Improvement of thermal stability of endo-β-1,4-xylanase from Aspergillus niger by introducing N-glycosylation site

Abstract:

[Background] Xylan is the second most abundant polysaccharide in nature, and second only to cellulose. The structure of xylan is complex, and its complete degradation needs many xylanases. Endo-β-1,4-xylanase is the key enzyme in the hydrolysis process of xylan main chain. It has been widely used in feed, papermaking, energy, food and medicine industries. However, in practical application, due to the poor thermal stability of fungal xylanase, its application in industry is limited. [Objective] The purpose of this study is to improve the thermal stability of endo-β-1,4-xylanase (xynB) from Aspergillus niger. [Methods] Firstly, an N-glycosylation site was introduced into xynB by amino acid virtual mutation technology to obtain the mutants. Then the mutants and wild-type enzymes were expressed in Pichia pastoris SMD1168. Finally, the purified wild-type and mutant enzymes were characterized by enzymatic properties. [Results] Five candidate mutants were obtained by virtual mutation and screening. Four mutants were successfully expressed in Pichia pastoris SMD1168, and three of which were glycosylated. The mutants and wild-type enzyme showed wide pH tolerance, and the stability of xynBA92N/D94T at pH 4.0–11.0 was significantly better than that of the wild-type; xynBA92N/D94T, xynBG66N/A68T and xynBG66F/D67N/G69T, which were glycosylated, showed significantly higher thermal stability at 60–80 °C than that of the wild-type, and the residual enzyme activity of xynBG66N/A68T at 80 °C after incubation for 30 min was about 30% higher than that of the wild-type. [Conclusion] The method of this study can provide reference for the thermal stability molecular modification of other xylanases and other industrial enzymes.