Selection of Strain and Optimization of Mutanase Production in Submerged Cultures of Trichoderma harzianum

Abstract

Nineteen fungal strains belonging to different genera were tested for extracellular mutanase production in shaken flasks. The optimal enzymatic activity was achieved by Trichoderma harzianum F-470, a strain for which the mutanase productivity has not yet been published. Some of factors affecting the enzyme production in shaken flasks and aerated fermenter cultures have been standardized. Mandels mineral medium with initial pH 5.3, containing 0.25% mutan and inoculated with 10% of the 48-h mycelium, was the best for enzyme production. A slight mutanolytic activity was also found when sucrose, raffinose, lactose and melibiose were carbon sources. Application of optimized medium and cultural conditions, as well as use of a fermenter with automatic pH control set at pH 6.0 enabled to obtain a high mutanase yield (0.33 U/ml, 2.5 U/mg protein) in a short time (2–3 days). The enzyme in crude state was stable over a pH range of 4.5–6.0, and at temperatures up to 35 °C; its maximum activity was at 40 °C and at pH 5.5.

References

  1. 1.

    Chung, J. (1998) Characterization of mutanase produced by Microbispora rosea. J. Dent. Res. 77, 1322.

    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. 124, 1489–1501.

    CAS  PubMed  PubMed Central  Google Scholar 

  3. 3.

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

    CAS  Article  Google Scholar 

  4. 4.

    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. Agr. Biol. Chem. 39, 1391–1400.

    CAS  Google Scholar 

  5. 5.

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

    CAS  PubMed  Google Scholar 

  6. 6.

    Lamont, R. J., Jenkinson, H. F. (2000) Adhesion as an ecological determinant in the oral cavity. In: Kuramitsu, H. K., Ellen, R. P. (eds) Oral Bacterial Ecology: The Molecular Basis. Horizon Scientific Press, Wymondham, Norfolk, pp. 131–168.

    Google Scholar 

  7. 7.

    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 

  8. 8.

    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 

  9. 9.

    Monchois, V., Willemot, R. M., Monsan, P. (1999) Glucansucrases: mechanism of action and structure-function relationships. FEMS Microbiol. Rev. 23, 131–151.

    CAS  Article  Google Scholar 

  10. 10.

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

    CAS  Google Scholar 

  11. 11.

    Oppermann, R. V. (1979) Effect of chlorhexidine on acidogenicity of dental plaque in vivo. Scand. J. Dent. Res. 87, 302–308.

    CAS  PubMed  Google Scholar 

  12. 12.

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

    CAS  Article  Google Scholar 

  13. 13.

    Saunders, P. R., Siu, R. G. H., Genest, R. N. (1948) A cellulolytic enzyme preparation from Myrothecium verrucaria. J. Biol. Chem. 174, 697–703.

    CAS  PubMed  Google Scholar 

  14. 14.

    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 

  15. 15.

    Shukla, G. L., Madhu, Prabhu, K. A. (1989) Study of some parameters for the production of dex-tranase by Penicillium aculeatum. Enzyme Microb. Technol. 11, 533–536.

    CAS  Article  Google Scholar 

  16. 16.

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

    CAS  Google Scholar 

  17. 17.

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

    CAS  Article  Google Scholar 

  18. 18.

    Szczodrak, J., Trojanowski, J., Ilczuk, Z., Ginalska, G. (1982) Cellulolytic activity of moulds. I. Characteristics of the cellulases complex and xylanase of the strain Aspergillus terreus F-413. Acta Microbiol. Polon. 31, 257–270.

    CAS  Google Scholar 

  19. 19.

    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 

  20. 20.

    Wiater, A., Choma, A., Szczodrak, J. (1999) Insoluble glucans synthesized by cariogenic streptococci: a structural study. J. Basic Microbiol. 39, 265–273.

    CAS  Article  Google Scholar 

  21. 21.

    Yamashita, Y., Bowen, W. H., Burne, R. A., Kuramitsu, H. K. (1993) Role of the Streptococcus mutans gtf genes in caries induction in the specific-pathogen-free rat model. Infect. Immun. 61, 3811–3817.

    CAS  PubMed  PubMed Central  Google Scholar 

Download references

Author information

Affiliations

Authors

Corresponding author

Correspondence to J. Szczodrak.

Rights and permissions

This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made.

Reprints and Permissions

About this article

Cite this article

Wiater, A., Szczodrak, J. Selection of Strain and Optimization of Mutanase Production in Submerged Cultures of Trichoderma harzianum. BIOLOGIA FUTURA 53, 389–401 (2002). https://doi.org/10.1556/ABiol.53.2002.3.15

Download citation

Keywords

  • Trichoderma harzianum
  • mutanase production
  • shaken flask cultures
  • fermenter cultures
  • optimization