Enhancement of Mutanase Production in Trichoderma harzianum by Mutagenesis


Conidia of Trichoderma harzianum F-340, an active producer of fungal mutanase, were mutagenized with physical and chemical mutagens used separately or in combination. After mutagenesis, the drop in conidia viability ranged from 0.004% to 71%. Among the applied mutagens, nitrosoguanidine gave the highest frequency of cultures with enhanced mutanase activity (98%). In total, 400 clones were isolated, and preliminarily evaluated for mutanase activity in flask microcultures. Eight most productive mutants were then quantified for mutanase production in shake flask cultures. The obtained results fully confirmed a great propensity of all the tested mutants to synthesize mutanase, the activity of which increased from 59 to 107% in relation to the parental T. harzianum culture. The best mutanase-overproducing mutant (T. harzianum F-340-48), obtained with nitrosoguanidine, produced the enzyme activity of 1.36 U/ml (4.5 U/mg protein) after 4 days of incubation in shake flask culture. This productivity was almost twices higher than that achieved by the initial strain F-340, and, at present, is the best reported in the literature. The potential application of mutanase in dentistry is also discussed.


  1. 1.

    Balasubramanian, N., Juliet, G. A., Srikalaivani, P., Lalithakumari, D. (2003) Release and regeneration of protoplasts from the fungus Trichothecium roseum. Can. J. Microbiol. 49, 263–268.

    CAS  Article  Google Scholar 

  2. 2.

    Ebisu, S., Kato, K., Kotani, S., Misaki, A. (1975) Isolation and purification of Flavobacterium α-1,3- glucanase-hydrolyzing, insoluble, sticky glucan of Streptococcus mutans. J. Bacteriol. 35, 1489–1501.

    Google Scholar 

  3. 3.

    Fiedurek, J., Ilczuk, Z. (1978) Mutagenization of Aspergillus niger with pectinolytic activity under conditions of submerged culture (in Polish). Ann. Univ. Maria Curie-Sklodowska, Sec. C 33, 29–39.

    CAS  Google Scholar 

  4. 4.

    Fuglsang, C. C., Berka, R. M., Wahleithner, J. A., Kauppinen, S., Shuster, J. R., Rasmussen, G., Halkier, T., Dalbøge, H., Henrissat, B. (2000) Biochemical analysis of recombinant fungal mutanases. A new family of ?1,3-glucanases with novel carbohydrate-binding domains. J. Biol. Chem. 275, 2009–2018.

    CAS  Article  Google Scholar 

  5. 5.

    Guggenheim, B., Haller, R. (1972) Purification and properties of an ?-(1?3) glucanohydrolase from Trichoderma harzianum. J. Dent. Res. 51, 394–402.

    CAS  Article  Google Scholar 

  6. 6.

    Gyémánt, G., Kandra, L., Nagy, V., Somsák, L. (2003) Inhibition of human salivary ?-amylase by glucopyranosylidene-spiro-thiohydantoin. Biochem. Biophys. Res. Comm. 312, 334–339.

    Google Scholar 

  7. 7.

    Hare, M. D., Svensson, S., Walker, G. J. (1978) Characterization of extracellular, water-insoluble ?- D-glucans of oral streptococci by methylation analysis, and by enzymic synthesis and degradation. Carbohydr. Res. 66, 245–264.

    CAS  Article  Google Scholar 

  8. 8.

    Ilczuk, Z., Fiedurek, J., Paszczynski, A. (1983) Intensification of amylase synthesis with Aspergillus niger by way of multistage mutagenization. Starch/Stärke 35, 397–400.

    CAS  Article  Google Scholar 

  9. 9.

    Inoue, M., Egami, T., Yokogawa, K., Kotani, H., Morioka, T. (1975) Isolation, identification and some cultural conditions of Streptomyces species that produce water-insoluble polyglucan hydrolase. Agric. Biol. Chem. 39, 1391–1400.

    CAS  Google Scholar 

  10. 10.

    Inoue, M., Yakushiji, T., Mizuno, J., Yamamoto, Y., Tanii, S. (1990) Inhibition of dental plaque formation by mouthwash containing an endo-?-1,3 glucanase. Clin. Prevent. Dent. 12, 10–14.

    CAS  Google Scholar 

  11. 11.

    Kandra, L., Gyémánt, G. (2000) Examination of the active sites of human salivary ?-amylase (HSA). Carbohydr. Res. 329, 579–585.

    CAS  Article  Google Scholar 

  12. 12.

    Kandra, L., Gyémánt, G., Remenyik, J., Ragunath C., Ramasubbu, N. (2003) Subsite mapping of human salivary ?-amylase and the mutant Y151M. FEBS Lett. 544, 194–198.

    CAS  Article  Google Scholar 

  13. 13.

    Kandra, L., Gyémánt, G., Zajácz, A., Batta, G. (2004) Inhibitory effects of tannin on human salivary α-amylase. Biochem. Biophys. Res. Comm. 319, 1265–1271.

    CAS  Article  Google Scholar 

  14. 14.

    Kelstrup, J., Holm-Pedersen, P., Poulsen, S. (1978) Reduction of formation of dental plaque and gingivitis in humans by crude mutanase. Scand. J. Dent. Res. 86, 93–102.

    CAS  PubMed  Google Scholar 

  15. 15.

    Mandels, M., Parrish, F. W., Reese, E. T. (1962) Sophorose as an inducer of cellulase in Trichoderma viride. J. Bacteriol. 83, 400–408.

    CAS  PubMed  PubMed Central  Google Scholar 

  16. 16.

    Martin, K. (1950) Use of acid, rose bengal and streptomycin in the plate method for estimating soil fungi. Soil Sci. 38, 215–220.

    Article  Google Scholar 

  17. 17.

    Matsuda, S., Kawanami, Y., Takeda, H., Ooi, T., Kinoshita, S. (1997) Purification and properties of mutanase from Bacillus circulans. J. Ferment. Bioeng. 83, 593–595.

    CAS  Article  Google Scholar 

  18. 18.

    Nelson, N. (1944) A photometric adaptation of the Somogyi method for the determination of glucose. J. Biol. Chem. 153, 375–380.

    CAS  Google Scholar 

  19. 19.

    Quivey, R. G., Kriger, P. S. (1993) Raffinose-induced mutanase production from Trichoderma harzianum. FEMS Microbiol. Lett. 112, 307–312.

    CAS  Article  Google Scholar 

  20. 20.

    Schacterle, G. R., Pollack, R. L. (1973) A simplified method for the quantitative assay of small amounts of protein in biologic material. Anal. Biochem. 51, 654–655.

    CAS  Article  Google Scholar 

  21. 21.

    Somogyi, M. (1945) A new reagent for the determination of sugars. J. Biol. Chem. 160, 61–68.

    CAS  Google Scholar 

  22. 22.

    Stephen, E. R., Nasim, A. (1981) Production of protoplasts in different yeasts by mutanase. Can. J. Microbiol. 27, 550–553.

    CAS  Article  Google Scholar 

  23. 23.

    Szczodrak, J., Pleszczynska, M., Fiedurek, J. (1994) Penicillium notatum 1 a new source of dextranase. J. Ind. Microbiol. 13, 315–320.

    CAS  Article  Google Scholar 

  24. 24.

    Szczodrak, J. (1989) The use of cellulases from a β-glucosidase-hyperproducing mutant of Trichoderma reesei in simultaneous saccharification and fermentation of wheat straw. Biotechnol. Bioeng. 33, 1112–1116.

    CAS  Article  Google Scholar 

  25. 25.

    Szekeres, A., Kredics, L., Antal, Z., Kevei, F., Manczinger, L. (2004) Isolation and characterization of protease overproducing mutants of Trichoderma harzianum. FEMS Microbiol. Lett. 233, 215–222.

    CAS  Article  Google Scholar 

  26. 26.

    Tsuchiya, R., Fuglsang, C. C., Johansen, C., Aaslyng, D. (1998) Effect of recombinant mutanase and recombinant dextranase on plaque removal. J. Dent. Res. 77 (Sp. Iss. B), 2713.

    Google Scholar 

  27. 27.

    Wiater, A., Szczodrak, J., Pleszczynska, M., Próchniak, K. (2005) Production and use of mutanase from Trichoderma harzianum for effective degradation of streptococcal mutans. Braz. J. Microbiol. 36, 137–146.

    CAS  Article  Google Scholar 

  28. 28.

    Wiater, A., Szczodrak, J., Rogalski, J. (2001) Purification and characterization of an extracellular mutanase from Trichoderma harzianum. Mycol. Res. 103, 1357–1363.

    Article  Google Scholar 

  29. 29.

    Wiater, A., Szczodrak, J., Rogalski, J. (2004) Hydrolysis of mutant and prevention of its formation in streptococcal films by fungal ?-D-glucanases. Proc. Biochem. 39, 1481–1489.

    CAS  Article  Google Scholar 

  30. 30.

    Zaldivar M., Velásques, J. C., Contreras I., Pérez, L. A. (2001) Trichoderma aueroviride 7-121, a mutant with enhanced production of lytic enzymes: its potential use in waste cellulose degradation and/or biocontrol. E. J. Biotechnol. 4 (3), 160–168.

    Google Scholar 

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This work was financially supported by the BW/BS/BiNoZ/UMCS Research Programs.

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Correspondence to J. Szczodrak.

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Wiater, A., Szczodrak, J. & Pleszczyńska, M. Enhancement of Mutanase Production in Trichoderma harzianum by Mutagenesis. BIOLOGIA FUTURA 57, 123–132 (2006). https://doi.org/10.1556/ABiol.57.2006.1.12

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  • Trichoderma harzianum
  • mutanase
  • mutagenesis
  • mutanase-overproducing mutants
  • shake cultures