SCREENING OF ENDOPHYTIC FUNGI FOR THEIR ABILITY TO PRODUCE EXTRACELLULARCELLULASES
Objective: Screening endophytic isolates from woody perennial medicinal plants of Western Ghats for production of extracellular cellulases
Methods: Endophytes were isolated using normal microbiological methods and their colonization frequency and dominance were calculated using statistical methods. Efficiency of growth on complex cellulosic substrates was evaluated on media supplemented with specific substrates. Enzyme assays with identified endophytic fungi were carried from their secretome.
Forty endophytic fungal isolates were obtained using standard isolation methods from different medicinal plants from a biodiversity hotspot in the Western Ghats region of Karnataka, India. The isolated endophytic fungi were then identified based on their morphological characters. The percentage of dominant endophytes (ð·) was calculated based on the colonization frequency. Among the isolated fungi, F. solani and Talaromyces sp. was found to be highest, at5.5 and 5.6 respectively. Each of the identified fungi grown on CMC and seven among the 40 isolates were found to grow luxuriantly as measured by radial growth. The identities of these fungi were morphologically reconfirmed and were completely carbon drained by growing them on a low nutrient medium. These fungi were later evaluated for their growth on avicel and microcrystalline cellulose. Fusarium solani and Tarlaromyces sp. were significantly better in their growth when compared to other endoophytes tested. Further, the cellulosome complex of enzymes were analysed in the secretome of Fusarium solani and Talaromyces sp. Total filter paper activity of Fusarium solani was found to be FPU/ml, 76 FPU/ml and 70 FPU/ml at 24, 48 and 72 hours respectively. Similarly, Filter paper activity of Talaromyces sp. was found to be 89, 86 and 78 FPU/ml at 24h, 48h and 72h respectively. Endogluconase activity of Fusarium solani was found to be 63 CMCase, 60and 61 CMCase at 24, 48 and 72hours of incubation respectively, which was greater than Talaromyces sp. Similarly, Exogluconase and Beta-glucosidase activities were also found to be high in Talomyces sp. when compared to Fusarium solani at all the time intervals tested.
The results from the present study reveals that Fusarium sSolani and Talaromyces sp.are extremely potent producers of cellulases and can thus be used for eco-friendly and economic hydrolysis of biomass for biofuel purposes.
2. Fargione J, Plevin R J and Hill J. The ecological impact of biofuels Annu. Rev. Ecol. Evol. Syst. 2010;41 351â€“77.
3. Beringer T I M, Lucht W and Schaphoff S Bioenergy production potential of global biomass plantations under environmental and agricultural constraints GCB Bioenergy. 2011; 3 299â€“312.
4. Gabriel Morales, Juan A Melero, Jose Iglesias and Marta Paniagua. Advanced Biofuels from Lignocellulosic Biomass J Adv Chem Eng 2014; 4:e101. doi: 10.4172/2090-4568.1000e101.
5. Benedict C. Okeke, Rosine W. Hall, Ananda Nanjundaswamy, M. Sue Thomson,Yasaman Deravi, Leah Sawyer, Andrew Prescott. Selection and molecular characterization of cellulolyticâ€“xylanolyticfungi from surface soil-biomass mixtures from Black Belt sites Microbiological Research 2015; 175 24â€“33.
6. Diogo Robl, Priscila da Silva Delabona, Carla Montanari Mergel, Juan Diego Rojas, PatrÃcia dos Santos Costa, Ida Chapaval Pimentel, Vania Aparecida Vicente, JosÃ© Geraldo da Cruz Pradella and Gabriel Padilla. The capability of endophytic fungi for production of hemicellulases and related enzymes. BMC Biotechnology 2013; 13:94 doi:10.1186/1472-6750-13-94.
7. Midgley DJ, J ordon LA, Saleeba JA, McGee PA. Utilisation of carbon substrates by orchid and ericoid mycorrhizal fungi from Australian dry sclerophyll forests Mycorrhiza 2006; 16 , 175â€“182.
8. Zhang Y.B., Yuan L.J., Chen Z.J., Fu L., Lu J.H., Meng Q.F., He H., Yu X.X., Lin F., Teng L.R. Purification and characterization of beta-glucosidase from a newly isolated strain Tolypocladium cylindrosporum Syzx4. Chem. Res. Chin. Univ. 2009;27:557â€“561.
9. Jean-Guy Berrin,David Navarro,Marie CouturierCaroline OlivÃ©Sacha Grisel,Mireille Haon,Sabine TaussacChristian Lechat,RÃ©gis Courtecuisse,Anne Favel,Pedro M. Coutinho,and Laurence Lesage-Meessen. Exploring the Natural Fungal Biodiversity of Tropical and Temperate Forests toward Improvement of Biomass Conversion Volume 78 Number 18 Applied and Environmental Microbiology 2012; p. 6483â€“6490.
10. Schulz B, Boyle C. The endophytic continuum. Mycol Res 2005; 109: 661 â€“ 686.
11. Gusakov AV. 2011. Alternatives to Trichoderma reesei in biofuel production.Trends Biotechnol 2011; 29:419â€“425. doi:10.1016/j.tibtech..04.004.
12. Tuohy, M. G., Walsh, D. J., Murray, P.G., Claessens, M., Cuffee, M.M. and Savage, A. V. Kinetic parameters and mode of action of the cellobiohydrolases produced by Talaromyces emersonii. Biochem. Biopsy. Acta 2002; 1596:366-380.
13. Sideney B.Onofree,Zipora M.Q.Santosh, Francini Y.Kasimura.Cellulases Produced by Endophytic fungi Pycnoporus sanguiness(L) African Journal Of Agriculture Research 2015: Vol.10(13),pp.1557-1564.
14. Inoue H, Decker SR, Taylor LE, Yano S, Sawayama S. Identification and characterization of core cellulolytic enzymes from Talaromyces cellulolyticus (formerly Acremonium cellulolyticus) critical for hydrolysis of lignocellulosic biomass. Biotechnol Biofuels 2014; 7:151. doi:10.1186/s13068-014-0151-5.
15. Watanabe T. Cellulolytic enzymes IV. Fractionation of a cellulolytic enzyme system. Journal of Fermentation Technology 1968a ; 46, 299â€“302.
16. Watanabe T. Cellulolytic enzymes V. Some properties of cellulase fraction I. Journal of Fermentation Technology 1968b; 46, 303â€“307.
17. FÃ¤hnrich P, Irrgang K.Conversion of cellulose to sugars and cellobionic acid by the extracellular enzyme system of Chaetomium cellulolyticum. Biotechnology Letters 1982;12, 775â€“780.
18. Jiang C, Li SX, Luo FF, Jin K, Wang Q, Hao ZY, Wu LL, Zhao GC, Ma GF, Shen PH, Tang XL, Wu B. Biochemical characterization of two novel Î²-glucosidase genes by metagenome expression cloning. Bioresour Te