Abstract:[Background] Xylanase, the core enzyme responsible for the degradation of xylan, can promote the digestion and hydrolysis of lignocellulose. Therefore, it has been widely used in animal breeding fields. The GH10 xylanase CbXyn10C derived from Caldicellulosiruptor bescii has good thermal stability at 80 ℃ with an optimum temperature of 85 ℃. With this property, it demonstrates a promising prospect for application in the feed industry. [Objective] To improve the thermal stability of CbXyn10C for meeting the technological requirements of feed granulation, especially aquatic feed processing, and decipher its heat resistance mechanism. [Methods] Based on the crystal structure of CbXyn10C, mutations were designed by introducing rigid amino acids and rearranging the hydrophobic network. After a single-point mutant with increased specific activity at 100 ℃ was obtained, the thermal stability of the enzyme was further improved by the stacking of beneficial mutation sites. Molecular dynamics (MD) simulation was employed to decipher the mechanism of thermal stability improvement. [Results] Four single-point mutants A45P, T69P, F309V, and A325P with improved stability were obtained, among which the mutant A45P showed the greatest stability improvement. The stacking of other three mutation sites on A45P gradually improved the thermal stability of the enzyme without compromising the enzyme activity. The obtained four-point mutant A45P/F309V/A325P/T69P showed the best heat tolerance, and its optimum temperature and melting temperature Tm were increased by 5 ℃ and 6.8 ℃, respectively, compared with those of the wild type. MD simulation showed that mutations at the four sites introduced new hydrogen bond forces and optimized the hydrophobic network, resulting in a more stable structure and conformation. [Conclusion] This study promotes the application of xylanase in the feed industry and provides theoretical support for molecular modification for improving enzyme stability based on its structure.
