Biophysical and structural characterization of the oxalate decarboxylase from Bacillus pumilus 15.1

Bacillus pumilus 15.1 is a bacterial strain capable of producing parasporal inclusions during sporulation, whose main protein component has been identified as an oxalate decarboxylase (OxD). This enzyme belongs to the cupin superfamily, is manganese-dependent, and catalyzes the conversion of oxalate into formate and carbon dioxide. Due to its ability to organize into crystalline structures and its possible relationship with the entomopathogenic activity of the strain against the fruit fly, the OxD from B. pumilus 15.1 represents a protein of interest for its biophysical and structural characterization. In the present work, OxD was purified from sporulated biomass of B. pumilus 15.1 and characterized using different biophysical techniques. The obtainment of the purified protein was evaluated by SDS-PAGE, while the final soluble fraction was quantified by spectrophotometry at 280 nm. Likewise, the behavior of OxD in solution was analyzed by Dynamic Light Scattering (DLS) and Size Exclusion Chromatography (SEC). Secondary structure and thermal stability were studied by Circular Dichroism (CD) and Differential Scanning Calorimetry (DSC), respectively. The results showed that the purified OxD retained a detectable secondary structure and that its behavior in solution depended on pH conditions and buffer composition. In NaP buffer at pH 7.5, the protein showed behavior more compatible with species of smaller apparent size, whereas in HEPES/MES buffer, signals associated with larger species and a thermal transition shifted toward higher temperatures were observed. Overall, these results suggest that buffer composition influenced the hydrodynamic, oligomeric, and thermal state of OxD, providing useful information for understanding its stability and structural behavior in solution.