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Cloning and characterization of α‐ L ‐arabinofuranosidase and bifunctional α‐ L ‐arabinopyranosidase/β‐ D ‐galactopyranosidase from Bifidobacterium longum H‐1
Author(s) -
Lee J.H.,
Hyun Y.J.,
Kim D.H.
Publication year - 2011
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/j.1365-2672.2011.05128.x
Subject(s) - bifidobacterium longum , recombinant dna , bifidobacterium breve , enzyme , biology , microbiology and biotechnology , affinity chromatography , escherichia coli , bifidobacterium , actinomycetaceae , biochemistry , chemistry , fermentation , gene , lactobacillus
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.