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

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 T_1/2^15min 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.