Enhancing thermal tolerance of a fungal GH11 xylanase guided by B-factor analysis and multiple sequence alignment.

Xylanases, capable of hydrolyzing xylans which are abundant in nature, have been employed as important biocatalyst in many industrial processes. Xylanases with advantageous properties, especially excellent thermostability, are in high demand in industry. In this study, we aim to improve the thermostability of XynCDBFV, a fungal GH11 xylanase. To achieve this aim, we discovered residues 87-QNSS-90 with pronounced flexibility based on B-factor analysis, identified highly conserved residues 87-RGHT-90 in GH11 xylanases by multiple sequence alignment, and constructed four single mutants by substituting residues from 87 to 90 by site-directed mutagenesis. Temperature stability measurements showed promising enhancement of thermostability for all four single mutants, and the thermal tolerant ability from strong to weak is N88 G, S90 T, S89H, Q87R, XynCDBFV. Four single mutants all retained higher than 50% activities after incubation at the optimal temperature 60℃ for 1 h, while the retained activity for XynCDBFV was only 20.94% at the same condition. N88 G retained greater than 60% residual activity after incubation at 65℃ for 1 h, while the residual activity of XynCDBFV decreased rapidly, losing all activity after 45 min of incubation. Molecular dynamics simulations and structural analysis were applied to explore the heat-resistant mechanisms for mutants: novel hydrogen bonding interaction were discovered and accounted for the improved thermostability. Enzyme activity of the single mutants compromised with their thermostability and combined mutations displayed antagonistic effect due to the closed contact of the mutated residues. This study confirms that combining B-factor analysis and multiple sequence alignment is an effective strategy for obtaining a thermostable enzyme, and the negative findings help to recognize limitations in xylanase engineering for preferable properties.