Biotechnology Letters

, Volume 31, Issue 7, pp 1065–1071

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

  • Shigeaki Ito
  • Shigeki Hamada
  • Hiroyuki Ito
  • Hirokazu Matsui
  • Tadahiro Ozawa
  • Hidenori Taguchi
  • Susumu Ito
Original Research Paper

DOI: 10.1007/s10529-009-9979-3

Cite this article as:
Ito, S., Hamada, S., Ito, H. et al. Biotechnol Lett (2009) 31: 1065. doi:10.1007/s10529-009-9979-3

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.

Keywords

Cellobiose 2-epimerase Epilactose Homology modeling Prebiotics Ruminococcusalbus Site-directed mutagenesis 

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  • Shigeaki Ito
    • 1
  • Shigeki Hamada
    • 1
  • Hiroyuki Ito
    • 1
  • Hirokazu Matsui
    • 1
  • Tadahiro Ozawa
    • 2
  • Hidenori Taguchi
    • 3
  • Susumu Ito
    • 3
  1. 1.Laboratory of Biochemistry, Research Faculty of AgricultureHokkaido UniversitySapporoJapan
  2. 2.Tochigi Research Laboratories of Kao CorporationTochigiJapan
  3. 3.Creative Research Initiative ‘Sousei’ (CRIS), Hokkaido UniversitySapporoJapan

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