Purification, characterization, cloning, and expression of a novel xyloglucan-specific glycosidase, oligoxyloglucan reducing end-specific cellobiohydrolase

J Biol Chem. 2002 Dec 13;277(50):48276-81. doi: 10.1074/jbc.M208443200. Epub 2002 Oct 8.

Abstract

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.

MeSH terms

  • Amino Acid Sequence
  • Base Sequence
  • Cellulase / chemistry
  • Cellulase / genetics
  • Cellulase / metabolism*
  • Cellulose 1,4-beta-Cellobiosidase
  • Cloning, Molecular
  • DNA, Complementary
  • Geotrichum / enzymology
  • Glucans*
  • Glycoside Hydrolases / metabolism*
  • Molecular Sequence Data
  • Nuclear Magnetic Resonance, Biomolecular
  • Phylogeny
  • Polysaccharides / metabolism*
  • Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
  • Substrate Specificity
  • Xylans*

Substances

  • DNA, Complementary
  • Glucans
  • Polysaccharides
  • Xylans
  • xyloglucan
  • Glycoside Hydrolases
  • Cellulase
  • Cellulose 1,4-beta-Cellobiosidase

Associated data

  • GENBANK/AB089343