title: Cloning and characterization of α-L-arabinofuranosidase and bifunctional α-L-arabinopyranosidase/β-D-galactopyranosidase from Bifidobacterium longum H-1.
abstract: OBJECTIVE
  This study focused on the cloning, expression and characterization of recombinant α-l-arabinosidases from Bifidobacterium longum H-1.
  
  
  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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Life and Nanopharmaceutical Sciences, Kyung Hee University, Seoul, Korea.
authors.forename: J H
authors.initials: JH
authors.lastname: Lee
authors.forename: Y-J
authors.initials: YJ
authors.lastname: Hyun
authors.forename: D-H
authors.initials: DH
authors.lastname: Kim
mesh_terms: D001644:Bifidobacterium / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D005690:Galactose / Q000378:metabolism; D015723:Gene Library; D036145:Ginsenosides / Q000378:metabolism*; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism*
chemical_list: D036145:Ginsenosides; D011994:Recombinant Proteins; C044462:ginsenoside Rc; D006026:Glycoside Hydrolases; D001616:beta-Galactosidase; C019713:alpha-N-arabinofuranosidase; D005690:Galactose