Cloning, expression and characterization of thermostable YdaP from Bacillus licheniformis 9A

Bacillus licheniformis YdaP gene encodes for the enzyme pyruvate oxidase that catalyses the oxidative decarboxylation of pyruvate to acetate and carbon dioxide. The YdaP form of this enzyme was purified about 48.6-fold to homogeneity in three steps. The enzyme was recovered in a soluble form and it demonstrated significant activity on pyruvate using 2, 6-dichlorophenolindophenol (DCPIP) as an artificial electron acceptor. HPLC analysis of YdaP-enzyme catalysed conversion of pyruvate showed acetate as the sole product, confirming the putative identity of pyruvate oxidase. Analysis of the substrate specificity showed that the YdaP enzyme demonstrated preference for short chain oxo acids, activated by 1% Triton X-100. The YdaP substrate-binding pocket from the YdaP protein differed substantially from the equivalent site in the all characterized pyruvate oxidases, suggesting that the B. licheniformis YdaP might accept different substrates. The thermostability and pH activity of the YdaP enzyme were determined, with optimal at 50 oC and pH 5.8, respectively. The amino acid residues forming the catalytic cavity were identified as Gln460 to Ala480. The YdaP substrate-binding pocket from the B. licheniformis YdaP protein differed substantially from the equivalent site in the L. plantarum POX structure, suggesting that the B. licheniformis YdaP might accept different substrates.