title: Characterization of a Paenibacillus woosongensis beta-Xylosidase/alpha-Arabinofuranosidase produced by recombinant Escherichia coli.
abstract: A gene encoding the beta-xylosidase/alpha-arabinofuranosidase (XylC) of Paenibacillus woosongensis was cloned into Escherichia coli. This xylC gene consisted of 1,425 nucleotides, encoding a polypeptide of 474 amino acid residues. The deduced amino acid sequence exhibited an 80% similarity with those of both Clostridium stercorarium beta-xylosidase/alpha-N-arabinosidase and Bacillus cellulosilyticus alpha-arabinofuranosidase, belonging to the glycosyl hydrolase family 43. The structural gene was subcloned with a Cterminal His-tag into a pET23a(+) expression vector. The His-tagged XylC, purified from a cell-free extract of a recombinant E. coli BL21(DE3) Codon Plus carrying a xylC gene by affinity chromatography, was active on paranitrophenyl- alpha-arabinofuranoside (pNPA) as well as paranitrophenyl- beta-xylopyranoside (pNPX). However, the enzymatic activities for the substrates were somewhat incongruously influenced by reaction pHs and temperatures. The enzyme was also affected by various chemicals at different levels. SDS (5 mM) inhibited the enzymatic activity for pNPX, while enhancing the enzymatic activity for pNPA. Enzyme activity was also found to be inhibited by addition of pentose or hexose. The Michaelis constant and maximum velocity of the purified enzyme were determined for hydrolysis of pNPX and pNPA, respectively.
authors.affiliation: Department of Food Science and Biotechnology, Woosong University, Daejeon 300-718, Korea.
authors.forename: Yeon A
authors.initials: YA
authors.lastname: Kim
authors.forename: Ki-Hong
authors.initials: KH
authors.lastname: Yoon
mesh_terms: D001089:Arabinose / Q000031:analogs & derivatives / Q000378:metabolism; D002846:Chromatography, Affinity; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D004791:Enzyme Inhibitors / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D006027:Glycosides / Q000378:metabolism; D006601:Hexoses / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D056507:Paenibacillus / Q000201:enzymology*; D010429:Pentoses / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D012967:Sodium Dodecyl Sulfate / Q000378:metabolism; D013696:Temperature; D014995:Xylosidases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D004269:DNA, Bacterial; D004791:Enzyme Inhibitors; D006027:Glycosides; D006601:Hexoses; D010429:Pentoses; D011993:Recombinant Fusion Proteins; C008646:4-nitrophenyl beta-D-xyloside; D012967:Sodium Dodecyl Sulfate; C017567:4-nitrophenyl-alpha-L-arabinofuranoside; D001089:Arabinose; D006026:Glycoside Hydrolases; D014995:Xylosidases; C026579:exo-1,4-beta-D-xylosidase; C019713:alpha-N-arabinofuranosidase