# Characterization of a Thermophilic and Acidophilic GH78 α-L-Rhamnosidase from Thermotoga sp. 2812B Capable of Efficiently Hydrolyzing a Variety of Natural Flavonoid Diglycosides

## TL;DR

A new heat- and acid-tolerant enzyme from Thermotoga sp. 2812B efficiently breaks down various plant flavonoid glycosides, making it a promising biocatalyst for food and pharmaceutical applications.

## Contribution

The discovery and characterization of a novel thermophilic and acidophilic GH78 α-L-rhamnosidase with broad substrate specificity and high conversion efficiency.

## Key findings

- TsRha shows optimal activity at 90 °C and pH 5.0 with remarkable thermostability.
- The enzyme efficiently hydrolyzes multiple natural flavonoid diglycosides with ≥99.1% conversion.
- TsRha has broad substrate selectivity for various glycosidic bonds and aglycone subgroups.

## Abstract

α-L-Rhamnosidase can specifically hydrolyze plant natural glycosides and holds significant potential for biocatalytic applications in functional foods, healthy products, and pharmaceutical industries. Herein, a novel thermophilic and acidophilic α-L-rhamnosidase TsRha from Thermotoga sp. 2812B belonging to glycoside hydrolase family 78 was identified by genome mining and comprehensively characterized by bioinformatics, computer-aided structural analysis, and biochemical characterization. TsRha possesses a domain architecture comprising one catalytic (α/α)6-barrel domain and four β-sheet domains. TsRha displayed optimal activity at 90 °C and pH 5.0, remarkable thermostability at 80 °C, and considerable tolerance to organic solvents. TsRha exhibited broad substrate selectivity and might efficiently hydrolyze a series of natural flavonoid glycosides with various glycosidic bonds (α-1, α-1, 2, α-1, 6) from different aglycone subgroups (flavanone, flavone, flavonol, and dihydrochalcone). Moreover, it demonstrated high conversion efficiencies toward a variety of natural flavonoid diglycosides rutin, naringin, naringin dihydrochalcone, hesperidin, and troxerutin, achieving ≥99.1% conversion within 20~100 min. The excellent properties including high activity, thermophilicity, acidophilicity, good thermostability, broad substrate spectrum will make the α-L-rhamnosidase TsRha a promising biocatalyst for the efficient production of rare and high-value flavonoid glucosides with improved bioavailability and bioactivity.