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

Abstract

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 K m values and high catalytic efficiencies (k cat/K m) 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.

Methods for the extraction, storage, amplification and sequencing of DNA from environmental samples

“…not using commercial kits) were used in the majority of studies (e.g. Callejas et al 2011;Barberan & Casamayor 2014;Mao et al 2014) DNA in a number of studies, but only twice since 2014 (Merlin et al 2014;Wasaki et al 2015).…”

Characterisation of a novel cellobiose 2‐epimerase from thermophilic Caldicellulosiruptor obsidiansis for lactulose production

“…In this study, mannobiose was the favoured substrate of Caob‐CE, indicating that it is a mannobiose 2‐epimerase as well. Recently, Wasaki et al obtained tens of ce ‐like genes by using PCR‐based metagenomic approach and identified two of them with the result that the enzyme actually showed higher substrate specificity toward mannobiose than cellobiose and thus should be named mannobiose 2‐epimerase . The authors further proposed that CE, actually mannobiose 2‐epimerase, perhaps participates in the mannan catabolic pathway in a wide range of bacteria …”

“…CE catalyses hydroxyl epimerisation at the C‐2 position of the glucose moiety of cellobiose (4‐ O‐β ‐ d ‐glucopyranosyl‐ d ‐glucose) to generate 4‐ O‐β ‐ d ‐glucopyranosyl‐ d ‐mannose (Glc‐Man) . It has a wide substrate specificity and catalyses the reversible epimerisation reactions between lactose and epilactose (4‐ O‐β ‐ d ‐galactopyranosyl‐ d ‐mannose) and between mannobiose (4‐ O‐β ‐g d ‐mannopyranosyl‐ d ‐mannose) and 4‐ O‐β ‐ d ‐mannopyranosyl‐ d ‐glucose (Man‐Glc) . CE occurs in a wide range of microorganisms and is considered to play a role in mannan catabolic pathway .…”

Current Status and Potential Applications of Underexplored Prokaryotes

“…The International Society of Rare Sugar () has defined rare sugars as monosaccharides and their derivatives that rarely exist in nature [145]. These fine chemicals (i.e., D-allose, D-psicose, D-gulose, D-sorbose, and L-ribose) are valuable and have numerous applications in food products and sweeteners, pharmaceuticals, and agriculture [145,146,147]. The conversion of natural sugars to rare sugars involves several enzymatic reactions involving isomerases, epimerases, and oxidoreductases [145].…”

“…The majority of enzymes already applied in the industry have been sourced from cultured bacteria. To the best of our knowledge, attempts of finding underexplored culturable prokaryotes as well as gene-mining using a metagenomic approach to produce rare sugar are scarce [147].…”