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Objective The mesophilic salt-tolerant xylanase XynRBM26, a member of the GH10 family, holds significant application value in industrial fields such as animal feed. This study improved the thermostability of this enzyme by protein modification via rational design, aiming to lay a foundation for the industrial application of XynRBM26 preparations. Methods The bioinformatics software FoldX was used to conduct positionscan of the 3D structure predicted by AlphaFold 2.0 for XynRBM26. The mutants with free energy changes less than -0.5 kcal/mol were selected to construct an initial electronic library. According to the Pro effect and screening principles for potential mutants, an electronic library composed of Pro mutations was subsequently established. Finally, site-directed mutagenesis was employed to construct mutant genes, and positive mutants were screened after heterologous expression, purification, and experimental verification. Results After screening of the initial potential mutants, a small and precise mutant library consisting of 21 Pro effect mutants was constructed. All the mutants were experimentally validated, and positive single-point mutants D222P, V182P, D344P, and A352P with significantly increased Tm values were screened out. Through subsequent stacking screening, a three-point stacked Pro effect mutant with superior properties was obtained. The combination of this mutant with the experimentally screened positive mutant G115D produced the most stable mutant M4 (G115D-D222P-D344P-A352P). Compared with wild-type XynRBM26, M4 showed increases of 6.5 °C and 5 °C in Tm and optimal temperature, respectively. Moreover, M4 presented the half-life (t1/2) at 55 °C 7.5-fold longer than the wild type, and its relative activity at the optimal temperature was 3.44 folds that of the wild type. Conclusion Screening thermostable mutants of the salt-tolerant xylanase XynRBM26 of the GH10 family based on the Pro effect and two different effect superimposing strategies is an effective approach.
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| 科 Family | 属数 Number of genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) | 属 Genus | 种数 Number of species | 占总种数比例 Percentage of total species (%) |
|---|---|---|---|---|---|---|
| 鹅膏菌科Amanitaceae | 2 | 11 | 5.26 | 鹅膏菌属 Amanita | 10 | 4.78 |
| 小菇科 Mycenaceae | 2 | 12 | 5.74 | 丝盖伞属 Inocybe | 5 | 2.39 |
| 多孔菌科 Polyporaceae | 8 | 14 | 6.70 | 蜡蘑属 Laccaria | 5 | 2.39 |
| 红菇科 Russulaceae | 3 | 23 | 11.00 | 小皮伞属 Marasmius | 6 | 2.87 |
| 小菇属 Mycena | 11 | 5.26 | ||||
| 光柄菇属 Pluteus | 5 | 2.39 | ||||
| 红菇属 Russula | 17 | 8.13 | ||||
| 栓菌属 Trametes | 5 | 2.39 |
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