Enzyme Sources for Urinary Oxalate Measurements

An alternative source of oxalate oxidase (Oxox) was to be purified for use in a biosensor system to detect oxalate in patients suffering from primary hyperoxaluria (PH). Oxox has been isolated from a number of sources, however there is significant variation in the information available. Prior to the purification of Oxox, the properties of commercially available Sigma Aldrich Ltd. Oxox were researched. An optimum enzymatic assay, substrate specificity, optimum buffer and optimum pH for Sigma Oxox were investigated prior to its purification. The purification of the enzyme led to the finding that Sigma Oxox was unstable in a number of salt buffers, and insoluble. To determine the ability of this enzyme to detect oxalate, amperometric analysis was undertaken, which demonstrated the Sigma Oxox based sensors’ reusability and reproducibility. Oxox from barley roots was successfully purified using a five step purification protocol, the findings obtained were in accordance with those published. The barley root extracts, after each purification procedure, were analysed amperometrically and highlighted the requirement for purification to optimise the analytical signal. Over 140 sources were screened from a number of plant families, to detect the presence of Oxox activity. Three possible sources of Oxox were identified by level of Oxox activity and chosen for further purification: cabbage, carrot and mint leaves. The presence of Oxox in different varieties of these sources varied, illustrating the dissimilarities in Oxox, confirming the diversity of the data available on Oxox. Oxox from cabbage, carrot and mint leaves were purified using the standardised protocol employed during the purification of Sigma and barley root Oxox. The studies undertaken led to the finding that the components of interest possessed dissimilar properties to documented Oxox. Cabbage, carrot and mint leaves oxalate oxidative components were found to be smaller than 700 kDa, extremely thermally stable, did not possess strong positively or negatively charged groups. The identity of the oxalate oxidising components being Oxox was disproved. The possibility of the oxidising components being cofactors, or novel oxalate oxidising components were addressed, however, no distinct conclusion could be made.