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Sequence- and structure-guided improvement of the catalytic performance of a GH11 family xylanase from Bacillus subtilis.

ABSTRACT: Xylanases produce xylooligosaccharides (XOS) from xylan and have thus attracted increasing attention for their usefulness in industrial applications. Previously, we demonstrated that the GH11 xylanase XynLC9 from Bacillus subtilis formed xylobiose and xylotriose as major products with negligible production of xylose when digesting corncob-extracted xylan. Here, we aimed to improve the catalytic performance of XynLC9 via protein engineering. Based on sequence and structural comparisons of XynLC9 with the xylanases Xyn2 from Trichoderma reesei and Xyn11A from Thermobifida fusca, we identified the N-terminal residues 5-YWQN-8 in XynLC9 as engineering hotspots and subjected this sequence to site-saturation and iterative mutagenesis. The mutants W6F/Q7H and N8Y possessed a 2.6- and 1.8-fold higher catalytic activity than XynLC9, respectively, and both mutants were also more thermostable. Kinetic measurements suggested that W6F/Q7H and N8Y had lower substrate affinity, but a higher turnover rate (k_cat), which resulted in increased catalytic efficiency compared to wild type XynLC9. Furthermore, the W6F/Q7H mutant displayed a 160% increase in the yield of XOS from corncob-extracted xylan. Molecular dynamics simulations revealed that the W6F/Q7H and N8Y mutations led to an enlarged volume and surface area of the active site cleft, which provided more space for substrate entry and product release, and thus accelerated the catalytic activity of the enzyme. The molecular evolution approach adopted in this study provides the design of a library of sequences that captures a meaning functional diversity in a limited number of protein variants.

SUBMITTER: Wang L

PROVIDER: S-EPMC8546418 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description: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 (Km, 0.22 and 0.59 µM, respectively). Catalytic efficiency values (kcat/Km) 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.

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Sequence- and structure-guided improvement of the catalytic performance of a GH11 family xylanase from Bacillus subtilis.

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