Applied Microbiology and Biotechnology

, Volume 92, Issue 5, pp 961–969 | Cite as

Identification of potential cell wall component that allows Taka-amylase A adsorption in submerged cultures of Aspergillus oryzae

  • Hiroki Sato
  • Yoshiyuki Toyoshima
  • Takahiro Shintani
  • Katsuya GomiEmail author
Biotechnologically Relevant Enzymes and Proteins


We observed that α-amylase (Taka-amylase A; TAA) activity in the culture broth disappeared in the later stage of submerged cultivation of Aspergillus oryzae. This disappearance was caused by adsorption of TAA onto the cell wall of A. oryzae and not due to protein degradation by extracellular proteolytic enzymes. To determine the cell wall component(s) that allows TAA adsorption efficiently, the cell wall was fractionated by stepwise alkali treatment and enzymatic digestion. Consequently, alkali-insoluble cell wall fractions exhibited high levels of TAA adsorption. In addition, this adsorption capacity was significantly enhanced by treatment of the alkali-insoluble fraction with β-glucanase, which resulted in the concomitant increase in the amount of chitin in the resulting fraction. In contrast, the adsorption capacity was diminished by treating the cell wall fraction with chitinase. These results suggest that the major component that allows TAA adsorption is chitin. However, both the mycelium and the cell wall demonstrated the inability to allow TAA adsorption in the early stage of cultivation, despite chitin content in the cell wall being identical in both early and late stages of cultivation. These results suggest the existence of unidentified factor(s) that could prevent the adsorption of TAA onto the cell wall. Such factor(s) is most likely removed or diminished from the cell wall following longer cultivation periods.


Aspergillus oryzae α-Amylase Cell wall Chitin Submerged culture Protein production 

Supplementary material

253_2011_3422_Fig9_ESM.jpg (85 kb)

(JPEG 84 kb)

253_2011_3422_MOESM1_ESM.tif (6 mb)
High resolution image (TIFF 6184 kb)


  1. Adachi T (1954) The new method of α-amylase assay of sake-koji (Aspergillus oryzae). J Ferment Technol 32:448–451Google Scholar
  2. Archer DB, Mackenzie DA, Jeenes DJ, Roberts IN (1992) Proteolytic degradation of heterologous proteins expressed in Aspergillus niger. Biotechnol Lett 14:357–362CrossRefGoogle Scholar
  3. Archer DB, Jeenes DJ, Mackenzie DA (1994) Strategies for improving heterologous protein production from filamentous fungi. Antonie Van Leeuwenhoek 65:245–250CrossRefGoogle Scholar
  4. Bartnicki-Garcia S (1968) Cell wall chemistry, morphogenesis, and taxonomy of fungi. Annu Rev Microbiol 22:87–108CrossRefGoogle Scholar
  5. Blix G (1948) The determination of hexosamines according to Elson and Morgan. Acta Chem Scand 2:467–473CrossRefGoogle Scholar
  6. Bowman SM, Free SJ (2006) The structure and synthesis of the fungal cell wall. BioEssays 28:799–808CrossRefGoogle Scholar
  7. Brandhorst T, Klein B (2000) Cell wall biogenesis of Blastomyces dermatitidis: evidence for a novel mechanism of cell surface localization of a virulence-associated adhesin via extracellular release and reassociation with cell wall chitin. J Biol Chem 275:7925–7934CrossRefGoogle Scholar
  8. Christensen T, Woeldike H, Boel E, Mortensen SB, Hjortshoej K, Thim L, Hansen MT (1988) High level expression of recombinant genes in Aspergillus oryzae. Bio/Technology 6:1419–1422CrossRefGoogle Scholar
  9. Fontaine TC, Simenel G, Dubreucq O, Adam M, Delepierre J, Lemoine CE, Vorgias M, Diaquin M, Latgé JP (2000) Molecular organization of the alkali-insoluble fraction of Aspergillus fumigatus cell wall. J Biol Chem 275:27594–27607Google Scholar
  10. Hartland RP, Vermeulen CA, Klis FM, Sietsma JH, Wessels JHG (1994) The linkage of (1–3)-β-glucan to chitin during cell wall assembly in Saccharomyces cerevisiae. Yeast 10:1591–1599CrossRefGoogle Scholar
  11. Jin FJ, Watanabe T, Juvvadi PR, Maruyama J, Arioka M, Kitamoto K (2007) Double disruption of the proteinase genes, tppA and pepE, increases the production level of human lysozyme by Aspergillus oryzae. Appl Microbiol Biotechnol 75:1059–1068CrossRefGoogle Scholar
  12. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685CrossRefGoogle Scholar
  13. Latgé JP (2007) The cell wall: a carbohydrate armour for the fungal cell. Mol Microbiol 66:279–290CrossRefGoogle Scholar
  14. Latgé JP, Calderone R (2006) The fungal cell wall. In: Kües U, Fischer R (eds) The Mycota I. Growth, differentiation and sexuality. Springer, Berlin, pp 73–104CrossRefGoogle Scholar
  15. Oda K, Kakizono D, Yamada O, Iefuji H, Akita O, Iwashita K (2006) Proteomic analysis of extracellular proteins from Aspergillus oryzae grown under submerged and solid-state culture conditions. Appl Environ Microbiol 72:3448–3457CrossRefGoogle Scholar
  16. Tonomura K, Tanabe O (1964) Localization of cell-bound α-amylase in Aspergillus oryzae demonstrated by fluorescent-antibody technique. J Bacteriol 87:226–227Google Scholar
  17. Tonomura K, Futai F, Tanabe O (1962) Cell bound α-amylase in Aspergillus oryzae (1) Accumulation of α-amylase within the cell. Agric Biol Chem 26:10–15Google Scholar
  18. Tonomura K, Futai F, Tanabe O (1963) Binding of α-amylase to the cell wall of Aspergillus oryzae. Biochim Biophys Acta 78:802–805CrossRefGoogle Scholar
  19. Tsuchiya K, Tada S, Gomi K, Kitamoto K, Kumagai C, Jigami Y, Tamura G (1992) High level expression of the synthetic human lysozyme gene in Aspergillus oryzae. Appl Microbiol Biotechnol 38:109–114CrossRefGoogle Scholar
  20. Tsuchiya K, Gomi K, Kitamoto K, Kumagai C, Tamura G (1993) Secretion of calf chymosin from the filamentous fungus Aspergillus oryzae. Appl Microbiol Biotechnol 40:327–332CrossRefGoogle Scholar
  21. van den Hombergh JP, van de Vondervoort PJ, Fraissinet-Tachet L, Visser J (1997) Aspergillus as a host for heterologous protein production: the problem of proteases. Trends Biotechnol 15:256–263CrossRefGoogle Scholar
  22. Wessels JGH, Sietsma JH (1981) Fungal cell walls: a survey. In: Tanner W, Loewus FA (eds) Encyclopedia of plant physiology, vol. 13B. Springer, Berlin, pp 352–394Google Scholar
  23. Yabuki M, Fukui S (1970) Presence of binding site for α-amylase and of masking protein for this site on mycelial cell wall of Aspergillus oryzae. J Bacteriol 104:138–144Google Scholar
  24. Yamada O, Lee BR, Gomi K (1997) Transformation system for Aspergillus oryzae with double auxotrophic mutations, niaD and sC. Biosci Biotechnol Biochem 61:1367–1369CrossRefGoogle Scholar
  25. Yoon J, Kimura S, Maruyama J, Kitamoto K (2009) Construction of quintuple protease gene disruptant for heterologous protein production in Aspergillus oryzae. Appl Microbiol Biotechnol 82:691–701CrossRefGoogle Scholar
  26. Yoon J, Maruyama J, Kitamoto K (2011) Disruption of ten protease genes in the filamentous fungus Aspergillus oryzae highly improves production of heterologous proteins. Appl Microbiol Biotechnol 89:747–759CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Hiroki Sato
    • 1
  • Yoshiyuki Toyoshima
    • 1
    • 2
  • Takahiro Shintani
    • 1
  • Katsuya Gomi
    • 1
    Email author
  1. 1.Laboratory of Bioindustrial Genomics, Graduate School of Agricultural ScienceTohoku UniversitySendaiJapan
  2. 2.Yamasa Shoyu Co. Ltd.ChoshiJapan

Personalised recommendations