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RESEARCH PRODUCT

Gene Cloning, Transcriptional Analysis, Purification, and Characterization of Phenolic Acid Decarboxylase from Bacillus subtilis

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Phenolic acids, also called substituted cinnamic acids, are important lignin-related aromatic acids and natural constituents of plant cell walls. These acids (particularly ferulic, p-coumaric, and caffeic acids) bind the complex lignin polymer to the hemicellulose and cellulose in plants (1) or are generally esterified with tartaric acid (for example, in grape must, wine, and cider) and can be released as free acids during wine making by some cinnamoyl esterase activities (9). Most often, free phenolic acids are metabolized by different microorganisms into 4-vinyl derivatives and then are eventually reduced into 4-ethyl derivatives (5, 6). Some of these volatile phenols, particularly vinyl and ethyl guaiacol (generated from ferulic acid), are useful aromatic chemicals (12) or contribute naturally to aroma in wine (10) and other fermented foods and beverages. Other volatile phenols, such as ethyl and vinyl phenols (from p-coumaric acid), are most often considered phenolic off-flavors and are responsible for alterations in organoleptic properties. Previously, only three bacterial phenolic acid decarboxylases (PADs) have been purified and characterized (4, 8, 13). Two of these enzymes have been cloned and sequenced, a ferulate decarboxylase (FDC) from Bacillus pumilus (5) and a p-coumarate decarboxylase (PDC) from Lactobacillus plantarum (17). Although these enzymes exhibit 66% amino acid sequence identity, they differ in structure, biochemical characteristics, and substrate specificity. They are also different from the phenylacrylic decarboxylase cloned from Saccharomyces cerevisiae (7), which exhibited very low activity with ferulic and p-coumaric acids. The substrate specificity of these bacterial decarboxylases (ferulic and p-coumaric acids for FDC and p-coumaric and caffeic acids for PDC) is an obstacle for production of aroma compounds from crude or partially purified substrates, which always contain these two acids. It was our goal to screen new microorganisms in order to isolate decarboxylases with different substrate specificities and to better characterize this enzyme family. A comparison of amino acid sequences should help identify regions that specify substrate specificity and residues essential for catalysis. The results presented here are a first step toward obtaining recombinant enzymes with appropriate substrate specificities for aroma production and toward engineering genetically modified starters for vegetable fermentation and wine making. In the course of our screening, we found that Bacillus subtilis was able to decarboxylate ferulic, p-coumaric, and caffeic acids. We describe the cloning and the results of a transcriptional analysis of a pad gene that encodes a PAD. Purification and characterization of the stable recombinant enzyme overexpressed in Escherichia coli confirmed that B. subtilis PAD can metabolize all three phenolic acids; to our knowledge, this is a novel substrate specificity for an enzyme belonging to the PAD family. The PAD examined, which exhibits extensive similarity to FDC in amino acid sequence and differs from FDC in enzymatic characteristics, should be useful in experiments to determine substrate specificity and in catabolic site characterization studies in which site-directed mutagenesis is used.