title: Paenibacillus sp. 598K 6-α-glucosyltransferase is essential for cycloisomaltooligosaccharide synthesis from α-(1 → 4)-glucan.
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 by13C 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.
authors.affiliation: Food Research Institute, National Agriculture and Food Research Organization (NARO), 2-1-12 Kannondai, Tsukuba, 305-8642, Japan.|Department of Agricultural and Life Sciences, Faculty of Agriculture, Shinshu University, 8304 Minami-minowa, Kami-ina, Nagano, 399-4598, Japan.
authors.forename: Hitomi
authors.initials: H
authors.lastname: Ichinose
authors.affiliation: Food Research Institute, National Agriculture and Food Research Organization (NARO), 2-1-12 Kannondai, Tsukuba, 305-8642, Japan.|Department of Biological Production, Faculty of Bioresource Sciences, Akita Prefectural University, Akita City, Akita, 010-0195, Japan.
authors.forename: Ryuichiro
authors.initials: R
authors.lastname: Suzuki
authors.affiliation: Food Research Institute, National Agriculture and Food Research Organization (NARO), 2-1-12 Kannondai, Tsukuba, 305-8642, Japan.|Laboratory of Biotechnology, Research Institute of Green Science and Technology, Shizuoka University, Shizuoka City, Shizuoka, 422-8529, Japan.
authors.forename: Takatsugu
authors.initials: T
authors.lastname: Miyazaki
authors.affiliation: Food Research Institute, National Agriculture and Food Research Organization (NARO), 2-1-12 Kannondai, Tsukuba, 305-8642, Japan.
authors.forename: Keitarou
authors.initials: K
authors.lastname: Kimura
authors.affiliation: Advanced Analysis Center, National Agriculture and Food Research Organization (NARO), 2-1-2 Kannondai, Tsukuba, 305-8602, Japan.
authors.forename: Mitsuru
authors.initials: M
authors.lastname: Momma
authors.affiliation: Structural Biology Research Center, Photon Factory, Institute of Materials Structure Science, High Energy Accelerator Research Organization (KEK), Tsukuba, Ibaraki, 305-0801, Japan.
authors.forename: Nobuhiro
authors.initials: N
authors.lastname: Suzuki
authors.affiliation: Advanced Analysis Center, National Agriculture and Food Research Organization (NARO), 2-1-2 Kannondai, Tsukuba, 305-8602, Japan.
authors.forename: Zui
authors.initials: Z
authors.lastname: Fujimoto
authors.affiliation: Research Faculty of Agriculture, Hokkaido University, Kita-9 Nisi-9, Kita-ku, Sapporo, 060-8589, Japan.
authors.forename: Atsuo
authors.initials: A
authors.lastname: Kimura
authors.affiliation: Food Research Institute, National Agriculture and Food Research Organization (NARO), 2-1-12 Kannondai, Tsukuba, 305-8642, Japan. funane@affrc.go.jp.
authors.forename: Kazumi
authors.identifier: http://orcid.org/0000-0002-4325-8891
authors.initials: K
authors.lastname: Funane
mesh_terms: D001407:Bacillus / Q000201:enzymology; D002853:Chromatography, Liquid; D003470:Culture Media / Q000737:chemistry; D005936:Glucans / Q000378:metabolism*; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006868:Hydrolysis; D013058:Mass Spectrometry; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000737:chemistry; D056507:Paenibacillus / Q000201:enzymology* / Q000235:genetics; D013213:Starch / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D003470:Culture Media; D005936:Glucans; D009844:Oligosaccharides; C021705:maltooligosaccharides; D013213:Starch; D005964:Glucosyltransferases; C489364:glucosyltransferase I; C087517:cycloisomaltooligosaccharide glucanotransferase
keywords: 6-α-Glucosyltransferase; Cycloisomaltooligosaccharide; Glycoside hydrolase family 31; Paenibacillus sp. 598K; Starch