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

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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.

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Acknowledgments

Mr. Tomohiro Hirose and Mr. Akira Miyao at the Center for Instrumental Analysis, Hokkaido University, determined the N-terminal amino acid sequences of mD1 and mD2. This research was partly supported by the Special Coordination Funds for Promoting Science and Technology and by the Platform for Drug Discovery, Informatics, and Structural Life Science from the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.

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Authors and Affiliations

  1. Graduate School of Biosphere Science, Hiroshima University, Kagamiyama 1-7-1, Higashihiroshima, 739-8521, Japan

    Jun Wasaki

  2. Creative Research Initiative ‘Sousei’ (CRIS), Hokkaido University, N21W10, Kita-ku, Sapporo, 001-0021, Japan

    Hidenori Taguchi, Takeshi Senoura & Hiroshi Akasaka

  3. National Food Research Institute, National Agriculture and Food Research Organization, 2-1-12 Kannondai, Tsukuba, 305-8642, Japan

    Jun Watanabe

  4. Department of Bioscience and Biotechnology, Faculty of Agriculture, University of the Ryukyus, 1 Senbaru, Nishihara, Okinawa, 903-0213, Japan

    Kazuki Kawaguchi, Yosuke Komata, Kiyotoshi Hanashiro & Susumu Ito

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  1. Jun Wasaki
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  2. Hidenori Taguchi
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Correspondence to Jun Wasaki.

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Wasaki, J., Taguchi, H., Senoura, T. et al. Identification and distribution of cellobiose 2-epimerase genes by a PCR-based metagenomic approach. Appl Microbiol Biotechnol 99, 4287–4295 (2015). https://doi.org/10.1007/s00253-014-6265-7

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  • DOI: https://doi.org/10.1007/s00253-014-6265-7

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