Cloning and characterization of α- L-arabinofuranosidase and bifunctional α- L-arabinopyranosidase/β- D-galactopyranosidase from Bifidobacterium longum H-1.

Aims: This study focused on the cloning, expression and characterization of recombinant α- l-arabinosidases from Bifidobacterium longum H-1. Methods and Results: α- l-Arabinofuranosidase (AfuB-H1) and bifunctional α- l-arabinopyranosidase/β- d-galactosidase (Apy-H1) from B. longum H-1 were identified by Southern blotting, and their recombinant enzymes were overexpressed in Escherichia coli BL21 (DE3). Recombinant AfuB-H1 (rAfuB-H1) was purified by single-step Ni2+-affinity column chromatography, whereas recombinant Apy-H1 (rApy-H1) was purified by serial Q-HP and Ni2+-affinity column chromatography. Enzymatic properties and substrate specificities of the two enzymes were assessed, and their kinetic constants were calculated. According to the results, rAfuB-H1 hydrolysed p-nitrophenyl-α- l-arabinofuranoside (pNP-αL-Af) and ginsenoside Rc, but did not hydrolyse p-nitrophenyl-α- l-arabinopyranoside (pNP-αL-Ap). On the other hand, rApy-H1 hydrolysed pNP-αL-Ap, p-nitrophenyl-β- d-galactopyranoside (pNP-βD-Ga) and ginsenoside Rb2. Conclusions: Ginsenoside-metabolizing bifidobacterial rAfuB-H1 and rApy-H1 were successfully cloned, expressed, and characterized. rAfuB-H1 specifically recognized the α- l-arabinofuranoside, whereas rApy-H1 had dual functions, that is, it could hydrolyse both β- d-galactopyranoside and α- l-arabinopyranoside. Significance and Impact of the Study: These findings suggest that the biochemical properties and substrate specificities of these recombinant enzymes differ from those of previously identified α- l-arabinosidases from Bifidobacterium breve K-110 and Clostridium cellulovorans. [ABSTRACT FROM AUTHOR]