Abstract: The aim of this study was to evaluate the enzymatic properties of recombinant α-L-rhamnosidases PRHA2 and PRHA3 from Pediococcus acidilactici AS1.2696. The whole genome sequence of P. acidilactici AS1.2696 was analyzed and the two genes encoding α-L-rhamnosidase were amplified by PCR and ligated into expression vector pSE, yielding recombinant plasmids pSE-prha2 and pSE-prha3. The recombinant plasmids were separately transformed and expressed in E.coli XL-blue. The recombinant proteins PRHA2 and PRHA3 were purified by Ni-NTA affinity column chromatography and their enzymatic characteristics were investigated in detail. The results showed the optimal pH and temperature of PRHA2 were 5.0 and 60 ℃, respectively. Its Km and Vmax values were (3.039 ± 0.581) mmol/L and (2.032 ± 0.186) μmol/(min·mg), respectively. The optimal pH and temperature of PRHA3 were 5.5 and 45 ℃, and its Km and Vmax values were (2.797 ± 0.132) mmol/L and (113.35 ± 1.485) μmol/(min·mg), respectively. PRHA2 and PRHA3 could not only hydrolyze the artificial substrate p-nitrophenyl-α-L-rhamnopyranoside (pNPR) but could also hydrolyze the α-1,6 glucosidic linkages of the natural substrates hesperidin and rutin. PRHA2 had hydrolytic activity on 4-nitrophenyl-β-L-arabinoside (pNPA). PRHA3 weakly hydrolyzed the glucosidic linkages at the C-7 position of icariin, icariside I, epimedin A, epimedin B and epimedin C. In addition, PRHA3 could hydrolyze the outer rhamnosidic linkage to the C-3 position of epimedin C through α-Rha(2→1)α-Rha, leading to potential applications in the food and medicinal fields.

Key words: Pediococcus acidilactici AS1.2696, α-L-rhamnosidase, cloning and expression, enzymatic properties