Purification, characterization, cloning, and expression of a novel xyloglucan-specific glycosidase, oligoxyloglucan reducing end-specific cellobiohydrolase.
A novel oligoxyloglucan-specific glycosidase, oligoxyloglucan reducing end-specific cellobiohydrolase (OXG-RCBH), with a molecular mass of 97 kDa and a pI of 6.1, was isolated from the fungus Geotrichum sp. M128. Analysis of substrate specificity using various xyloglucan oligosaccharide structures revealed that OXG-RCBH had exoglucanase activity. It recognized the reducing end of oligoxyloglucan and released two glucosyl residue segments from the main chain. The full-length cDNA encoding OXG-RCBH was cloned and sequenced, and it had a 2436-bp open reading frame encoding an 812amino acid protein. The deduced protein showed approximately 35% identity to members of glycoside hydrolase family 74. The cDNA encoding OXG-RCBH was then expressed in Escherichia coli. Although the recombinant protein was expressed as an inclusion body, renaturation was successful, and enzymatically active recombinant OXG-RCBH was obtained.
Yaoi
Katsuro
K
Institute for Biological Resources and Functions, National Institute of Advanced Industrial Science and Technology, Tsukuba Central 6, 1-1-1 Higashi, Ibaraki 305-8566, Japan. k-yaoi@aist.go.jp
Mitsuishi
Yasushi
Y
D000595:Amino Acid Sequence; D001483:Base Sequence; D002480:Cellulase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase; D003001:Cloning, Molecular; D018076:DNA, Complementary; D005848:Geotrichum / Q000201:enzymology; D005936:Glucans; D006026:Glycoside Hydrolases / Q000378:metabolism*; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010802:Phylogeny; D011134:Polysaccharides / Q000378:metabolism*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013379:Substrate Specificity; D014990:Xylans
D018076:DNA, Complementary; D005936:Glucans; D011134:Polysaccharides; D014990:Xylans; C029353:xyloglucan; D006026:Glycoside Hydrolases; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase