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Endophytes from Australian Native Plants As Novel Sources of Bioactive Compounds for Industrial, Environmental and Medicinal Applications

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Endophytes from Australian Native Plants As Novel Sources of Bioactive Compounds for Industrial, Environmental and Medicinal Applications Endophytes from Australian native plants as novel sources of bioactive compounds for industrial, environmental and medicinal applications A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy By Bita Zaferanloo Department of Chemistry & Biotechnology, School of Science, Faculty of Science, Engineering and Technology Swinburne University of Technology, Australia March 2014 Abstract Natural products, including endophytic fungi, have received increased interest over the past decade for their therapeutic potential. Endophytes are fungi (or bacteria) which live in the intercellular spaces of plant tissues, for all or part of their lifecycle. The endophyte-plant relationship is symbiotic and it is believed that endophytic fungi live mutualistically with the host plant with no apparent symptoms. The asymptomatic nature of these fungi demonstrates enormous diversity and abundance, with nearly 30,000 land plant species in symbiosis with a multitude of species of endophytes. The ability of these microorganisms to colonize host plants in extreme, specialized environments has seen the development of complex relationships forming between endophyte and host. This subsequently increases the fitness of both plant and endophyte by the adaptive evolution of specialised survival mechanisms. It is suggested that these fungal endophytes offer a potentially enormous source of novel bioactive compounds that could be useful in medicine and other diverse applications, including industry and environment. In this work, the antibacterial, anticancer and enzyme activities of endophytic fungi from two Australian native plants Eremophila longifolia and E. maculata were investgiated. The rationale behind this search for new bioactive compounds and effective enzymes was based on the result of accumulated knowledge collected by indigenous Australians about the potentially medicinal properties of these two Australian native plants. In addition, the use of plants which are well-adapted to the harsh, dry climate of the Australian desert increased the likelihood of finding novel compounds. Following screening for bioactive compounds, investigation of the particular enzymes secreted by selected endophytic fungi was carried out to better understand how they survive in their natural habitat; furthermore, new enzymes with potential for industrial applications can be revealed. Twenty-six fungal strains were isolated from Eremophila longifolia and E. maculata. These were identified and screened for the production of antibacterial compounds against four different bacteria including Escherichia coli, Salmonella typhimurium, Staphylococcus aureus and Bacillus cereus using disk diffusion assays. EM-4 (Preussia sp.) showed the broadest range of antibacterial activity against all bacteria; however, EL-11 (Leptosphaerulina sp.) and EL-13 exhibited the highest ii antibacterial activities against Bacillus cereus with minimum inhibitory concentrations of 1.6 and 0.01 µg/ml, respectively. Fungal extracts were also screened against two human cancer cell lines (T-cell acute lymphoblastic leukemia MOLT-4 and PreB697 cells), at concentrations ranging from 250.00–0.11 μg/mL. The endophytes that demonstrated potent cytotoxicity (>70%) were further screened against adherent lung cancer (A549) and a primary foreskin fibroblast cell lines. The prominent metabolites potentially contributing to the cytotoxicity were investigated by means of HPLC and comparing samples against toxic metabolites previously identified from related endophytes: Alternariol (AOH) and Alternariol methyl-ether (AME). The toxic endophytes EM-6, EM-7, EM-9 (all Alternaria sp.) and EL-14 (Preussia minima) showed 50% inhibition of proliferation (EC50) at concentrations < 103 μg/mL, with EM-6 demonstrating the greatest cytotoxicity. EM-6, EM-7 and EM-9 all contained AME and AOH metabolites at varying concentrations; however, EM-6 and EL-14 showed additional unidentified peaks which may represent novel cytotoxic compounds. These data emphasise the importance of endophytic fungi as novel anti-cancer agents. Moreover, these endophytes were screened for enzyme activities using agar plate assays; over two-thirds of the isolates from Eremophila longifolia secreted amylase and over one-thirds proteases, while many isolates from E. maculata produced xylanase. The enzyme activities of seven isolates were characterised using liquid cultures. Three samples with the highest activities were comprehensively characterised by liquid enzyme assays and zymography. Their activities were compared with standard amylase, protease and xylanase producers, including Aspergillus oryzae (ATCC 10124), Fusarium oxysporum (FRR 3414) and Aspergillus niger (ATCC 10577), respectively. Three isolates, EL-14 (Preussia minima), EL-17 (Alternaria alternate) and EM-4 (Preussia sp.), were high secretors of protein and displayed the broadest selected enzyme activities under different conditions. The amylase(s) from EL-14 Preussia minima sustained activity at cool temperatures and alkaline conditions. The protease(s) from EL-17 Alternaria alternata displayed optimal activities under neutral to alkaline conditions. The xylanase(s) from EM-4 Preussia sp. revealed versatility at lower temperatures under neutral to alkaline conditions. Some of the enzymes hold potential for application in the production of detergents, dairy and ethanol-based biofuels. iii Finally, the α-amylase produced by EL-14 was purified to homogeneity through fractional acetone precipitation and Sephadex G-200 gel filtration, followed by DEAE- Sepharose ion exchange chromatography and electroelution. The purified α-amylase showed a molecular mass of 70 kDa which was confirmed by zymography. Temperature and pH optima were 25°C and pH 9, respectively. The enzyme was activated and stabilized mainly by the metal ions manganese and calcium. Enzyme activity was also studied using different carbon and nitrogen sources. It was observed that enzyme activity was highest (138 U/mg) with starch as the carbon source and L- asparagine as the nitrogen source. Bioreactor studies proved that enzyme activity remained stable in a larger cultivation volume, which is relevant to potential scale-up of enzyme production. Following in-gel digestion of the purified protein by trypsin, a 9- mer peptide was sequenced and analysed by LC-ESI-MS/MS. The partial amino acid sequence of the purified enzyme presented similarity to α-amylase from Magnaporthe oryzae. In the secretome analysis of the endophytic fungus, a complex array of enzymes integral to plant biomass degradation was identified, including enzymes that could be of value to industry in the future. These results confirm the value of endophytes from Australian native plants as novel sources of bioactive compounds for industrial, environmental and medicinal applications. Further investigation is warranted to determine the structures and properties of these bioactive compounds. iv Acknowledgements I am very thankful to Swinburne University and the funding provided by Swinburne University Postgraduate Research Award for making this research possible. There are also many people I would like to thank for making my PhD candidature such a rewarding and fulfilling experience. First and foremost, I am extremely grateful to my supervisor Professor Enzo Palombo. The inspiration and motivation he provided was a vital driving force behind the project and it helped me to achieve the best and strive towards my goals, whatever came my way. I would also like to thank Dr Peter Mahon for co-supervising the project, for always finding time to have a chat and keeping me on track. Ali, words seem inadequate to fully express my gratitude to you. Your support has been invaluable, your compassion and sense of humour have helped me to put things in perspective and kept me sane. Thank you. A special thank you to Ngan, Soula, Chris, Savithri and the Swinburne Research Higher Degrees staff especially Aimii Treweek for their kind assistance, always delivered with a smile and a lot of patience! Thank you to Nick, Paul and Ching-Seng for their professional advice and support from the mass spectrometry lab, Bio21 Institute, Melbourne University, during my experimental work in their lab. Special thanks to Dr Sally Coulthard and her group, including Stephanie Pepper, from the Institute of Cellular Medicine, Newcastle University Medical School, for their valuable collaboration in an in-depth insight on the anticancer aspect of the project. Thanks to Irene Hatzinisiriou for helping with Imaging at Monash University. Thank you too to all my friends in the laboratory who have made coming to university so enjoyable. Although I can’t thank you all individually I would like to especially thank Hayden, Abdullah, Shahanee, Elisa, Sarah, Saifone, Shanthi, Rue, Rebecca, Babu, Hamid, Dhivya, Avinash, Vanu, Jafar, Rashida, Rasika, Jun, Ha, Snehal and everyone else I met along the way! To Kaylass, thank you for the many times you have patiently taught me skills in the lab. Special thanks to Robyn Peterson and Mahmoud Ghorbani v for kindly helping me with expert advice. Thank you Narges and Azadeh, for our great friendly conversations and cups of tea. Thank you to all my Masters students for their contribution in this project. Finally I would like to express my deepest gratitude to my parents and my family. The support you provided me through difficult years helped me to turn my life around and to achieve “the impossible”. It goes without saying, but I’ll say it here:
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