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

Abstract

The Bacillus licheniformis ydaP gene encodes for a pyru­vate oxidase that catalyses the oxidative decarboxyla­tion of pyruvate to acetate and CO2. The YdaP form of this enzyme was purified about 48.6-folds to homoge­neity in three steps. The enzyme was recovered in a soluble form and demonstrated significant activity on pyruvate using 2, 6-dichlorophenolindophenol (DCPIP) as an artificial electron acceptor. HPLC analysis of the 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; however, it was activated by 1% Triton X-100. The YdaP substrate-bind­ing pocket from the YdaP protein differed substantially from the equivalent site in all of the so far character­ized pyruvate oxidases, suggesting that the B. licheni­formis YdaP might accept different substrates. This could allow more accessibility of large substrates into the active site of this enzyme. The thermostability and pH activity of the YdaP enzyme were determined, with optimums at 50ºC and pH 5.8, respectively. The amino acid residues forming the catalytic cavity were identi­fied as Gln460 to Ala480.