Improving the thermostability of a fungal GH11 xylanase via site-directed mutagenesis guided by sequence and structural analysis

Nanyu Han; Huabiao Miao; Junmei Ding; Junjun Li; Yuelin Mu; Junpei Zhou; Zunxi Huang

doi:10.1186/s13068-017-0824-y

Improving the thermostability of a fungal GH11 xylanase via site-directed mutagenesis guided by sequence and structural analysis

Biotechnol Biofuels. 2017 May 23:10:133. doi: 10.1186/s13068-017-0824-y. eCollection 2017.

Authors

Nanyu Han^#^{1

2}, Huabiao Miao^#¹, Junmei Ding^{1

2}, Junjun Li^{1

2}, Yuelin Mu^{1

2}, Junpei Zhou^{1

2}, Zunxi Huang^{1

2}

Affiliations

¹ School of Life Sciences, Yunnan Normal University, Kunming, 650500 China.
² Key Laboratory of Enzyme Engineering, Yunnan Normal University, Kunming, 650500 China.

^# Contributed equally.

Abstract

Background: Xylanases have been widely employed in many industrial processes, and thermophilic xylanases are in great demand for meeting the high-temperature requirements of biotechnological treatments. In this work, we aim to improve the thermostability of XynCDBFV, a glycoside hydrolase (GH) family 11 xylanase from the ruminal fungus Neocallimastix patriciarum, by site-directed mutagenesis. We report favorable mutations at the C-terminus from B-factor comparison and multiple sequence alignment.

Results: C-terminal residues 207-NGGA-210 in XynCDBFV were discovered to exhibit pronounced flexibility based on comparison of normalized B-factors. Multiple sequence alignment revealed that beneficial residues 207-SSGS-210 are highly conserved in GH11 xylanases. Thus, a recombinant xylanase, Xyn-MUT, was constructed by substituting three residues (N207S, G208S, A210S) at the C-terminus of XynCDBFV. Xyn-MUT exhibited higher thermostability than XynCDBFV at ≥70 °C. Xyn-MUT showed promising improvement in residual activity with a thermal retention of 14% compared to that of XynCDBFV after 1 h incubation at 80 °C; Xyn-MUT maintained around 50% of the maximal activity after incubation at 95 °C for 1 h. Kinetic measurements showed that the recombinant Xyn-MUT had greater kinetic efficiency than XynCDBFV (K_m, 0.22 and 0.59 µM, respectively). Catalytic efficiency values (k_cat/K_m) of Xyn-MUT also increased (1.64-fold) compared to that of XynCDBFV. Molecular dynamics simulations were performed to explore the improved catalytic efficiency and thermostability: (1) the substrate-binding cleft of Xyn-MUT prefers to open to a larger extent to allow substrate access to the active site residues, and (2) hydrogen bond pairs S208-N205 and S210-A55 in Xyn-MUT contribute significantly to the improved thermostability. In addition, three xylanases with single point mutations were tested, and temperature assays verified that the substituted residues S208 and S210 give rise to the improved thermostability.

Conclusions: This is the first report for GH11 recombinant with improved thermostability based on C-terminus replacement. The resulting Xyn-MUT will be an attractive candidate for industrial applications.

Keywords: B-factor; C-terminus replacement; MD simulation; Site-directed mutagenesis; Thermostability; Xylanase.