Identification and distribution of cellobiose 2-epimerase genes by a PCR-based metagenomic approach

Cellobiose 2-epimerase (CE) catalyzes the reversible epimerization of cellobiose to 4-O-β-D-glucopyranosyl-D-mannose. By using a PCR-based metagenomic approach, 71 ce-like gene fragments were obtained from wide-ranging environmental samples such as sheep rumen, soils, sugar beet extracts, and anaerobic sewage sludge. The frequency of isolation of the fragments similar to known sequences varied depending on the nature of the samples used. The ce-like genes appeared to be widely distributed in environmental bacteria belonging to the phyla Bacteroidetes, Chloroflexi, Dictyoglomi, Firmicutes, Proteobacteria, Spirochaetes, and Verrucomicrobia. The phylogenetic analysis suggested that the cluster of CE and CE-like proteins was functionally and evolutionarily separated from that of N-acetyl-D-glucosamine 2-epimerase (AGE) and AGE-like proteins. Two ce-like genes containing full-length ORFs, designated md1 and md2, were obtained by PCR and expressed in Escherichia coli. The recombinant mD1 and mD2 exhibited low Kₘvalues and high catalytic efficiencies (kcₐₜ/Kₘ) for mannobiose compared with cellobiose, suggesting that they should be named mannobiose 2-epimerase, which is involved in a new mannan catabolic pathway we proposed.