Abstract
The cellobiose 2-epimerase from Ruminococcus albus (RaCE) catalyzes the epimerization of cellobiose and lactose to 4-O-β-d-glucopyranosyl-d-mannose and 4-O-β-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 (α/α)6 core barrel structure.
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Acknowledgements
This study was supported in part by national projects Special Coordination Funds for Promoting Science and Technology and Knowledge Cluster Initiative (2nd Stage, Sapporo Biocluster Bio-S), The Ministry of Education, Science, Sports and Culture of Japan.
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Ito, S., Hamada, S., Ito, H. et al. Site-directed mutagenesis of possible catalytic residues of cellobiose 2-epimerase from Ruminococcus albus . Biotechnol Lett 31, 1065–1071 (2009). https://doi.org/10.1007/s10529-009-9979-3
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DOI: https://doi.org/10.1007/s10529-009-9979-3