Rational molecular design for improving digestive enzyme resistance of beta-glucosidase from Trichoderma viride based on inhibition of bound state formation

Hao Wang; Xiangna Lin; Shuang Li; Jianlin Lin; Chunfang Xie; Daling Liu; Dongsheng Yao

doi:10.1016/j.enzmictec.2019.109465

Rational molecular design for improving digestive enzyme resistance of beta-glucosidase from Trichoderma viride based on inhibition of bound state formation

Enzyme Microb Technol. 2020 Feb:133:109465. doi: 10.1016/j.enzmictec.2019.109465. Epub 2019 Nov 6.

Authors

Hao Wang¹, Xiangna Lin², Shuang Li¹, Jianlin Lin³, Chunfang Xie¹, Daling Liu⁴, Dongsheng Yao⁵

Affiliations

¹ Institute of Microbial Biotechnology, Jinan University, Guangzhou City, Guangdong Province, 510632, China; Department of Bioengineering, Jinan University, Guangzhou City, Guangdong Province, 510632, China.
² Institute of Microbial Biotechnology, Jinan University, Guangzhou City, Guangdong Province, 510632, China; National Engineering Research Center of Genetic Medicine, Guangzhou City, Guangdong Province, 510632, China.
³ Institute of Microbial Biotechnology, Jinan University, Guangzhou City, Guangdong Province, 510632, China.
⁴ Institute of Microbial Biotechnology, Jinan University, Guangzhou City, Guangdong Province, 510632, China; Department of Bioengineering, Jinan University, Guangzhou City, Guangdong Province, 510632, China. Electronic address: tldl@jnu.edu.cn.
⁵ Institute of Microbial Biotechnology, Jinan University, Guangzhou City, Guangdong Province, 510632, China; National Engineering Research Center of Genetic Medicine, Guangzhou City, Guangdong Province, 510632, China. Electronic address: tdsyao@jnu.edu.cn.

PMID: 31874695
DOI: 10.1016/j.enzmictec.2019.109465

Abstract

Beta-glucosidase (BGL1) is widely used in animal feed industries. However, degradation caused by digestive enzymes in the intestine hampers its application. Improving the resistance of feed enzymes against proteases is crucial in livestock farming. To improve the resistance of beta-glucosidase against pepsin and trypsin, a rational molecular design based on the inhibition of bound-state formation and secondary design was developed. The strategy includes: (1) prediction of the interaction surface of the pepsin-BGL1 complex structure, (2) prediction of key amino acids affecting the formation of the complex, (3) optimization of pepsin-resistant mutants by structural evaluation, (4) secondary molecular design based on pepsin-resistant mutants, and optimization of pepsin and trypsin-resistant mutants. Two BGL1 protein mutants (BGL1^Q627C and BGL1^{Q627C/R543H/R646W}) were constructed, and then mutated and wild-type BGL1s were expressed in Pichia pastoris. The half-life of BGL1^Q627C and BGL1^{Q627C/R543H/R646W} were 1.36 and 1.51 times that of the wild type upon pepsin exposure, respectively. For trypsin resistance, the half-life were 0.93 and 1.53 times that of the wild type, respectively. Compare to those of the wild type, most of the basic enzymatic properties of both mutants were not significantly changed except for increased Michaelis constants. The rational design method can be used as a guide for modifying other feed enzymes.

Keywords: Beta-glucosidase 1; Pepsin resistance; Secondary design; Trypsin resistance; molecular simulation.

MeSH terms

Hydrolysis
Pepsin A / metabolism*
Trichoderma / enzymology*
Trichoderma / genetics
Trypsin / metabolism*
beta-Glucosidase / genetics*
beta-Glucosidase / metabolism

Substances

beta-Glucosidase
Trypsin
Pepsin A