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2013 | 60 | 1 | 123-128
Article title

Purification and properties of an α-(1 → 3)-glucanase (EC from Trichoderma harzianum and its use for reduction of artificial dental plaque accumulation

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Extracellular α-(1 → 3)-glucanase (mutanase, EC produced by Trichoderma harzianum CCM F-340 was purified to homogeneity by ultrafiltration followed by ion exchange and hydrophobic interaction chromatography, and final chromatofocusing. The enzyme was recovered with an 18.4-fold increase in specific activity and a yield of 4.3%. Some properties of the α-(1 → 3)-glucanase were investigated. The molecular mass of the enzyme is 67 kDa, as estimated by SDS/PAGE, its isoelectric point 7.1, and the carbohydrate content 3%. The pH and temperature optima are 5.5 and 45°C, respectively. The enzyme is stable over a pH range of 4.5-6.0 and up to 45°C for 1 h. The Km and Vmax under standard assay conditions are 0.73 mg/ml and 11.39 x 10-2 µmol/min/mg protein, respectively. The enzyme activity is stimulated by addition of Mg2+ and Na+, and significantly inhibited by Hg2+. The α-(1 → 3)-glucanase preparation preferentially catalyzed the hydrolysis of various streptococcal mutans and fungal α-(1 → 3)-glucans. The 20-residue N-terminal sequence of the enzyme is identical with those of other α-(1 → 3)-glucanases from the genus Trichoderma, and highly similar to those from other fungi. The purified α-(1 → 3)-glucanase was effective in preventing artificial dental plaque formation. The easy purification from fermentation broth and high stability, and the effective inhibition of oral biofilm accumulation make this α-(1 → 3)-glucanase highly useful for industrial and medical application.
Physical description
  • Department of Industrial Microbiology, Faculty of Biology and Biotechnology, Maria Curie-Skłodowska University, Lublin, Poland
  • Department of Industrial Microbiology, Faculty of Biology and Biotechnology, Maria Curie-Skłodowska University, Lublin, Poland
  • Department of Biochemistry, Faculty of Biology and Biotechnology, Maria Curie-Skłodowska University, Lublin, Poland
  • Department of Biochemistry, Faculty of Biology and Biotechnology, Maria Curie-Skłodowska University, Lublin, Poland
  • Department of Industrial Microbiology, Faculty of Biology and Biotechnology, Maria Curie-Skłodowska University, Lublin, Poland
  • Ait-Lahsen H, Soler A, Rey M, de la Cruz J, Monte E, Llobell A (2001) An antifungal exo-α(1 → 3)-glucanase (AGN13.1) from the biocontrol fungus T. harzianum. Appl Environ Microbiol 67: 5833-5839.
  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-54.
  • Calo L, García I, Gotor C, Romero LC (2006) Leaf hairs influence phytopathogenic fungus infection and confer an increased resistance when expressing a Trichoderma α-1,3-glucanase. J Exp Bot 57: 3911-3920.
  • Coenye T, Nelis HJ (2010) In vitro and in vivo model systems to study microbial biofilm formation. J Microbiol Methods 83: 89-105.
  • Dubois M, Gilles KA, Hamilton JK, Rebers PA, Smith F (1956) Colorimetric method for determination of sugar and related substances. Anal Chem 28: 350-356.
  • Fuglsang CC, Berka RM, Wahleitner JA, Kauppinen S, Shuster JR, Rasmussen G, Halkier T, Dalbøge H, Henrissat B (2000) Biochemical analysis of recombinant fungal mutanases. J Biol Chem 275: 2009-2018.
  • Guggenheim B, Haller R (1972) Purification and properties of an a-(1 → 3)-glucanohydrolase from Trichoderma harzianum. J Dent Res 51: 394-402.
  • Hasegawa S, Nordin JH, Kirkwood S (1969) Enzymes that hydrolyze fungal cell wall polysaccharides. I. Purification and properties of an endo-a-D-(1-3)-glucanase from Trichoderma viride. J Biol Chem 244: 5460-5470.
  • Kiho T, Yoshida I, Katsuragawa M, Sakushima M, Usui S, Ukai S (1994) Polysaccharides in fungi XXXIV: A polysaccharide from fruiting bodies of Amanita muscaria and the antitumor activities of its carboxymethylated product. Biol Pharm Bull 17: 1460-1462.
  • Kopec LK, Vacca-Smith AM, Bowen WH (1997) Structural aspects of glucans formed in solution and on the surface of hydroxyapatite. Glycobiology 7: 929-934.
  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685.
  • Ledder RG, Madhwani T, Sreenivasan PK, De Vizio W, McBain AJ (2009) An in vitro evaluation of hydrolytic enzymes as dental plaque control agents. J Med Microbiol 58: 482-491.
  • Mandels M, Parrish FW, Reese ET (1962) Sophorose as an inducer of cellulase in Trichoderma viride. J Bacteriol 83: 400-408.
  • Nelson N (1944) A photometric adaptation of the Somogyi method for the determination of glucose. J Biol Chem 153: 375-380.
  • Pleszczyńska M, Wiater A, Jodkowska E, Rusyan E, Dubielecka M, Małkiewicz K, Paduch R, Walasik K, Szczodrak J (2011) The effects of microbial mutanases on dental plaque aggregation and periodontal parameters. Pol J Environ Stud 20: 91-96.
  • Pleszczyńska M, Wiater A, Skowronek M, Szczodrak J (2012) Purification and characterization of mutanase produced by Paenibacillus curdlanolyticus MP-1. Prep Biochem Biotech 42: 335-347.
  • Sanz L, Montero M, Redondo J, Llobell A, Monte E (2005) Expression of an α-1,3-glucanase during mycoparasitic interaction of Trichoderma asperellum. FEBS J 272: 493-499.
  • Somogyi M (1945) A new reagent for the determination of sugars. J Biol Chem 160: 61-68.
  • Takehara T, Inoue M, Morioka T, Yokogawa K (1981) Purification and properties of endo-α-1,3-glucanase from a Streptomyces chartreusis strain. J Bacteriol 145: 729-735.
  • Wiater A, Janczarek M, Pleszczyńska M, Szczodrak J (2011) Identification and characterization of the Trichoderma harzianum gene encoding α-1,3-glucanase involved in streptococcal mutan degradation. Pol J Microbiol 60: 293-301.
  • Wiater A, Pleszczyńska M, Próchniak K, Szczodrak J (2012) Structural diversity of streptococcal mutans synthesized under different culture and environmental conditions and its effect on mutanase synthesis. Molecules 17: 11800-11815.
  • Wiater A, Szczodrak J, Rogalski J (2001) Purification and characterization of an extracellular mutanase from Trichoderma harzianum. Mycol Res 105: 1357-1363.
  • Wiater A, Szczodrak J, Rogalski J (2004) Hydrolysis of mutan and prevention of its formation in streptococcal films by fungal α-D-glucanases. Process Biochem 39: 1481-1489.
  • Wiater A, Szczodrak J, Pleszczyńska M (2005) Optimization of conditions for the efficient production of mutan in streptococcal cultures and post-culture liquids. Acta Biol Hung 56: 137-150.
  • Wiater A, Szczodrak J, Pleszczyńska M (2008) Mutanase induction in Trichoderma harzianum by cell wall of Laetiporus sulphureus and its application for mutan removal from oral biofilms. J Microbiol Biotechnol 18: 1335-1341.
  • Yamashita Y, Bowen WH, Burne RA, Kuramitsu HK (1993) Role of the Streptococcus mutans gtf genes in caries induction in the specific-pathogen-free rat model. Infect Immun 61: 3811-3817.
  • Zonneveld BJM (1972) A new type of enzyme, an exo-splitting a-1,3 glucanase from non-induced cultures of Aspergillus nidulans. Biochim Biophys Acta 258: 541-547.
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