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

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.

Fig. 1

Comparison of normalized B-factors after pairwise sequence alignment. a Normalized B-factors were compared between XynCDBFV (black) and NFX (red). b Pairwise sequence alignment between XynCDBFV and NFX

Fig. 2

Conformation of residues from 207 to 210 in two xylanases. a Conformation of 207-NGGA-210 in the XynCDBFV crystal structure. b Conformation of 207-SSGS-210 in the NFX crystal structure

Fig. 3

Multiple sequence alignment of GH11 xylanases from different organisms. Sequence logo from multiple sequence alignment from all fungal GH 11 xylanases (a), bacterial GH11 xylanases (b), fungal and bacterial GH11 xylanases (c) from the NCBI protein database

Fig. 4

SDS-PAGE analysis of the recombinant xylanases. Lanes 1 and 2 correspond to purified Xyn-MUT and XynCDBFV from P. pastoris, respectively; lane M corresponds to standard protein molecular mass markers

Fig. 5

Residual activities of xylanases incubated in different thermal conditions. Residual activities of XynCDBFV and Xyn-MUT expressed in P. pastoris incubated at 70 °C (a), 80 °C (b), and 95 °C (c) for 1 h

Fig. 6

Open and closed states of substrate-binding sites in two xylanases. a Schematic structure of XynCDBFV colored by secondary structure, β-strand in yellow, α-helix in red and loop in green. b Minimal distance distribution between W32 and P151 in XynCDBFV and Xyn-MUT at 65 and 80 °C

Fig. 7

Statistics of hydrogen bond interactions with substituted residues in two xylanases. a Number of hydrogen bonds interacted with substituted residues in XynCDBFV (upper) and Xyn-MUT (lower) over the whole simulations. Number of hydrogen bonds in three pairs N/S207-N205 (b), G/S208-N205 (c), and A/S210-A55 (d) as a function of simulation time in XynCDBFV and Xyn-MUT at 65 and 80 °C

Fig. 8

Residual activities of XynCDBFV and single mutants incubated at 65 °C. Residual activities of XynCDBFV and three single mutants (N207S, G208S, and A210S) expressed in E. coli incubated at 65 °C for 20 min

Fig. 9

DSSP analysis of two xylanases at different temperatures during the simulations. Analysis of secondary structure propensities of XynCDBFV at 65 °C (a) and 80 °C (b) during the simulations; analysis of secondary structure propensities of Xyn-MUT at 65 °C (c) and 80 °C (d)

Fig. 10

Conformations of 207-NGGA-210 and 86-KQNSSN-91 in XynCDBFV crystal structure