Site-directed mutagenesis of possible catalytic residues of cellobiose 2-epimerase from Ruminococcus albus

Biotechnol Lett. 2009 Jul;31(7):1065-71. doi: 10.1007/s10529-009-9979-3. Epub 2009 Mar 29.


The cellobiose 2-epimerase from Ruminococcus albus (RaCE) catalyzes the epimerization of cellobiose and lactose to 4-O-beta-D-glucopyranosyl-D-mannose and 4-O-beta-D-galactopyranosyl-D-mannose (epilactose). Based on the sequence alignment with N-acetyl-D-glucosamine 2-epimerases of known structure and on a homology-modeled structure of RaCE, we performed site-directed mutagenesis of possible catalytic residues in the enzyme, and the mutants were expressed in Escherichia coli cells. We found that R52, H243, E246, W249, W304, E308, and H374 were absolutely required for the activity of RaCE. F114 and W303 also contributed to catalysis. These residues protruded into the active-site cleft in the model (alpha/alpha)(6) core barrel structure.

MeSH terms

  • Amino Acid Sequence
  • Amino Acid Substitution / genetics*
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Catalytic Domain
  • Cellobiose / metabolism*
  • Escherichia coli / genetics
  • Gene Expression
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed*
  • Mutant Proteins / biosynthesis
  • Protein Structure, Tertiary
  • Racemases and Epimerases / chemistry
  • Racemases and Epimerases / genetics*
  • Racemases and Epimerases / metabolism*
  • Ruminococcus / enzymology*
  • Ruminococcus / genetics
  • Sequence Alignment


  • Bacterial Proteins
  • Mutant Proteins
  • Cellobiose
  • Racemases and Epimerases