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Isoquercetin is a flavonoid with antioxidant, anti-inflammatory, and immunomodulatory activities. However, the low content in plants poses a challenge to the large-scale production of isoquercetin by the extraction method.[Objective] α-L-rhamnosidase can specifically hydrolyze the terminal L-rhamnose residues of natural glycosides. In this study, we screened the strains capable of efficiently and specifically transforming rutin to produce isoquercetin with rutin as the sole carbon source and applied the α-L-rhamnosidase to the production of isoquercetin, aiming to provide new elements for the large-scale production of isoquercetin. [Methods] The selective culture medium with rutin as the sole carbon source was used to screen and identify the strains that can specifically hydrolyze rutin into isoquercetin. The transcriptome analysis was carried out to obtain highly efficient and specific α-L-rhamnosidase, the domain composition of which was determined by structural simulation. The enzymatic properties and substrate specificity of the α-L-rhamnosidase were studied. Furthermore, the hydrolysis effect of the enzyme heterologously expressed in Pichia pastoris in a 5 L fermenter was determined. [Results] AfRhase had five domains, including one α-domain (domain A) and four β-domains (domains N, E, F, and C). With rutin as the substrate, the recombinant enzyme AfRhase showcased the best performance at 55 ℃ and pH 4.5. AfRhase had a wide range of substrates including rutin, hesperidin, naringin, and epimedin C. In a 5 L fermenter for scaled-up production of isoquercetin, P. pastoris expressing AfRhase generated 61 g isoquercetin by hydrolyzing 120 g crude rutin (purity of 70%), with the molar conversion rate of 95.4% and production efficiency of 2.0 mmol/(L·h). [Conclusion] This study for the first time discovered a highly efficient and specific α-L-rhamnosidase from Aspergillus sp. XT-1 for the production of isoquercetin from rutin and heterologously expressed this enzyme in P. pastoris. The domain composition, enzymatic properties, substrate specificity, and hydrolysis efficiency in a 5 L fermenter of this enzyme were determined. In conclusion, this study broadened the function of a fungus-derived α-L-rhamnosidase for the transformation of rutin and laid a foundation for the industrial production of isoquercetin.
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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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