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Purification, characterization, cDNA cloning, and expression of a xyloglucan endoglucanase from Geotrichum sp. M128 1
Author(s) -
Yaoi Katsuro,
Mitsuishi Yasushi
Publication year - 2004
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/s0014-5793(04)00068-7
Subject(s) - xyloglucan , geotrichum , complementary dna , biochemistry , glycosyl , cellulase , glucanase , open reading frame , peptide sequence , glycoside hydrolase , hydrolase , biology , molecular cloning , chemistry , microbiology and biotechnology , cell wall , enzyme , gene
A novel xyloglucan‐specific endo‐β‐1,4‐glucanase (XEG), xyloglucanase, with a molecular mass of 80 kDa and a p I of 4.8, was isolated from the fungus Geotrichum sp. M128. It was found to be an endoglucanase active toward xyloglucan and not active toward carboxymethylcellulose, Avicel, or barley 1,3‐1,4‐β‐glucan. Analysis of the precise substrate specificity using various xyloglucan oligosaccharide structures revealed that XEG has at least four subsites (−2 to +2) and specifically recognizes xylose branching at the +1 and +2 sites. The full‐length cDNA encoding XEG was cloned and sequenced. It consists of a 2436‐bp open reading frame encoding a 776‐amino acid protein. From its deduced amino acid sequence, XEG can be classified as a family 74 glycosyl hydrolase. The cDNA encoding XEG was then expressed in Escherichia coli , and enzymatically active recombinant XEG was obtained.