Abstract:α-L-Rhamnosidase can specifically hydrolyze α-L-rhamnose on glycosides and has been widely used in food, medicine and other industrial fields. This research aimed to obtain high-temperature-tolerant α-L-rhamnosidase, and to improve the efficiency of industrial production and reduce the cost of application. In this study, the α-L-rhamnosidase gene screened from the thermophilic bacterium Sulfolobus islandicus was cloned and heterogeneously expressed in Escherichia coli. In addition, the enzymatic properties of recombinant SisRha was determined, and the enzymatic preparation process of isoquercetin was subsequently optimized. The results showed that the recombinant SisRha was most active at 90 ℃with excellent thermal stability, and the enzyme activity was hardly lost after incubation at 80 ℃ for 120 min. The optimum reaction pH of the enzyme was 5.5, and the enzyme was quite stable in the pH range of pH 4.5~7.0. The Km, Vmax and kcat values of the recombinant enzyme SisRha against pNPR as the artificial substrate were (0.15±0.03) mmol/L, (6.26±0.51) U/mg and (10.48±0.86) s-1, respectively. The specific enzymatic activities of SisRha on the substrates pNPR, rutin, epimedin C, naringin, hesperidin and icariin were 25.06, 11.89, 6.74, 3.14, 2.77 ,1.68 U/mg, respectively. The optimum process of isoquercetin conversed from rutin by the recombinant SisRha was as follows: 2 mmol/L rutin could be almost converted with a molar conversion rate of 98.56% using 0.4 U/mL enzyme dosage of the recombinant SisRha under 85 ℃ at pH 5.0 for 1 hour incubation. This study enriched the current resources of α-L-rhamnosidase, established a theoretical foundation for the study of α-L-rhamnosidase derived from thermophilic bacteria, and provided a method of converting rutin to isoquercetin at high temperature.