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 812-amino acid protein. The deduced protein showed similar to35% 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.