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Current Genetics

, 48:195 | Cite as

Characterization of the Lentinula edodes exg2 gene encoding a lentinan-degrading exo-β-1,3-glucanase

  • Yuichi SakamotoEmail author
  • Ken-ichiro Minato
  • Masaru Nagai
  • Masashi Mizuno
  • Toshitsugu Sato
Research Article

Abstract

Lentinan, an antitumor substance purified from Lentinula edodes, is degraded during post-harvest preservation as a result of increased glucanase activity. We isolated an exo-β-1,3-glucanase encoding gene, exg2, from L. edodes which is a homologue of an exo-glucanase-encoding gene conserved in ascomycetous fungi. The exg2 gene was cloned as an approximately 2.4-kbp cDNA, and as a genomic sequence of 3.9-kbp. The product of the exg2 gene is predicted to contain 759 amino acids with a molecular weight of 79 kDa and a pI value of 4.6. The putative N-terminus of EXG2 is identical to the N-terminal sequences of lentinan-degrading enzymes, GNase I and II, and a custom-made anti-EXG2 peptide anti-serum cross-reacted with purified GNase I and II. Transcription and translation of exg2 was low in the gills of mature fruiting bodies, but increased after harvesting. We conclude that the exg2 gene is a lentinan-degrading enzyme-encoding-gene in L. edodes.

Keywords

Cell wall lysis Exo-glucanase Fruiting body differentiation Lentinan degradation Lentinula edodes 

References

  1. Balance FJ (1990) Transformation systems for filamentous fungi and an overview of fungal gene structure. In: Leong SA, Berka RM (eds) Molecular industrial mycology. Marcel Dekkerm, New York, pp 1–29Google Scholar
  2. Chambers RS, Broughton MJ, Cannon RD, Carne A, Emerson GW, Sullivan PA (1993) An exo-β-(1,3)-glucanase of Candida albicans: purification of the enzyme and molecular cloning of the gene. J Gen Microbiol 139:325–334PubMedGoogle Scholar
  3. Chihara G, Maeda Y, Hamuro J, Sasaki T, Fukuoka F (1969) Inhibition of mouse sarcoma 180 by polysaccharides from Lentinus edodes (Berk.) Sing. Nature 222:687–688PubMedCrossRefGoogle Scholar
  4. Cohen-Kupiec R, Broglie KE, Friesem D, Broglie RM, Chet I (1999) Molecular characterization of a novel β-1,3-exo-glucanase related to mycoparasitism of Trichoderma harzianum. Gene 226:147–154CrossRefPubMedGoogle Scholar
  5. Correa J, Vazquez de Aldana CR, Segundo PS, del Rey F (1992) Genetic mapping of 1,3-β-glucanase-encoding genes in Saccharomyces cerevisiae. Curr Genet 22:283–288CrossRefPubMedGoogle Scholar
  6. Donzelli BGG, Lorito M, Scala F, Harman GE (2001) Cloning, sequence and structure of a gene encoding an antifungal glucan 1,3-β-glucosidase from Trichoderma atroviride (T. harziamum). Gene 277:199–208CrossRefPubMedGoogle Scholar
  7. Eastwood DC, Kingsnorth CS, Jones HE, Burton KS (2001) Genes with increased transcript levels following harvest of the sporophore of Agarcus bisporus have multiple physiological roles. Mycol Res 105:1223–1230Google Scholar
  8. Giczey G, Kerényi Z, Fülöp L, Hornok L (2001) Expression of cmg1, an exo-β-1,3-glucanase gene from Coniothyrium minitans, increses during sclerotial parasitism Appl Environ Microbiol 67: 865–871PubMedCrossRefGoogle Scholar
  9. Gurr SJ, Unkles SEU, Kinghorn JR (1987) The structure and organization of nuclear genes of filamentous fungi. In: Kinghorn JR (eds) Gene structure in eukaryotic microbes. IRL Press, London, pp 93–139Google Scholar
  10. Henrissat B (1991) A classification of glycosyl hydrolases based on amino acid sequence similarities. Biochem J 280:309–316PubMedGoogle Scholar
  11. Hirano T, Sato T, Okawa K, Kanda K, Yaegashi K, Enei H (1999) Isolation and characterization of the glyceraldehyde-3-phosphate dehydrogenase gene of Lentinus edodes. Biosci Biotechnol Biochem 63:1223–1227CrossRefPubMedGoogle Scholar
  12. Kanda K, Sato T, Ishii S, Enei H, Ejiri S (1996) Purification and properties of tyrosinase isozymes from the gill of Lentinus edodes fruiting body. Biosci Biotechnol Biochem 60: 1273–1278PubMedCrossRefGoogle Scholar
  13. Kuranda MJ, Robbins RW (1987) Cloning and heterologous expression of glycosidase genes from Saccharomyces cerevisiae. Proc Natl Acad Sci USA 84:2585–2589PubMedCrossRefGoogle Scholar
  14. Kües U (2000) Life history and developmental processes in the basidiomycete Coprinus cinereus. Microbiol Mol Biol Rev 64:316–353CrossRefPubMedGoogle Scholar
  15. Larriba G, Andaluz E, Cueva R, Basco RD (1995) Molecular biology of yeast exoglucanases. FEMS Microbiol Lett 125:121–126CrossRefPubMedGoogle Scholar
  16. Minamide T, Habu T, Ogata K (1980a) Effect of storage temperature on keeping freshness of mushrooms after harvest. J Jpn Soc Food Sci Technol 27:281–287Google Scholar
  17. Minamide T, Tsuruta M, Ogata K (1980b) Studies on keeping freshness of Shii-take mushroom (Lentinus edodes (Bark) Sing.) after harvest. J Jpn Soc Food Sci Technol 27:498–504Google Scholar
  18. Minato K, Mizuno M, Terai H, Tsuchida H (1999) Autolysis of lentinan, an antitumor polysaccharide, during storage of Lentinus edodes, Shiitake mushroom. J Agric Food Chem 47:1530–1532CrossRefPubMedGoogle Scholar
  19. Minato K, Kawakami S, Nomura K, Tsuchida H, Mizuno M (2004) An exo β-1,3-glucanase synthesized de novo degrades lentinan during storage of Lentinule edodes and diminishes immunomodulating activity of the mushroom. Carbohydr Polym 56:279–286CrossRefGoogle Scholar
  20. Morikawa K, Takeda R, Yamazaki M, Mizuno D (1985) Induction of tumoricidal activity of polymorphonuclear leukocytes by a linear β-1,3-d-glucan and other immunomodulators in murine cells. Cancer Res 45:1496–1501PubMedGoogle Scholar
  21. Muthukumar G, Suhng S-H, Magee PT, Jewell RD, Primerano DA (1993) The Saccharomyces cerevisiae SPR1 gene encodes a spolulation-specific exo-1,3-β-glucanase which contributes to ascospore thermoresistance. J Bacteriol 175:386–394PubMedGoogle Scholar
  22. Nagai M, Kawata M, Watanabe H, Ogawa M, Saito K, Takesawa T, Kanda K, Sato T (2003) Important role of fungal intracellular laccase for melanin synthesis: purification and characterization of an intracellular laccase from Lentinula edodes fruit bodies. Microbiol 149:2455–2462CrossRefGoogle Scholar
  23. Nelson N (1944) A photometric adaptation of the somogyi method for the determination of glucose. J Biol Chem 153:375–380Google Scholar
  24. Nikolskaya AN, Pitkin JW, Schaeffer HJ, Ahn J-H, Walton JD (1998) EXG1p, a novel exo-β-1,3-glucanase from the fungus Cochliobolus carbonum, contains a repeated motif present in other proteins that interact with polysaccharides. Biochem Biophys Acta 1425:632–636PubMedGoogle Scholar
  25. Oda K, Kasahara S, Yamagata Y, Abe K, Nakajima T (2002) Cloning and expression of the exo-β-d-1,3-glucanse gene (exgS) from Aspergillus saitoi. Biosci Biotechnol Biochem 66:1587–1590CrossRefPubMedGoogle Scholar
  26. Rotem Y, Yarden O, Sztejnberg A (1999) The mycoparasite Ampelomyces quisqualis expresses exgA encoding and exo-β-1,3-glucanase in culture and during mycoparasitism. Phytopathol 89:631–638CrossRefGoogle Scholar
  27. Sakamoto Y, Irie T, Sato T (2005) Isolation and characterization of a fruiting body specific exo-β-1,3-glucanase-encoding gene, exg1, from Lentinula edodes. Curr Genet 47:244–252CrossRefPubMedGoogle Scholar
  28. Schaeffer HJ, Leykam J, Walton JD (1994) Cloning and targeted gene disruption of EXG1, encoding exo-β-1,3-glucanase, in the phytopathogenic fungus Cochliobolus carbonum. Appl Environ Microbiol 60:594–598PubMedGoogle Scholar
  29. Shida M, Ushioda Y, Nakajima T, Matsuda K (1981) Structure of the alkali-insoluble skeletal glucan of Lentinus edodes. J Biochem 90:1093–1100PubMedGoogle Scholar
  30. van de Rhee MD, Mendes Werten OMW, Huizing HJ, and Mooibroek H (1996) Highly efficient homologous integration via tandem exo-β-1,3-glucanase genes in the common mushroom, Agaricus bisporus. Curr Genet 30:166–173CrossRefPubMedGoogle Scholar

Copyright information

© Springer-Verlag 2005

Authors and Affiliations

  • Yuichi Sakamoto
    • 1
    Email author
  • Ken-ichiro Minato
    • 2
  • Masaru Nagai
    • 1
    • 4
  • Masashi Mizuno
    • 3
  • Toshitsugu Sato
    • 1
  1. 1.Iwate Biotechnology Research CenterKitakami-shiJapan
  2. 2.Miyagi Agriculture CollegeTaihaku, SendaiJapan
  3. 3.Faculty of Agriculture, Kobe UniversityNada-kuJapan
  4. 4.Institute for Environmental ScienceObuchi, Rokkasho-mura, Kamikita-gunJapan

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