title: Cloning and characterization of an exo-xylogucanase from rumenal microbial metagenome.
abstract: A novel exo-glucanase gene (xeg5B) was isolated from a rumenal microbial metagenome, cloned, and expressed in E. coli. The 1548 bp gene coded for a protein of 516 amino acids, which assumed an (a/b)(8) fold typical of glycoside hydrolase (GH) family 5. The protein molecule consisted of a loop segment blocking one end of the active site, which potentially provided the enzyme with exo-acting property. The recombinant enzyme showed exclusive specificity towards only xyloglucan and oligoxyloglucan substrates with no detectable activity on unsubstituted linear glucans, CMC, laminarin, and lichenan. The major end products of exhaustive hydrolysis were XX (tetrasaccharide) and XG (trisaccharide). The hydrolysis of tamarind xyloglucan followed the Michaelis-Menten kinetics, yielding K(m) and V(max) of 2.12+/-0.13 mg/ml and 0.17+/-0.01 mg/ml/min (37 degrees C, pH 6.0), respectively.
authors.affiliation: Western Regional Research Center, USDA-ARS, 800 Buchanan Street, Albany, CA 94710, USA. dwsw@pw.usda.gov
authors.forename: Dominic D W S
authors.initials: DD
authors.lastname: Wong
authors.forename: Victor J
authors.initials: VJ
authors.lastname: Chan
authors.forename: Amanda A
authors.initials: AA
authors.lastname: McCormack
authors.forename: Sarah B
authors.initials: SB
authors.lastname: Batt
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002417:Cattle; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D005936:Glucans / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D054892:Metagenome / Q000235:genetics*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000378:metabolism; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012417:Rumen / Q000382:microbiology*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014312:Trisaccharides / Q000378:metabolism; D014990:Xylans / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D005936:Glucans; D009844:Oligosaccharides; D011994:Recombinant Proteins; D014312:Trisaccharides; D014990:Xylans; C029353:xyloglucan; D006026:Glycoside Hydrolases; C072818:xyloglucan endo(1-4)-beta-D-glucanase