Rational Redesign of Chitosanase to Enhance Thermostability and Catalytic Activity to Produce Chitooligosaccharides with a Relatively High Degree of Polymerization.

Chitooligosaccharides (hdpCOS) with a high degree of polymerization (hdp, DP 4-10) generally have greater biological activities than those of low-DP (ldp, DP 2-3) COS. Chitosanase from Bacillus amyloliquefaciens KCP2 (Csn46) can degrade chitosan to more hdpCOS at high temperature (70 °C), but low thermal stability at this temperature makes it unsuitable for industrial application; the wild-type enzyme can only produce COS (DP 2-4) at lower temperatures. Several thermostable mutants were obtained by modifying chitosanase using a comprehensive strategy based on a computer-aided mutant design. A combination of four beneficial single-point mutations (A129L/T175 V/K70T/D34G) to Csn46 was selected to obtain a markedly improved mutant, Mut4, with a half-life at 60 °C extended from 34.31 to 690.80 min, and the specific activity increased from 1671.73 to 3528.77 U/mg. Mut4 produced COS with DPs of 2-4 and 2-7 at 60 and 70 °C, respectively. Therefore, Mut4 has the potential to be applied to the industrial-scale preparation of hdpCOS with high biological activity.