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Objective To clone and express a gene encoding the β-xylosidase from Enterobacter cloacae GX-3, a putative member of the glycoside hydrolase family 3, systematically characterize the recombinant enzyme, and improve the xylose tolerance by molecular engineering of key amino acid residues involved in xylose binding. Methods On the basis of the whole-genome sequencing data of E. cloacae GX-3, primers were designed to amplify the β-xylosidase gene annotated as GH3. The target gene was amplified by PCR and cloned into the pQE30 expression vector, and the resulting recombinant plasmid was transformed into Escherichia coli M15 for induced expression. The recombinant enzyme was purified by nickel-affinity chromatography, and its enzymatic properties were studied. Site-directed mutagenesis was conducted on amino acid residues associated with xylose tolerance. Results The β-xylosidase gene belonging to the GH3 family was successfully cloned from E. cloacae GX-3 and heterologously expressed in E. coli M15. Substrate specificity analysis revealed that the recombinant enzyme EXYL was a multifunctional enzyme exhibiting β-xylosidase, β-glucosidase, and α-L-arabinofuranosidase activities. EXYL showed the optimal performance with the substrate of pNPX and at pH 5.5 and 45 ℃. The Km and Vmax values of this enzyme were (0.73±0.06) mmol/L and (130.00±6.85) μmol/(mg·min), respectively. The inhibition constant (Ki) for xylose was (51.95±2.36) mmol/L. When EXYL acted on xylooligosaccharides (X3-X5), the main products were xylose and xylobiose, each accounting for approximately 50% of the yield. Site-directed mutagenesis of xylose tolerance-related residues yielded positive mutants W138C and W138A, which showed 2.38-fold and 1.83-fold improvements in xylose tolerance, respectively. Conclusion This study provides insights into the multifunctional activities of β-xylosidases and offers new strategies for enhancing the xylose tolerance of β-xylosidases in the GH3 family.
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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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