RHA-P: isolation, expression and characterization of a bacterial alfa-L-rhamnosidase from Novosphingobium sp. PP1Y.

Alfa-l-Rhamnosidases (-RHAs) are a group of glycosyl hydrolases of biotechnological potential in indus-trial processes, which catalyze the hydrolysis of -l-rhamnose terminal residues from several naturalcompounds. A novel –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-RHA activity was isolated from the native microorganism and the corresponding orf was identifiedin the completely sequenced and annotated genome of strain PP1Y. The coding gene was expressed inEscherichia coli, strain BL21(DE3), and the recombinant protein, rRHA-P, was purified and characterizedas an inverting monomeric glycosidase of ca. 120 kDa belonging to the GH106 family. A biochemical char-acterization of this enzyme using pNPR as substrate was performed, which showed that rRHA-P had amoderate 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 -RHAs described in literature. Moreover, rRHA-P wasable to hydrolyze natural glycosylated flavonoids (naringin, rutin, neohesperidin dihydrochalcone) con-taining -l-rhamnose bound to -d-glucose with either -1,2 or -1,6 glycosidic linkages. Data presentedin this manuscript strongly support the potential use of RHA-P as a biocatalyst for diverse biotechnological applications.