802; Thermostability improvement