Rational design of a thermophilic β-mannanase fromBacillus subtilis TJ-102 to improve its thermostability.

Xiang-Chao Wang, Sheng-Ping You, Jia-Xing Zhang, Ye-Mei Dai, Cheng-Yu Zhang, Wei Qi, Tong-Yi Dou, Rong-Xin Su, Zhi-Min He

Journal: Enzyme and microbial technology 2019;118():50-56

PMID: 30143199

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Abstract

A rational design method to improve β-mannanase (ManTJ102) thermostability was developed successfully in this study. The flexible area of residues 330-340 in ManTJ102 was firstly selected from analysis of molecular dynamics simulation and then the critical amino acid residue (Ala336Pro) with the lowest mutation energy was determined by virtual mutation, whose mutant was named as Mutant336. Afterward, the dynamics transition temperature (T) of ManTJ102 and Mutant336 was evaluated by simulated annealing and heating, and Mutant336 with higher T was implemented for experimental verification of the enzyme thermostability. As a result, the half-life of Mutant336 activity was 120 min at 60 °C, which was 24-fold of ManTJ102, and the irreversible thermal denaturation constant of Mutant336 was only about 2/5 of ManTJ102, indicating that Mutant336 has better thermostability than ManTJ102. Furthermore, Mutant336 has much higher β-mannanase activity and specific activity than ManTJ102. Therefore, Mutant336 was more suitable to further research for applications.

Address: Chemical Engineering Research Center, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China.; Chemical Engineering Research Center, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China; State Key Laboratory of Chemical Engineering, Tianjin University, Tianjin 300072, PR China; Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300072, PR China; Tianjin Key Laboratory of Membrane Science and Desalination Technology, Tianjin University, Tianjin 300072, PR China. Electronic address: [email protected].; School of Life Science and Medicine, Dalian University of Technology, NO. 2 Dagong Road, New District of Liaodong Bay, Panjin 124221, PR China.; Chemical Engineering Research Center, School of Chemical Engineering and Technology, Tianjin University, Tianjin 300072, PR China; State Key Laboratory of Chemical Engineering, Tianjin University, Tianjin 300072, PR China; Collaborative Innovation Center of Chemical Science and Engineering (Tianjin), Tianjin 300072, PR China; Tianjin Key Laboratory of Membrane Science and Desalination Technology, Tianjin University, Tianjin 300072, PR China.

DOI: 10.1016/j.enzmictec.2018.07.005

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MeSH Terms: Amino Acid Sequence, Bacillus subtilis, Enzyme Stability, Molecular Dynamics Simulation, Mutation, Protein Conformation, Temperature, beta-Mannosidase