Improving the thermostability and activity of Paenibacillus pasadenensis chitinase through semi-rational design

• Published in: International journal of biological macromolecules 2020-05, Vol.150, p.9-15
• Main Authors: Xu, Pei, Ni, Zi-Fu, Zong, Min-Hua, Ou, Xiao-Yang, Yang, Ji-Guo, Lou, Wen-Yong
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Amino Acids; Catalysis; Chitin - chemistry; Chitinase; Chitinases - chemistry; Chitinases - genetics; Chromatography, High Pressure Liquid; Consensus substitution; Disulfide bonds; Enzyme Stability; Hydrogen-Ion Concentration; Kinetics; Molecular Dynamics Simulation; Molecular Weight; N,N′-diacetylchitobiose; Paenibacillus - enzymology; Paenibacillus - genetics; Protein Conformation; Protein Engineering; Structure-Activity Relationship; Substrate Specificity; Temperature; Thermostability
• ISSN: 0141-8130; 1879-0003
• DOI: 10.1016/j.ijbiomac.2020.02.033

Chitinase is a promising biocatalyst for chitin biotransformation in the field of recalcitrant biomass degradation. Excellent catalytic performance is conducive to its commercial utilization. In this work, sequence- and structure-based semi-rational design was performed to evolve the thermostability and activity of a previously identified chitinase PpChi1 from Paenibacillus pasadenensis CS0611. After combinational mutagenesis, the mutant S244C-I319C/T259P with disulfide bond introduction and proline substitution exhibited higher specific activity at higher temperature, 26.3-fold in half-life value at 50 °C, and a 7.9 °C rise in half-inactivation temperature T1/215min compared to the wild-type enzyme. The optimal reaction temperature of the mutant was shifted from 45 °C to 52.5 °C. Molecular dynamic simulation and structure analysis confirmed that these improvements of the mutant were attributed to its stabilized folding form, possibly caused by the decreased entropy of unfolding. This work gives an initial insight into the effect of conserved proline residues in thermostable chitinases and proposes a feasible approach for improving chitinase thermostability to facilitate its application in chitin hydrolysis to valuable oligosaccharides. •Sequence- and structure-based mutation was performed to engineer chitinase.•Mutant S244C-I319C/T259P showed 26.3-fold increase in half-life at 50 °C.•MD simulation indicated the mutant had more stable active structure.•Efficient preparation of (GlcNAc)2 using this mutant was feasible.