Purification and characterization of a N-acetylglucosaminidase produced by Talaromyces emersonii during growth on algal fucoidan

  • Elaine O’Connell
  • Patrick Murray
  • Charles Piggott
  • Franck Hennequart
  • Maria Tuohy
Article

Abstract

A β-N-acetylglucosaminidase produced by a novel fungal source, the moderately thermophilic aerobic ascomycete Talaromyces emersonii, was purified to apparent homogeneity. Submerged fermentation of T. emersonii, in liquid medium containing algal fucoidan as the main carbon source, yielded significant amounts of extracellular N-acetylglucosaminidase activity. The N-acetylglucosaminidase present in the culture-supernatant was purified by hydrophobic interaction chromatography and preparative electrophoresis. The enzyme is a dimer with molecular weight and pI values of 140 and 3.85, respectively. Substrate specificity studies confirmed the glycan specificity of the enzyme for N-acetylglucosamine. Michaelis-Menten kinetics were observed during enzyme-catalyzed hydrolysis of the fluorescent substrate methylumbelliferyl-β-D-N-acetylglucosaminide at 50°C, pH 5.0 (Km value of 0.5 mM). The purified N-acetylglucosaminidase displayed activity over broad ranges of pH and temperature, yielding respective optimum values of pH 5.0 and 75°C. The T. emersonii enzyme was less susceptible to inhibition by N-acetylglucosamine and other related sugars than orthologs from other sources. The enzyme was sensitive to Hg2+, Co2+ and Fe3+.

Keywords

β-D-N-acetylglucosaminidase Fucoidan Algae Fungi Aspergillus niger Schizophyllum commune 

Abbreviations

4-MU-GlcNAc

Methylumbelliferyl-N-acetylglucosaminide

References

  1. Berteau O, Mulloy B (2003) Sulfated fucans, fresh perspectives: structures, functions, and biological properties of sulfated fucans and an overview of enzymes active toward this class of polysaccharide. Glycobiology 13:29R–40RPubMedCrossRefGoogle Scholar
  2. Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254PubMedCrossRefGoogle Scholar
  3. Carvalho M, Harada L, Gidlund M, Ketelhuth D, Boschcov P, Quintão E (2002) Macrophages take up triacylglycerol-rich emulsions at a faster rate upon co-incubation with native and modified LDL: An investigation on the role of natural chylomicrons in atherosclerosis. J Cell Biochem 84:309–323PubMedCrossRefGoogle Scholar
  4. Chizov A, Dell A, Morris H, Haslam S, McDowell R, Shashkov A, Nifant’ev N, Khatuntseva E, Usov A (1999) A study of fucoidan from the brown seaweed Chorda filum. Carbohydr Res 320:108–119CrossRefGoogle Scholar
  5. Elving P, Markowitz J, Rosenthal I (1956) Preparation of buffer systems of constant ionic strength. Anal Chem 28:1179–1180CrossRefGoogle Scholar
  6. Furukawa S, Fujikawa T, Koga D, Ide A (1992) Purification and some properties of exo-type fucoidanases from Vibrio sp. N-5. Biosci Biotech Biochem 56:1829–1834Google Scholar
  7. Gilleran CT (2004) An Environmentally-Benign Biotechnological Strategy to Utilize Carbohydrate Rich Waste. PhD Dissertation, National University of Ireland, GalwayGoogle Scholar
  8. Jones C, Kosman D (1980) Purification, properties, kinetics, and mechanism of β-N-acetylglucosamidase from Aspergillus niger. J Biol Chem 255:11861–11869PubMedGoogle Scholar
  9. Kitamura K, Matsuo M, Yasui T (1992) Enzymatic degradation of fucoidan by fucoidanase from the hepatopancreas of Patinopecten yessoensis. Biosci Biotech Biochem 56:490–494Google Scholar
  10. Kloareg B, Quatrano RS (1988) Structure of the cell-walls of marine-algae and ecophysiological functions of the matrix polysaccharides. Oceanogr Mar Biol 26:259–315Google Scholar
  11. Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  12. Lisboa De Marco J, Valadares-Inglis M (2004) Purification and characterization of an N-acetylglucosaminidase produced by a Trichoderma harzianum strain which controls Crinipellis pernicosa. Appl Microbiol Biotechnol 64:70–75PubMedCrossRefGoogle Scholar
  13. Lydon A (2005) Molecular characterization of the gene encoding a thermostable β-galactosidase from Talaromyces emersonii. PhD Dissertation, National University of Ireland, GalwayGoogle Scholar
  14. Lynn K (1990) Chitinase and chitobiases from the American lobster (Homarus americanus). Compar Biochem Physiol 96B:761–766CrossRefGoogle Scholar
  15. Maruyama H, Tamauchi H, Iizuka M, Nakano T (2006) The role of NK cells in antitumor activity of dietary fucoidan from Undaria pinnatifida sporophylls (Mekabu). Planta Med 15:1415–1417CrossRefGoogle Scholar
  16. Maruyama H, Tanaka M, Hashimoto M, Inoue M, Sasahara T (2007) The suppressive effect of Mekabu fucoidan on an attachment of Cryptosporidium parvum oocysts to the intestinal epithelial cells in neonatal mice. Life Sci 80:775–781PubMedCrossRefGoogle Scholar
  17. Matsubara K, Xue C, Zhao X, Mori M, Sugawara T, Hirata T (2005) Effects of middle molecular weight fucoidans on in vitro and ex vivo angiogenesis of endothelial cells. Int J Mol Med 15:695–699PubMedGoogle Scholar
  18. Miller GL (1959) Use of dinitrosalicylate acid reagent for the determination of reducing sugar. Anal Chem 31:426–428CrossRefGoogle Scholar
  19. Moloney A, Hackett T, Considine P, Coughlan M (1983) Isolation of mutants of Talaromyces emersonii CBS 814.70 with enhanced cellulose activity. Enzyme Microbial Technol 5:260–264CrossRefGoogle Scholar
  20. Murray P, Grassick A, Laffey C, Cuffe M, Higgins T, Savage A, Planas A, Tuohy M (2001) Isolation and characterization of a thermostable endo-β-glucanase activity on 1,3–1,4– β-D-glucans from the aerobic fungus Talaromyces emersonii CBS 814.70. Enzyme Microb Tech 29:90–98CrossRefGoogle Scholar
  21. Nagumo T, Nishino T (1996) Fucan sulfates and their anticoagulant activities. In: Dumitriu S (ed) Polysaccharides in Medicinal Applications. Maecel Dekker, New YorkGoogle Scholar
  22. Nishino T, Nishioka C, Ura H, Nagumo T (1994) Isolation and partial characterization of a novel amino sugar-containing fucan sulfate from commercial Fucus vesiculosus fucoidan. Carbohydr Res 225:213–224CrossRefGoogle Scholar
  23. Pomin V, Pereira M, Valente A, Tollefsen M, Pavao M, Mourao P (2005) Selective cleavage and anticoagulant activity of a sulfated fucan: stereospecific removal of a 2-sulfate ester from the polysaccharide by mild acid hydrolysis, preparation of oligosaccharides, and heparin cofactor II-dependent anticoagulant activity. Glycobiology 15:369–381PubMedCrossRefGoogle Scholar
  24. Ponce NM, Pujol CA, Damonte EB, Flores ML, Stortz CA (2003) Fucoidans from the brown seaweed Adenocystis utricularis: extraction methods, antiviral activity and structural studies. Carbohydr Res 338:153–165PubMedCrossRefGoogle Scholar
  25. Senni K, Gueniche F, Foucault-Bertaud A, Igondjo-Tchen S, Fioretti F, Colliec-Jouault S, Durand P, Guezennec J, Godeau G, Letourneur D (2006) Fucoidan a sulfated polysaccharide from brown algae is a potent modulator of connective tissue proteolysis. Arch Biochem Biophys 445:56–64PubMedCrossRefGoogle Scholar
  26. Thompson R (2004) Biochemical and structural studies on glycosyl hydrolases from a thermophilic eukaryote. PhD Dissertation, National University of Ireland, GalwayGoogle Scholar
  27. Tuohy M, Coughlan M (1992) Production of thermostable xylan-degrading enzymes by Talaromyces emersonii. Biores Technol 39:131–137CrossRefGoogle Scholar
  28. Tuohy M, Walsh D, Murray P, Claeyssens M, Cuffe M, Savage A, Coughlan M (2002) Kinetic parameters and mode of action of the cellobiohydrolases produced by Talaromyces emersonii. Biochim Biophys Acta 1596:336–380Google Scholar
  29. Ulhoa C, Peberdy J (1991) Purification and characterization of an extracellular chitobiase from Trichoderma harzianum. Cur Microbiol 23:285–289CrossRefGoogle Scholar
  30. Xue C-H, Fang Y, Lin H, Chen L, Li Z-J, Deng D, Lu C-X (2001) Chemical characters and antioxidative properties of sulphated polysaccharides from Laminaria japonica. J Appl Phycol 13:67–70CrossRefGoogle Scholar
  31. Yabuki M, Mizushina K, Amatatsu T, Ando A, Fujii T, Shimada M, Yamashida M (1986) Purification and characterization of chitinase and chitobiase produced by Aeromonas hydyrophila subsp. Anaerogenes A52. J Gen Appl Microbiol 32:25–32CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2007

Authors and Affiliations

  • Elaine O’Connell
    • 1
  • Patrick Murray
    • 1
  • Charles Piggott
    • 1
  • Franck Hennequart
    • 1
  • Maria Tuohy
    • 1
  1. 1.Molecular Glycobiotechnology Group, Department of BiochemistryNational University of IrelandGalwayIreland

Personalised recommendations