UBC Theses and Dissertations
A novel method, and its applications, to study alcohol oxidase from Auxiliary Activity Family 5 based on direct monitoring of enzymatic reactions Xia, Fan
Auxiliary Activity Family 5 (AA5) carbohydrate-active enzymes are mononuclear copper radical oxidases (CROs) capable of oxidizing a variety of alcohols to their corresponding aldehydes without organic cofactors. Classically, AA5 characterization is performed using a horseradish peroxidase (HRP) coupled assay using ABTS (2.2’-azino-bis(3-ehtylbenthiazoline-6-sulphonic acid) as a colorimetric indicator. However, this HRP-ABTS coupled assay indirectly monitors reaction progress – it measures a small portion of reactant conversion and provides no information about the extent of oxidation (percentage conversion) or potential product inhibition. Therefore, we developed an alternative approach to assess alcohol oxidation by AA5 enzymes that allows direct monitoring of reaction progress by incorporating tandem reaction progress analysis. This new approach collects time-resolved information about active chemical species and facilitates the interrogation and optimization of the system. CgrAlcOx, an AA5 oxidase from the phytopathogenic fungal species Colletotrichum graminicola, was previously characterized and displayed activity on a wide variety of aromatic and aliphatic alcohols. Reaction monitoring using the HRP-ABTS assay in this previous study was used as a benchmark to develop our new approach. We tested different instruments and identified high performance liquid chromatography coupled with an ultraviolet (UV) detector as the best way to directly monitor, on-line, the depletion of alcohols and the formation of aldehydes in alcohol oxidation catalyzed by CgrAlcOx. Reaction conditions were optimized, and the kinetics of aromatic alcohol oxidation were determined to support the applicability of this approach for monitoring enzymatic reactions. This new approach also allowed the detailed study of CgrAlcOx, including the screening of new substrates and the detection of product inhibition; previous assays did not permit the latter. Lastly, this new approach was combined with the HRP-ABTS coupled assay in a complementary way to overcome the inability of the previous assay to characterize reaction inhibitors. This allowed the discovery of new inhibitors of CgrAlcOx, such as benzylamine and benzyl mercaptan. In addition, using this combinatorial approach, butanethiol was identified as both an inhibitor and a substrate towards CgrAlcOx.
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