Isolation and Characterization of Bioactive Molecules from Endophytic Fungi

Abstract

Endophytes are ubiquitous organisms residing in the internal tissues of the plants and are capable of producing a wide range of secondary metabolites with the biological activities of interest for application in environment, agriculture, medicine and food industry. In the present study, endophytic fungi were isolated from Taxus baccata and were identified based on their morphological characteristics and internal transcribed spacer of rDNA sequence analysis. The extracts of these fungi were tested for anticancer and antioxidant activities. Some of the putative compounds involved in these biological activities were identified. An attempt has also been made to examine the potential of thermotolerant endophytic fungi, isolated from the plants growing in the Western Ghats of India, to produce extracellular enzymes. A total of 25 endophytic fungi were isolated from the bark of T. baccata and screened for antimicrobial and cytotoxic activities. Among the 25 isolates, four isolates (T1, T2, T4 and T6) showed more antimicrobial and cytotoxic activities compared to other isolates. These four isolates were selected for further studies. Further, these isolates were identified based on their morphological characteristics and internal transcribed spacer (ITS) sequence analyses. Based on the morphological and ITS sequence analysis, isolate T1 was identified as Diaporthe sp., isolate T2 as Marasmius scorodonius, isolate T5 as Microdiplodia sp. and isolate T6 as Fusarium tricinctum. The effect of these fungal crude extracts were tested on the growth of bacteria such as Staphylococcus aureus, Bacillus megaterium, Escherichia coli and Pseudomonas aeruginosa and Candida albicans. These isolates showed antibacterial and antifungal activities. The effect of crude fungal extracts on the growth of cancer cell lines was evaluated by MTT assay. Out of the four endophytes, Diaporthe sp. (T1) and F. tricinctum (T6) have shown cytotoxic effect against Human breast cancer cell lines (MCF-7) and human cervical cancer cell lines (HeLa) and the cytotoxic effect significantly increased with increase in the concentration of the fungal extract. The antioxidant effect of T1 and T6 significantly increased with increase in concentration. The extracts of Diaporthe sp. (T1) and Fusarium tricinctum (T6) were used to identify the bioactive compounds produced by these fungi. Some of the putative compounds identified from the crude extracts of Diaporthe sp. (T1) are cytosporones, cytochalasins and docetaxel. Three cytochalasins such as trichalasin E, F and H were identified based on their m/z values of eluting peaks. These compounds are being reported for the first time from the endophytic fungus Diaporthe sp (T1). Some of the important compounds produced by F. tricinctum (T6) includes, gniditrin, 7-hydroxy heptaphylline, tirandamycin A, fumitremorgin C, paclitaxel and 10-deacetyl baccatin III. These compounds have been reported to have the cytotoxic, antimicrobial and antioxidant activities. Production of extracellular enzymes by nine thermotolerant fungal endophytes such as Bartalinia sp., Chaetomella sp., Curvularia sp., Exserohilum rostratum, Leptosphaerulina sp., Montagnulaceae sp., Pestalotiopsis microspora and Phoma sp., were determined by digestion of suspended or dissolved substrates in agar plates. The results showed that each isolate was able to produce one or the other extracellular enzymes. All isolates except Curvularia sp., produced cellulase when CMC was amended in the medium. Maximum cellulase was produced by P. microspora and Phoma sp. Maximum laccase activity was observed in Phoma sp., followed by Curvularia sp and Montagnulaceae sp., which showed moderate activity. Only three isolates, Bartalinia sp., Leptospaherulina sp., and Montagnulaceae sp., showed positive to amylase production and the maximum activity was observed in Bartalinia sp. Most of the organisms were able to produce lipase and the maximum lipase activity was recorded with Montagnulaceae sp., and Leptosphaerulina sp. None of the isolates showed positive to pectinase and tyrosinase activities. Exserohilum rostratum, Montagnulaceae sp., and P. microspora showed little protease activity while other isolates failed to produce protease enzyme. Maximum L-asparginase activity was recorded in Curvularia sp. and moderate levels in E. rostratum, Leptosphaerulina sp., and P. microspora. Cellulase producing endophytic fungi P. microspora and Phoma sp. and lipase producing Montagnulaceae sp. were further tested for their ability to produce these enzymes in liquid culture. Cellulase activity was determined at different time intervals by growing the fungi in CMC amended medium. Maximum cellulase activity was observed in P. microspora and Phoma sp., at pH range of 5-10. The enzyme activity increased with increase in temperature with both the isolates. The enzyme was more stable for 60 min when incubated at 100oC and the thermal stability decreased with increase in time. The lipase activity was determined in endophytic fungus Montagnulacae sp. by incubating the culture at different time intervals. The enzyme activity increased up to 16 days and decreased thereafter. The maximum activity of lipase was observed at pH of 11.0. The enzyme activity was lower at pH range of 4.0 to 9.0 and at higher pH 12.0. The lipase activity increased with increase in temperature and the maximum activity was determined at 100oC. These results suggested that the thermotolerant endophytic fungi can be potential source for different enzyme activities with capacities to tolerate extreme pH and temperature conditions. From these results, it can be concluded that endophytic fungi are novel and important microbial resource for producing bioactive compounds. The endophytic fungi Diaaporthe sp. (T1) and F. tricinctum (T6) used in this study were able to produce different bioactive compounds which have cytotoxic, antioxidant and immunomodulatory activities. Some of the enzymes such as cellulose and lipase produced by the endophytic fungal isolates showed high tolerance to different pH and temperature which can be used as a source of industrial biocatalysts.