Highly thermostable and promiscuous β-1,3-xylanasen designed by optimized ancestral sequence reconstruction

Abstract

The ancestor of β-1,3-xylanases (AncXyl09) were reconstructed by the optimized ancestral sequences reconstruction strategy to solve the poor catalytic performances of existing β-1,3-xylanases. The results showed that the half-life at 50 °C was 65.08 h, indicating good thermostability. The large number of hydrogen bonds and the disulfide bonds were the major attributes related with the thermal stability of Anxyl09. Interestingly, AncXyl09 could hydrolyze lichen besides the original substrate of β-1, 3-xylan, which is the first reported β-1,3-xylanase with substrate promiscuity. Moreover, the hydrolytic products are mainly disaccharides, the content of β-1,3-xylobiose and lichoridiose more than 70% as determined by high performance liquid chromatography (HPLC), which could significantly facilitate the separation and purification of oligosaccharides. The successful design of AncXyl09 was the representative of the semi-rationally engineered β-1, 3-xylanase, which will shield a new light on the β-1,3-xylanase engineering, active oligosaccharide preparation and marine algae resource utilization.

Keywords: 3-Xylanase; Ancestor protein reconstruction; Oligosaccharide antioxidant activity; Promiscuous β-1; Thermal stability.