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Paenibacillus sp. 598K 6-α-glucosyltransferase is essential for cycloisomaltooligosaccharide synthesis from α-(1 → 4)-glucan

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Abstract

Paenibacillus sp. 598K produces cycloisomaltooligosaccharides (cyclodextrans) from starch even in the absence of dextran. Cycloisomaltooligosaccharide glucanotransferase synthesizes cycloisomaltooligosaccharides exclusively from an α-(1 → 6)-consecutive glucose chain consisting of at least four molecules. Starch is not a substrate of this enzyme. Therefore, we predicted that the bacterium possesses another enzyme system for extending α-(1 → 6)-linked glucoses from starch, which can be used as the substrate for cycloisomaltooligosaccharide glucanotransferase, and identified the transglucosylation enzyme Ps6GT31A. We purified Ps6GT31A from the bacterial culture supernatant, cloned its corresponding gene, and characterized the recombinant enzyme. Ps6GT31A belongs to glycoside hydrolase family 31, and it liberates glucose from the non-reducing end of the substrate in the following order of activity: α-(1 → 4)-> α-(1 → 2)- > α-(1 → 3)- > α-(1 → 6)-glucobiose and maltopentaose > maltotetraose > maltotriose > maltose. Ps6GT31A catalyzes both hydrolysis and transglucosylation. The resulting transglucosylation compounds were analyzed by high-performance liquid chromatography and mass spectrometry. Analysis of the initial products by 13C nuclear magnetic resonance spectroscopy revealed that Ps6GT31A had a strong α-(1 → 4) to α-(1 → 6) transglucosylation activity. Ps6GT31A elongated α-(1 → 6)-linked glucooligosaccharide to at least a degree of polymerization of 10 through a successive transglucosylation reaction. Eventually, cycloisomaltooligosaccharide glucanotransferase creates cycloisomaltooligosaccharides using the transglucosylation products generated by Ps6GT31A as the substrates. Our data suggest that Ps6GT31A is the key enzyme to synthesize α-(1 → 6)-glucan for cycloisomaltooligosaccharide production in dextran-free environments.

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Acknowledgements

We thank Dr. H. Ono and his staff for NMR measurements and Dr. M. Kameyama and her staff for MS measurements. We also thank Dr. M. Kobayashi for his gift of the glucans from L. mesenteroides NRRL B-1299 and B-1355. The authors would like to thank Enago (www.enago.jp) for the English language review.

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Author notes

  1. Hitomi Ichinose

    Present address: Department of Agricultural and Life Sciences, Faculty of Agriculture, Shinshu University, 8304 Minami-minowa, Kami-ina, Nagano, 399-4598, Japan

  2. Ryuichiro Suzuki

    Present address: Department of Biological Production, Faculty of Bioresource Sciences, Akita Prefectural University, Akita City, Akita, 010-0195, Japan

  3. Takatsugu Miyazaki

    Present address: Laboratory of Biotechnology, Research Institute of Green Science and Technology, Shizuoka University, Shizuoka City, Shizuoka, 422-8529, Japan

Authors and Affiliations

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

    Hitomi Ichinose, Ryuichiro Suzuki, Takatsugu Miyazaki, Keitarou Kimura & Kazumi Funane

  2. Advanced Analysis Center, National Agriculture and Food Research Organization (NARO), 2-1-2 Kannondai, Tsukuba, 305-8602, Japan

    Mitsuru Momma & Zui Fujimoto

  3. Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), Tsukuba, Ibaraki, 305-0801, Japan

    Nobuhiro Suzuki

  4. Research Faculty of Agriculture, Hokkaido University, Kita-9 Nisi-9, Kita-ku, Sapporo, 060-8589, Japan

    Atsuo Kimura

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  1. Hitomi Ichinose
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Correspondence to Kazumi Funane.

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Funding

This study was supported in part by the Program for Promotion of Basic and Applied Researches Innovations in Bio-oriented Industry (BRAIN, Japan); the Science and Technology Research Promotion Program for Agriculture, Forestry, Fisheries, and Food Industry; and JSPS KAKENHI Grant Number 26450133. The NARO Bio-oriented Technology Research Advancement Institution (BRAIN) provided funding to A.K., K.F., and Z.F. under grant number 25000A. The Agriculture, Forestry and Fisheries Research Council (AFFRC) provided funding to A.K. and K.F. under grant number 26062B. The Japan Society for the Promotion of Science (JSPS) provided funding to K.F. under grant number 26450133.

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The authors declare that they have no competing interests.

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This article does not describe any studies on human participants or animals performed by any of the authors.

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Ichinose, H., Suzuki, R., Miyazaki, T. et al. Paenibacillus sp. 598K 6-α-glucosyltransferase is essential for cycloisomaltooligosaccharide synthesis from α-(1 → 4)-glucan. Appl Microbiol Biotechnol 101, 4115–4128 (2017). https://doi.org/10.1007/s00253-017-8174-z

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