The Comparative Analysis of Wild-Type and F242Y/F242A Mutant Porcine D-Amino Acid Oxidase
Abstract
D-amino acid oxidase (DAO) is a flavoenzyme that utilizes D-amino acids from the peptidoglycan layer of bacterial cell walls to generate hydrogen peroxide, a known antibacterial agent. The objective of this study is to further explore the currently unknown potential of DAO as an antibacterial agent by examining its activity through active site mutations. In this study, F242Y and F242A porcine DAO mutants were created by site directed mutagenesis (SDM) and splicing by overlap extension (SOE) PCR respectively. Spectrophotometric assays were utilized to determine the effectiveness of wild type DAO in producing hydrogen peroxide with D-alanine, D-serine and L-alanine. It was determined that D-alanine, the natural substrate of DAO, contained the greatest hydrogen peroxide producing capability. D-serine also produced hydrogen peroxide but not to the extent of D-alanine, while L-alanine demonstrated no production. By utilizing the activity of wild type DAO as a baseline, we hope to create potent DAO mutants that are effective against a greater variety of peptidoglycan D-amino acids. Future implications of this research include the development of DAO injections or orally administered drugs that can combat currently resistant infectious diseases.
Keywords – Porcine D-Amino Acid Oxidase, mutagenesis methods, bactericidal abilities, peroxide production abilities, antibacterial properties.
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