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  • Title: Cloning and characterization of α-L-arabinofuranosidase and bifunctional α-L-arabinopyranosidase/β-D-galactopyranosidase from Bifidobacterium longum H-1.
    Author: Lee JH, Hyun YJ, Kim DH.
    Journal: J Appl Microbiol; 2011 Nov; 111(5):1097-107. PubMed ID: 21851513.
    Abstract:
    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 Ni(2+) -affinity column chromatography, whereas recombinant Apy-H1 (rApy-H1) was purified by serial Q-HP and Ni(2+) -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.
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