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Cleveland, Maria

University of British Columbia

The utilization and valorization of biomass has been proposed as a sustainable alternative to petroleum-based energy, chemicals and materials. Microbial enzymes can be used as selective catalysts for the depolymerization and functionalization of plant material. In this regard, the copper radical oxidases (CROs) from Auxiliary Activity Family 5 subfamily 2 (AA5_2) are attractive targets since they oxidize primary alcohols in a chemo-selective manner to the corresponding aldehydes without utilizing complex and expensive organic cofactors. AA5_2 was first defined by the archetypal galactose oxidase from Fusarium graminearum, however, other fungal AA5_2 members have recently been shown to comprise a wide range of specificities for aromatic, aliphatic and furan-based alcohols. This suggests a broader substrate scope of CROs for biocatalytic applications. However, only 10% of the annotated AA5_2 members have been characterized to date. The work presented in this thesis describes the biochemical characterization of fourteen fungal copper radical oxidases, recombinantly produced in Pichia pastoris, encompassing galactose oxidases, raffinose oxidases, broad spectrum alcohol oxidases, non-carbohydrate alcohol oxidases and aryl alcohol oxidases. Detailed product analysis was conducted of carbohydrates, furans and glycerol oxidized by these novel enzymes to further explore their potential applications. Of note, we observed direct oxidation of 5-hydroxymethylfurfural (HMF), an important platform chemical derived from biomass, to 5-formyl-2-furoic acid (FFCA) by six enzymes. One enzyme facilitated the direct one-pot conversion of HMF to 2,5-furandicarboxylic acid (FDCA), a building block for bio-plastic production. Through docking of galactose into homology models of these newly characterized enzymes, and comparing it to the archetypal galactose oxidase, key amino acid residues important for substrate specificity have been proposed. Finally, the potential of enzyme immobilization using the cross-linked enzyme aggregate (CLEA) strategy has been investigated for a specific alcohol oxidase to increase its utilization in biocatalysis. This work expands our understanding of the catalytic diversity of copper radical oxidases from AA5_2 and doubles the number of characterized AA5_2 members. Detailed sequence and enzymatic analysis highlights their structure-function relationships while product analysis informs their possible biotechnological applications in bio-products manufacturing. Furthermore, immobilization was shown to be a promising avenue for increasing the biocatalytic potential of CROs.

Text

2022-08-31

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/79415

Chemistry

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/