RHA-P: Isolation, expression and characterization of a bacterial α-l-rhamnosidase from Novosphingobium sp. PP1Y

α-l-Rhamnosidases (α-RHAs) are a group of glycosyl hydrolases of biotechnological potential in industrial processes, which catalyze the hydrolysis of α-l-rhamnose terminal residues from several natural compounds. A novel α–RHA activity was identified in the crude extract of Novosphingobium sp. PP1Y, a marine bacterium able to grow on a wide range of aromatic polycyclic compounds. In this work, this α-RHA activity was isolated from the native microorganism and the corresponding orf was identified in the completely sequenced and annotated genome of strain PP1Y. The coding gene was expressed in Escherichia coli, strain BL21(DE3), and the recombinant protein, rRHA-P, was purified and characterized as an inverting monomeric glycosidase of ca. 120 kDa belonging to the GH106 family. A biochemical characterization of this enzyme using pNPR as substrate was performed, which showed that rRHA-P had a moderate tolerance to organic solvents, a significant thermal stability up to 45 °C and a catalytic efficiency, at pH 6.9, significantly higher than other bacterial α-RHAs described in literature. Moreover, rRHA-P was able to hydrolyze natural glycosylated flavonoids (naringin, rutin, neohesperidin dihydrochalcone) containing α-l-rhamnose bound to β-d-glucose with either α-1,2 or α-1,6 glycosidic linkages. Data presented in this manuscript strongly support the potential use of RHA-P as a biocatalyst for diverse biotechnological applications.