Sequence- and structure-guided improvement of the catalytic performance of a GH11 family xylanase from Bacillus subtilis
| Autor: | Marcelo Monteiro Pedroso, Gerhard Schenk, Zhen Gao, Bingfang He, Bin Wu, Kun Cao, Lijuan Wang |
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| Rok vydání: | 2021 |
| Předmět: |
Mutation
Missense catalytic activity Bacillus Bacillus subtilis Xylose enzyme catalysis Biochemistry Catalysis RMSF root mean square fluctuation Enzyme catalysis chemistry.chemical_compound Bacterial Proteins GH glycoside hydrolase enzyme mutation DS Discovery Studio Xylobiose Molecular Biology Trichoderma reesei Endo-1 4-beta Xylanases biology Active site protein engineering molecular docking Cell Biology Protein engineering biology.organism_classification XOS xylooligosaccharides GH11 xylanase Amino Acid Substitution chemistry xylooligosaccharides biology.protein Xylanase Research Article |
| Zdroj: | The Journal of Biological Chemistry |
| ISSN: | 0021-9258 |
| Popis: | Xylanases produce xylooligosaccharides 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 the 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 the 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 (kcat), which resulted in increased catalytic efficiency than WT XynLC9. Furthermore, the W6F/Q7H mutant displayed a 160% increase in the yield of xylooligosaccharides 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 functional diversity in a limited number of protein variants. |
| Databáze: | OpenAIRE |
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