A Hyperthermostable Archaeal GH78 Rhamnosidase Efficiently Hydrolyzes Flavonoid Glycosides for Juice Debittering.

α-L-Rhamnosidases are a class of glycosyl hydrolases (GHs) that catalyze the hydrolysis of terminal α-L-rhamnose residues from diverse glycoconjugates. While extensively characterized in bacterial and fungal sources, no archaeal α-L-rhamnosidases have been characterized to date. Herein, we report the identification and characterization of the first thermostable archaeal α-L-rhamnosidase (ArRha), derived from the metagenomic data set of Pisciarelli solfatara hot spring. ArRha, classified in glycoside hydrolase family GH78, efficiently hydrolyzes α-1,2 and α-1,6 rhamnosyl linkages in flavonoid glycosides with notable biological activities. The novel enzyme showed remarkable temperature stability, wide-range pH activity, organic solvent tolerance, and no metal dependence. Combined with a thermostable β-glucosidase, ArRha converts naringin to prunin and naringenin in sweet and blood orange juices, achieving >95% conversion within 2 h at 65 °C. This represents the first report of a hyperthermostable archaeal GH78 α-L-rhamnosidase with promising applications in industrial enzymatic juice debittering and sustainable flavonoid biotransformation.