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Biological and enzymatic mechanisms of polyester biodegradation by fungi
This dissertation has explored the microbial and enzymatic processes that occur during the biodegradation of aliphatic polyesters by the fungus Aspergillus fumigatus. The relationship between the chemical structure of polyesters, the enzymes secreted by fungi to hydrolyze polyesters, and the enzymatic mechanisms of polyester degradation were investigated. Naturally occurring bacterial polyesters such as poly($\beta$-hydroxybutyrate) and poly($\beta$-hydroxyvalerate), as well as synthetic polyesters, were found to be utilized as the sole carbon source in liquid fermentation cultures by a strain of A. fumigatus isolated from leaf compost. The enzyme secreted during fungal growth on PHB was isolated and characterized using enzyme kinetics, affinity reagents and inhibitors, SDS-PAGE, and IEF analysis to determine the biochemical characteristics of the eukaryotic PHB depolymerase. The substrate specificity of the PHB depolymerase was evaluated using in vitro weight loss methods which determined the enzymatic rates of hydrolysis for bacterial and synthetic polyester films. The rates of polyester degradation were analyzed as a function of copolymer composition, molecular weight, and crystalline properties. Molecular modeling of polyesters suggested a relationship between polyester conformation and the susceptibility to PHB depolymerase hydrolysis. Direct observations of enzyme-polymer interactions showed that the PHB depolymerase adsorbs strongly to PHB and other aliphatic polyesters by hydrophobic surface forces. The substrate binding behavior of the fungal PHB depolymerase was found to be non-specific, and is mediated by two independent enzyme domains. By applying cyclic and linear PHB analogs to enzymatic hydrolysis experiments, a new understanding of the enzymatic mechanism of PHB hydrolysis was developed. Oligolide hydrolysis by the bacterial and fungal depolymerases established that the PHB depolymerases do not require free chain ends for the initiation of polymer degradation, and hydrolyze substrates in an exo mode of activity, in addition to endo chain cleavage. Enzymes present in the supernatant of A. fumigatus grown on poly($\varepsilon$-caprolactone) hydrolyzed olive oil, indicating that a lipase is also secreted by this fungus which degrades synthetic polyesters. The isolation and identification of the polyester hydrolases from A. fumigatus demonstrated that different esterase enzymes are secreted to catalyze PHB and PCL biodegradation.
Scherer, Thomas Marc, "Biological and enzymatic mechanisms of polyester biodegradation by fungi" (1996). Doctoral Dissertations Available from Proquest. AAI9709649.