Enzyme activities of fungi isolated from koala faeces
thesisposted on 2022-03-28, 09:19 authored by Robyn Anne Peterson
Filamentous fungi secrete enzymes to break down complex substances in the environment into smaller molecules they can use for nutrition. Investigation of the enzymes secreted by a fungus can lead to a better understanding of how it survives in a natural habitat; furthermore, new enzymes with potential for industrial applications can be revealed. In this work, the enzyme activities of fungi from koala faeces were investigated. As a result of a diet of Eucalyptus leaves, koala faeces are composed of recalcitrant plant cell wall polymers (cellulose, hemicellulose, pectin and lignin). Consequently, fungi that grow on koala faeces hold high potential as sources of enzymes for efficient degradation of plant biomass. -- Thirty-seven fungal strains were isolated from koala faeces, identified, and screened for enzyme activities using agar plate assays; over two-thirds of the isolates secreted xylanases, endoglucanases, ligninases and proteases, and over one-third secreted amylases, mannanases and tannases. The enzyme activities of seven isolates were comprehensively characterised using liquid cultures, liquid enzyme assays and zymography. Two isolates, Gelasinospora cratophora A10 and Trichoderma atroviride A2, were high secretors of protein and heat-tolerant enzymes. The lipase(s) from Mariannaea camptospora A11 sustained activity at cool temperatures. The xylanase(s), mannanase(s), endoglucanase(s) and β-glucosidase(s) of Doratomyces stemonitis C8 displayed optimal activities under neutral to alkaline conditions. Some of the enzymes hold potential for application in the production of paper, textiles, detergents and ethanol-based biofuels. -- Finally, the secretome of D. stemonitis C8 was studied by gel electrophoresis and mass spectrometry. As the genome of D. stemonitis has not been sequenced, the secretome analysis required cross-species identification and de novo sequencing; furthermore, a new technique was developed to identify proteins directly from zymogram gels by mass spectrometry. In the first secretome analysis of a coprophilous 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.