Gene cloning and characterization of alpha-glucuronidase of Bacillus stearothermophilus no. 236.
The alpha-glucuronidase gene of Bacillus stearothermophilus No. 236 was cloned, sequenced, and expressed in Escherichia coli. The gene, designated aguA, encoded a 691-residue polypeptide with calculated molecular weight of 78,156 and pI of 5.34. The alpha-glucuronidase produced by a recombinant E. coli strain containing the aguA gene was purified to apparent homogeneity and characterized. The molecular weight of the alpha-glucuronidase was 77,000 by SDS-PAGE and 161,000 by gel filtration; the functional form of the alpha-glucuronidase therefore was dimeric. The optimal pH and temperature for the enzyme activity were pH 6.5 and 40 degrees C, respectively. The enzyme's half-life at 50 degrees C was 50 min. The values for the kinetic parameters of Km and Vmax were 0.78 mM and 15.3 U/mg for aldotriouronic acid [2-O-alpha-(4-O-methyl-alpha-D-glucopyranosyluronic)-D-xylobiose]. The alpha-glucuronidase acted mainly on small substituted xylo-oligomers and did not release methylglucuronic acid from intact xylan. Nevertheless, synergism in the release of xylose from xylan was found when alpha-glucuronidase was added to a mixture of endoxylanase and beta-xylosidase.
Choi
I D
ID
Graduate School of Biotechnology, Korea University, Seoul.
Kim
H Y
HY
Choi
Y J
YJ
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics; D001411:Geobacillus stearothermophilus / Q000201:enzymology / Q000235:genetics*; D006026:Glycoside Hydrolases / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D014312:Trisaccharides / Q000378:metabolism
D011994:Recombinant Proteins; D014312:Trisaccharides; C425553:aldotriouronic acid; D006026:Glycoside Hydrolases; C096734:alpha-glucuronidase