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Biochemical Characterization of Extracellular Cellulase from Tuber maculatum Mycelium Produced Under Submerged Fermentation

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Interaction of truffle mycelium with the host plant involves the excretion of extracellular enzymes. The ability of Tuber maculatum mycelium to produce an extracellular cellulase during submerged fermentation was demonstrated for the first time. T. maculatum mycelia were isolated and tested for extracellular cellulase production at variable pH on solid agar medium, and the highest activity was observed at pH 7.0. Furthermore, T. maculatum was subjected to submerged fermentation in basal salt medium for cellulase production. Under optimized conditions using sodium carboxymethyl cellulose (0.5 % w/v) as carbon source and an initial pH of 7.0, the enzyme production yielded 1.70 U/mL of cellulase in the cell-free supernatant after 7 days of incubation time. The optimum of the obtained cellulase’s activity was at pH 5.0 and a temperature of 50 °C. The enzyme showed good thermostability at 50 °C by retaining 99 % of its maximal activity over an incubation time of 100 min. The cellulase activity was inhibited by Fe2+ and slightly activated by Mn2+ and Cu2+ at 1 mM concentration. The results indicated that truffle mycelium is utilizing cellulosic energy source in the root system, and the optimal conditions are those existing in the acidic Finnish soil.

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  1. Lynd, L. R., Weimer, P. J., van Zyl, W. H., et al. (2002). Microbial cellulose utilization: fundamentals and biotechnology. Microbiology and Molecular Biology Reviews, 66, 506–577.

    Article  CAS  Google Scholar 

  2. Simair, A. A., Dahot, M. U., & Mangrio, S. M. (2010). Production of xylanase enzyme by Pleurotus eryngii and Flamulina velutipes grown on different carbon sources under submerged fermentation. World Applied Sciences Journal (Special Issue of Biotechnology and Genetic Engineering, 8, 47–49.

    Google Scholar 

  3. Kirk, O., Borchert, T. V., & Fuglsang, C. C. (2002). Industrial enzyme applications. Current Opinion in Biotechnology, 13, 345–51.

  4. Rana, S., & Kaur, M. (2012). Isolation and screening of cellulase producing microorganisms from degraded wood. International Journal of Pharmacy and Biological Sciences Fundamentals, 2, 10–15.

    Google Scholar 

  5. Iotti, M., Rubinib, A., Tisserant, E., Kholer, A., Paoloccib, F., & Zambonellia, A. (2012). Self/nonself recognition in Tuber melanosporum is not mediated by a heterokaryon incompatibility system. Fungal Biol, 261–275.

  6. Cullere, L., Ferreira, V., Chevret, B., Venturini, M. E., & Sanchez-Gimeno, A. C. (2010). Characterization of aroma active compounds in black truffle (Tuber melanosporum) and summer truffle (Tuber aestivum) by gas chromatography olfatometry. Food Chemistry, 122, 300–306.

    Article  CAS  Google Scholar 

  7. Frank, B. (2005). On the nutritional dependence of certain trees on root symbiosis with belowground fungi. Mycorrhiza, 15, 267–275.

    Article  CAS  Google Scholar 

  8. Roth-Bejerano, N., Mendlinger, S., & Kagan-Zur, V. (2004). Effect of calcium on growth of submerged Terfezia boudieri mycelium. Mycoscience, 45, 30–34.

    Article  CAS  Google Scholar 

  9. Ceccaroli, P., Saltarelli, R., Polidori, E., Barbieri, E., Guescini, M., Ciacci, C., & Stocchi, V. (2015). Sugar transporters in the black truffle Tuber melanosporum: from gene prediction to functional characterization. Fungal Genetics and Biology, 81, 52–61.

    Article  CAS  Google Scholar 

  10. Rejon-Palomares, A., Garcia-Garrido, J. M., Ocampo, J. A., & Garcia-Romera, I. (1996). Presence of xyloglucan-hydrolyzing glucanases (xyloglucanases) in arbuscular mycorrhizal symbiosis. Symbiosis, 21, 249–261.

    CAS  Google Scholar 

  11. Garcia-Barreda, S., Molina-Grau, S., & Reyna, S. (2015). Reducing the infectivity and richness of ectomycorrhizal fungi in a calcareous Quercus ilex forest through soil preparations for truffle plantation establishment: A bioassay study. Fungal Biol., 1–7.

  12. Marquez, A. A. T., Mendoza, M. G. D., Gonzalez, M. S. S., et al. (2007). Actividad fibrolíticade enzimas producidas por Trametes sp. EUM1, Pleurotus ostreatus IE8 y Aspergillus niger AD96.4 en fermentación sólida. Interciencia, 32, 780–785.

    Google Scholar 

  13. Okamoto, K., Yanagi, S. O., & Sakai, T. (2000). Purification and characterization of extracellular laccase from Pleurotus ostreatus. Mycoscience, 41, 7–13.

    Article  CAS  Google Scholar 

  14. Shah, V., & Nerud, F. (2002). Lignin degrading system of white-rot fungi and its exploitation for dye decolorization. Canadian Journal of Microbiology, 48, 857–870.

    Article  CAS  Google Scholar 

  15. Nadim, M., Deshaware, S., Saidi, N., Abd-Elhakeem, M. A., Ojamo, H., & Shamekh, S. (2015). Extracellular enzymatic activity of Tuber maculatum and Tuber aestivum mycelia. Advances in Microbiology, 5, 523–530.

    Article  Google Scholar 

  16. Martin, F., Kohler, A., Murat, C., et al. (2010). Perigord black truffle genome uncovers evolutionary origins and mechanisms of symbiosis. Nature, 464, 1033–1038.

    Article  CAS  Google Scholar 

  17. Shamekh, S., Donnini, D., Zambonelli, A., & Leisola, M. (2009). Wild Finnish truffles. Acta Botanica Yunnanica, 31, 69–71.

    Google Scholar 

  18. Mandels, M., & Sternberg, D. (1976). Recent advances in cellular technology. Journal of Fermentation Technology, 54, 267–286.

    CAS  Google Scholar 

  19. Ghose, T. K. (1987). Measurement of cellulase activities. International union of pure and applied chemistry, 59, 257–268.

    CAS  Google Scholar 

  20. Miller, G. L. (1959). Use of dinitrosalicylic acid reagent for determination of reducing sugar. Analytical Chemistry, 31, 426–428.

    Article  CAS  Google Scholar 

  21. Quimio, T. H. (1982). Tropical mushrooms: biological nature and cultivation methods. Chinese University Press.

  22. Mandels, M., & Reese, R. T. (1965). Inhibition of cellulase. Annual Review of Phytopathology, 3, 85–102.

    Article  CAS  Google Scholar 

  23. Karim, A., Nawaz, M. A., & Aman, A. (2015). Hyper production of cellulose degrading endo (1, 4) β-D glucanase from Bacillus lichenoformis KIBGE-IB2. Journal of Radiation Research and Applied Sciences, 8, 160–165.

    Article  CAS  Google Scholar 

  24. Yao, G., Li, Z., Wua, R., Qin, Y., Liu, G., & Qu, Y. (2016). Penicillium oxalicum poflbc regulates fungal asexual development and is important for cellulase gene expression. Fungal Genetics and Biology, 86, 91–102.

    Article  CAS  Google Scholar 

  25. Romero, M. D., Aguado, J., Gonzalez, L., & Ladero, M. (1999). Cellulase production by Neurospora crassa on wheat straw. Enzyme and Microbial Technology, 25, 244–250.

    Article  CAS  Google Scholar 

  26. Sippola, J., & Yli, H. (2004). Status of soil mapping in Finland. European soil bureau. Research Report, 9, 105–110.

    Google Scholar 

  27. Garraway, O. M., & Evans, C. R. (1984). Fungal nutrition and physiology. Singapore: John Wiley.

    Google Scholar 

  28. Jackeline, P. A., Aline Simoes da R. B., Phellippe, A. S., & Rodrigo, P. N. (2011). Production and partial characterization of cellulases from Trichoderma sp. IS-05 isolated from sandy coastal plains of northeast Brazil. Enzyme Research, 2011, 1–7.

  29. Mandels, M., & Weber, J. (1969). Cellulases and their applications. Advances in chemistry, 95, 391–414.

    Article  CAS  Google Scholar 

  30. Carter, B. L. A., & Bull, A. T. (1969). Studies of fungal growth and intermediary metabolism under steady and non-steady conditions. Biotechnology and Bioengineering, 11, 785–804.

    Article  Google Scholar 

  31. Gautam, S. P., Bundela, P. S., Pandey, A. K., Awasthi, M. K., & Sarsaiya, S. (2010). Effect of different carbon sources on production of cellulases by Aspergillus niger. Journal of Applied Sciences in Environmental Sanitation, 5, 295–300.

    CAS  Google Scholar 

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This study was conducted at Juva Truffle Center Finland. The financial support of Regional Council of Southern Savo, Finland, is appreciated and thanked. Authors are grateful for Mr. Antti Kinnunen for administrative service and Mrs. Heli Valtonen for her technical assistance.

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Correspondence to Salem Shamekh.

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Research Highlights

• Tuber maculatum mycelium is novel source for cellulase.

Mycelium utilizes cellulosic carbon as the energy source during its growth cycle.

Maximum cellulase activity (1.70 U/ml) was detected after 7 days of fermentation.

Cellulase showed optimum activity at 50 °C, pH 5, and thermostability at 50 °C.

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Bedade, D.K., Singhal, R.S., Turunen, O. et al. Biochemical Characterization of Extracellular Cellulase from Tuber maculatum Mycelium Produced Under Submerged Fermentation. Appl Biochem Biotechnol 181, 772–783 (2017).

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