Cloning and characterization of an exo-xylogucanase from rumenal microbial metagenome.
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.
Wong
Dominic D W S
DD
Western Regional Research Center, USDA-ARS, 800 Buchanan Street, Albany, CA 94710, USA. dwsw@pw.usda.gov
Chan
Victor J
VJ
McCormack
Amanda A
AA
Batt
Sarah B
SB
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
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