Naotake Konno

Iwate Biotechnology Institute/Iwate Biotechnology Research Centre, Kitakami, Iwate, Japan

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Publications (9)14.22 Total impact

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    ABSTRACT: Bud dormancy is an adaptive strategy that perennials use to survive unfavorable conditions. Gentians (Gentiana), popular alpine flowers and ornamentals, produce overwintering buds (OWBs) that can persist through the winter, but the mechanisms regulating dormancy are currently unclear. In this study, we conducted targeted metabolome analysis to obtain clues about the metabolic mechanisms involved in regulating OWB dormancy. Multivariate analysis of metabolite profiles revealed metabolite patterns characteristic of dormant states. The concentrations of gentiobiose [β-d-Glcp-(1→6)-d-Glc] and gentianose [β-d-Glcp-(1→6)-d-Glc-(1→2)-d-Fru] significantly varied depending on the stage of OWB dormancy, and the gentiobiose concentration increased prior to budbreak. Both activation of invertase and inactivation of β-glucosidase resulted in gentiobiose accumulation in ecodormant OWBs, suggesting that gentiobiose is seldom used as an energy source but is involved in signaling pathways. Furthermore, treatment with exogenous gentiobiose induced budbreak in OWBs cultured in vitro, with increased concentrations of sulfur-containing amino acids, GSH, and ascorbate (AsA), as well as increased expression levels of the corresponding genes. Inhibition of GSH synthesis suppressed gentiobiose-induced budbreak accompanied by decreases in GSH and AsA concentrations and redox status. These results indicate that gentiobiose, a rare disaccharide, acts as a signal for dormancy release of gentian OWBs through the AsA-GSH cycle.
    The Plant cell. 10/2014;
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    ABSTRACT: Lentinan from Lentinula edodes fruiting bodies (shiitake mushrooms) is a valuable β-glucan for medical purposes based on its anti-cancer activity and immunomodulating activity. However, lentinan content in fruiting bodies decreases after harvesting and storage due to an increase in glucanase activity. In this study, we downregulated the expression of an exo-β-1,3-glucanase, exg2, in L. edodes using RNA interference. In the wild-type strain, β-1,3-glucanase activity in fruiting bodies remarkably increased after harvesting, and 41.7% of the lentinan content was lost after 4 days of preservation. The EXG2 downregulated strain showed significantly lower lentinan degrading activity (60-70% of the wild-type strain) in the fruiting bodies 2-4 days after harvesting. The lentinan content of fresh fruiting bodies was similar in the wild-type and EXG2 downregulated strains, but in the downregulated strain, only 25.4% of the lentinan was lost after 4 days, indicating that downregulation of EXG2 enables keeping the lentinan content high longer.
    Journal of agricultural and food chemistry. 07/2014;
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    ABSTRACT: Three genes encoding glycoside hydrolase family 12 (GH12) enzymes from Lentinula edodes, namely Lecel12A, Lecel12B, and Lecel12C, were newly cloned by PCR using highly conserved sequence primers. To investigate enzymatic properties, recombinant enzymes encoded by L. edodes DNAs and GH12 genes from Postia placenta (PpCel12A and PpCel12B) and Schizophyllum commune (ScCel12A) were prepared in Brevibacillus choshinensis. Recombinant LeCel12A, PpCel12A, and PpCel12B, which were grouped in GH12 subfamily 1, preferentially hydrolyzed 1,3-1,4-β-glucan. By contrast, LeCel12B, LeCel12C, and ScCel12A, members of the subfamily 2, exhibited specific hydrolysis of xyloglucan. These results suggest that two subfamilies of GH12 are separated based on the substrate specificity. Transcript levels of L. edodes genes increased 72 h after growth of L. edodes mycelia cells in the presence of plant cell wall polymers such as xyloglucan, 1,3-1,4-β-glucan, and cellulose. These results suggest that L. edodes GH12 enzymes have evolved to hydrolyze 1,3-1,4-β-glucan and xyloglucan, which might enhance hyphal extension and nutrient acquisition.
    Journal of Agricultural and Food Chemistry 07/2013; · 3.11 Impact Factor
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    ABSTRACT: Polyporus brumalis ibrc05015 is a strain capable of high laccase (Lac) production. Among several inducers, 0.25 mM copper was most effective for Lac production. One of the Lacs induced by copper was PbLac1, and its transcription was induced within 60 min after copper addition. The promoter region of pblac1 contained six putative metal response elements and one Ace1 consensus cis-element. We cloned the P. brumalis PbAce1 transcription factor, a homologue of Saccharomyces cerevisiae transcription factor Ace1, which regulates metallothionein genes in response to excess copper. PbAce1 complemented the function of Ace1 in an S. cerevisiae Δace strain. The conserved N-terminal copper-fist DNA binding domain of PbAce1 was required for complementation. In the PbAce1 complemented Δace1 strain, the pblac1 promoter was constitutively expressed at a high level, independent of copper concentration. PbAce1 has two Cys-rich repeat motifs (PbC1 and PbC2), which are similar to the Cys-rich repeat domain in metallothionein proteins, and are uniquely conserved in the C-terminal domain of basidiomycetous Ace1 sequences. These C-terminal domains could be involved in copper sensing and concentration-dependent Lac production in basidiomycetous fungi.
    Fungal Biology 01/2013; 117(1):52-61. · 2.08 Impact Factor
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    ABSTRACT: Many carbohydrates are involved in the biofilm formation and activities of glucosyltransferases (Gtfs) of Streptococcus mutans, and the effects of various disaccharides and polysaccharides were investigated in this study, including the hot water-extracted glucan fraction of the Lentinula edodes fruiting body (HWG). HWG was found to inhibit the initial adhesion of S. mutans to saliva-coated hydroxyapatite (sHA), and also laminarin to inhibit glucan synthesis by Gtfs. However, sucrose-dependent biofilm formation by S. mutans was not inhibited by these materials. Interestingly, dextran was found to have an inhibitory effect on the sucrose-dependent biofilm formation. The data suggest that the presence of such an edible glucan as dextran in daily foods would act to some degree on S. mutans for suppressing the cariogenic activity.
    Bioscience Biotechnology and Biochemistry 12/2012; · 1.27 Impact Factor
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    ABSTRACT: We purified and cloned a β-N-acetylhexosaminidase, LeHex20A, with a molecular mass of 79 kDa from the fruiting body of Lentinula edodes (shiitake mushroom). The gene lehex20a gene had 1,659 nucleotides, encoding 553 amino acid residues. Sequence analysis indicated that LeHex20A belongs to glycoside hydrolase (GH) family 20, and homologues of lehex20a are broadly represented in the genomes of basidiomycetes. Purified LeHex20A hydrolyzed the terminal monosaccharide residues of β-N-acetylgalactosaminides and β-N-acetylglucosaminides, indicating that LeHex20A is a β-N-acetylhexosaminidase classified into EC 3.2.1.52. The maximum LeHex20A activity was observed at pH 4.0 and 50°C. The kinetic constants were estimated using chitooligosaccharides with degree of polymerization 2-6. GH20 β-N-acetylhexosaminidases generally prefer chitobiose among natural substrates. However, LeHex20A had the highest catalytic efficiency (kcat/Km) for chitotetraose, and the Km values for GlcNAc6 were 3.9-fold lower than for chitobiose. Furthermore, the enzyme partially hydrolyzed amorphous chitin polymers. These results indicate that LeHex20A can produce N-acetylglucosamine from long-chain chitomaterials.
    AMB Express. 06/2012; 2(1):29.
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    Food Quality, 04/2012; , ISBN: 978-953-51-0560-2
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    Yuichi Sakamoto, Keiko Nakade, Naotake Konno
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    ABSTRACT: The cell wall of the fruiting body of the mushroom Lentinula edodes is degraded after harvesting by enzymes such as β-1,3-glucanase. In this study, a novel endo-type β-1,3-glucanase, GLU1, was purified from L. edodes fruiting bodies after harvesting. The gene encoding it, glu1, was isolated by rapid amplification of cDNA ends (RACE)-PCR using primers designed from the N-terminal amino acid sequence of GLU1. The putative amino acid sequence of the mature protein contained 247 amino acid residues with a molecular mass of 26 kDa and a pI of 3.87, and recombinant GLU1 expressed in Pichia pastoris exhibited β-1,3-glucanase activity. GLU1 catalyzed depolymerization of glucans composed of β-1,3-linked main chains, and reaction product analysis by thin-layer chromatography (TLC) clearly indicated that the enzyme had an endolytic mode. However, the amino acid sequence of GLU1 showed no significant similarity to known glycoside hydrolases. GLU1 has similarity to several hypothetical proteins in fungi, and GLU1 and highly similar proteins should be classified as a novel glycoside hydrolase family (GH128).
    Applied and Environmental Microbiology 09/2011; 77(23):8350-4. · 3.95 Impact Factor
  • Naotake Konno, Yuichi Sakamoto
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    ABSTRACT: A β-1,6-glucanase, LePus30A, was purified and cloned from fruiting bodies of the basidiomycete Lentinula edodes. β-1,6-glucanases degrade β-1,6-glucan polysaccharides, a unique and essential component of fungal cell walls. The complementary DNA of LePus30A includes an open reading frame of 1,575 bp encoding an 18 amino acid signal peptide and the 506 amino acid mature protein. Sequence analysis indicated that LePus30A is a member of glycoside hydrolase family 30, and highly similar genes are broadly conserved among basidiomycetes. The purified LePus30A catalyzed depolymerization of β-1,6-glucan endolytically and was highly specific toward β-1,6-glucan polysaccharide. It is known that the cell walls of fruiting bodies of basidiomycetes are autodegraded after harvesting by means of enzymatic hydrolysis. The transcript level of LePus30A gene (lepus30a) was significantly increased in fruiting bodies after harvesting. Moreover, LePus30A showed hydrolyzing activity against the cell wall components of L. edodes fruiting bodies. These results suggest that LePus30A is responsible for the degradation of the cell wall components during fruiting body autolysis after harvest.
    Applied Microbiology and Biotechnology 04/2011; 91(5):1365-73. · 3.81 Impact Factor