The Study of Natural Isolates of Fusarium spp. Micromycetes – Ligninolytic Enzymes Producers

I.V. Darmov^*, E.I. Gorshunova^**, T.S. Tarasovа^***

Vyatka State University, Kirov, 610000 Russia

E-mail: ^*gitikx@mail.ru, ^**ekaterina250188@mail.ru, ^***tatyana.apple@icloud.com

Received December 28, 2016

Full text PDF

Abstract

It is known that many basidiomycetes, white rot agents of wood in particular, produce ligninolytic complex enzymes, the most important of which are laccase, manganese peroxidase, and lignin peroxidase. Using these basidiomycete enzymes, promising methods have been developed for disposal of logging and plant farming wastes, paper stock delignification, cloth bio-bleaching, and production of wooden fibreboards; however, these techniques are not commonly applied on an industrial scale. It should be noted that wood lignin can be also destructed by various micromycetes, such as Fusarium, Trichoderma, Aspergillus, Penicillium, etc. Nevertheless, the range of ligninolytic complex enzymes produced by them, as well as the level of their activity and localization in a cell, have been studied insufficiently. Thus, it is not possible to fully appreciate the ligninolytic potential of micromycetes. The purpose of this research is to evaluate the ability of natural isolates of Fusarium spp. to produce laccase, manganese peroxidase, and lignin peroxidase. Furthermore, the level of activity of these enzymes in the culture liquid and cell mass of producers has been determined.

The following natural isolates of micromycetes have been isolated and identified from natural environments of the Kirov region: F. culmorum, strain 3, F. sporotrichioides, strain 12, and F. solani, strain 52, all capable of degradation of wood lignin and carrying simultaneously genetic determinants of all the three major ligninolytic enzymes – laccase, manganese peroxidase, and lignin peroxidase. We have studied the dynamics of the activity of these enzymes in the culture liquid supernatants at deep micromycete cultivation. The enzyme activity has been determined by the spectrophotometric method. In terms of laccase and manganese peroxidase production, two micromycete strains – F. culmorum 3 and F. sporotrichioides 12 – have been almost as good as the basidiomycete T. versicolor. According to lignin peroxidase production under the same conditions of cultivation, F. sporotrichioides 12 has been 2.7 times better than T. versicolor. Regarding the ability to accumulate biomass in case of deep cultivation on rich and synthetical media, Fusarium has turned out to be 1.5–1.7 times better than T. versicolor. All micromycetes have been shown to secrete laccase, manganese peroxidase, and lignin peroxidase into the culture medium; the enzyme output in culture liquid has been up to 75.5–91.9%. Therefore, it has been demonstrated that Fusarium micromycetes are promising producers of ligninolytic enzymes, further investigation of their biotechnological potential is needed.

Keywords: micromycetes, Fusarium spp., deep cultivation, secretion of ligninolytic enzymes, laccase, manganese peroxidase, lignin peroxidase

Figure Captions

Fig. 1. Dynamics of laccase activity in the culture liquid supernatant of producing strains: 1 – F. culmorum 3; 2 – F. solani 52; 3 – F. sporotrichioides 12; 4 – T. versicolor (control). The Czapek–Dox culture medium with lignin.

Fig. 2. Dynamics of manganese peroxidase activity in the culture liquid supernatant of producing strains: 1 – F. culmorum 3; 2 – F. solani 52; 3 – F. sporotrichioides 12; 4 – T. versicolor (control). The Czapek–Dox culture medium with tannin.

Fig. 3. Dynamics of lignin peroxidase activity in the culture liquid supernatant of producing strains: 1 – F. culmorum 3; 2 – F. solani 52; 3 – F. sporotrichioides 12; 4 – T. versicolor (control). The oat decoction culture medium.

References

1. Rabinovich M.L., Bolobova A.V., Kondrashchenko V.I. Theoretical Foundations of the Biotechnology of Woody Composites. Vol. 1. Wood and Wood-Destroying Fungi. Moscow, Nauka, 2001. 264 p. (In Russian)
2. Fedorova Т.V., Shakhova N.V., Klein О.I., Glazunova O.A., Maloshenok L.G., Kulikova N.A., Psurtseva N.V., Koroleva О.V. Comparative analysis of the ligninolytic potential of basidiomycetes belonging to different taxonomic and ecological groups. Appl. Biochem. Microbiol., 2013, vol. 49, no. 6, pp. 570–580. doi: 10.1134/S0003683813060082.
3. Yaropolov A.I., Skorobogat'ko O.V., Vartanov S.S., Varfolomeyev S.D. Laccase: Properties, catalytic mechanism, and applicability. Appl. Biochem. Biotechnol., 1994, vol. 49, no. 3, pp. 257–280. doi: 10.1007/BF02783061.
4. Karhunen E., Kantelinen A., Niku-Paavola M.-L. Mn-dependent peroxidase from the lignin-degrading white rot fungus Phlebia radiata. Arch. Biochem. Biophys., 1990, vol. 279, no. 1, pp. 25–31. doi: 10.1016/0003-9861(90)90458-B.
5. Sarkanen S., Razal R.A., Piccariello T., Yamamoto E., Lewis N.G. Lignin peroxidase: Toward a clarification of its role in vivo. J. Biol. Chem., 1991, vol. 266, no. 6, pp. 3636–3643.
6. Stepanova E.V., Koroleva О.V., Vasilchenko L.G., Karapetyan K.N., Landesman E.O., Yavmetdinov I.S., Kozlov Yu.P., Rabinovich M.L. Fungal decomposition of oat straw during liquid and solid-state fermentation. Appl. Biochem. Microbiol., 2003, vol. 39, no. 1, pp. 65–74. doi: 10.1023/A:1021702211169.
7. Addleman K., Archibald F. Kraft pulp bleaching and delignification by dikaryons and monokaryons of Trametes versicolor. Appl. Environ. Microbiol., 1993, vol. 59, no. 1, pp. 266–273.
8. Solomatov V.I., Cherkasov V.D., Selyaev V.P., Rusakov V.A., Prytkova T.N., Trokhin V.I. Pressed pulp for preparing woody plates. Patent RF no. 2017770. Byull. Izobret., 1994, no. 15. 5 p. (In Russian)
9. Rusinova T.V. Development of a technology for laccase enzyme biosynthesis by basidiomycetes from the genus Trametes. Extended Abstract of Cand. Eng. Sci. Diss. Moscow, Gos. Nauchno-Issled. Inst. Biosint. Belkovykh Veshchestv, 2007. 20 p. (In Russian)
10. Rabinovich M.L., Bolobova A.V., Vasil'chenko L.G. Fungal decomposition of natural aromatic structures and xenobiotics: A review. Appl. Biochem. Microbiol., 2004, vol. 40, no. 1, pp. 1–17. doi: 10.1023/B:ABIM.0000010343.73266.08.
11. Mokrushina N.S., Tarasova T.S., Darmov I.V. Bioconversion of wood wastes by the method of composting with receiving organic fertilizer. Izv. Samar. Nauchn. Tsentra Ross. Akad. Nauk., 2009, vol. 11, no. 1, pp. 228–232. (In Russian)
12. Mokrushina N.S., Darmov I.V. Penicillium sizovae strain – decomposer of wood lignin. Patent RF no. 2355753. Byull. Izobret., 2009, no. 14. 7 p. (In Russian)
13. Shevchenko E.A., Bessolitsyna E.A., Darmov I.V. Identification of genes encoding ligninolytic enzymes in naturally occurring basidiomycete isolates. Appl. Biochem. Microbiol., 2013, vol. 49, no. 3, pp. 280–286. doi: 10.1134/S0003683813030150.
14. Litvinov M.A. Guide to Microscopic Soil Fungi. Leningrad, Nauka, 1967. 303 p. (In Russian)
15. Sutton D.A., Fothergill A.W., Rinaldi M.G. Guide to Clinically Significant Fungi. Baltimore, Williams and Wilkins, 1998. 471 p.
16. Bilai V.I. (Ed.) Methods of Experimental Mycology. Kiev, Naukova Dumka, 1973. 242 p. (In Russian)
17. Koroleva О.V.., Yavmetdinov I.S., Shleev S.V., Stepanova E.V., Gavrilova V.P. Isolation and study of some properties of laccase from the basidiomycetes Cerrena maxima. Biochemistry, 2001, vol. 66, no. 6, pp. 618–622. doi: 10.1023/A:1010299012591.
18. Paszczyński A., Crawford R.L., Huynh V.-B. Manganese peroxidase of Phanerochaete chrysosporium: Purification. Methods Enzymol., 1988, vol. 161, pp. 264–270. doi: 10.1016/0076-6879(88)61028-7.
19. Nikitina О.V., Shleev S.V., Gorshina E.S., Rusinova T.V., Serezhenkov V.A., Burbaev D.Sh., Belovolova L.V., Yaropolov A.I. Isolation and purification of enzymes from ligninolytic complex of the basidial fungus Trametes pubescens (Schumach.) Pilát and study of their properties. Biochemistry, 2005, vol. 70, no. 11, pp. 1274–1279. doi: 10.1007/s10541-005-0259-0.
20. Pavarina E.C., Durrant L.R. Growth of lignocellulosic-fermenting fungi on different substrates under low oxygenation conditions. Appl. Biochem. Biotechnol., 2002, vols. 98–100, pp. 663–677.
21. Obruca S., Marova I., Matouskova P., Haronikova A., Lichnova A. Production of lignocellulose-degrading enzymes employing Fusarium solani F-552. Folia Microbiol. (Prague), 2012, vol. 57, no. 3, pp. 221–227. doi: 10.1007/s12223-012-0098-5.

For citation: Darmov I.V., Gorshunova E.I., Tarasovа T.S. The study of natural isolates of Fusarium spp. micromycetes – ligninolytic enzymes producers. Uchenye Zapiski Kazanskogo Universiteta. Seriya Estestvennye Nauki, 2017, vol. 159, no. 1, pp. 72–84. (In Russian)

The content is available under the license Creative Commons Attribution 4.0 License.