GENE CLONING, TRANSCRIPTIONAL ANALYSIS, PURIFICATION, AND CHARACTERIZATION OF PHENOLIC-ACID DECARBOXYLASE FROM BACILLUS-SUBTILIS

Bacillus subtilis displays a substrate-inducible decarboxylating activ ity with the following three phenolic acids: ferulic, p-coumaric, and caffeic acids, Based on DNA sequence homologies between the Bacillus p umilus ferulate decarboxylase gene (fdc) (A, Zago, G, Degrassi, and C. V. Bruschi, Appl. Environ. Microbiol. 61:4484-4486, 1995) and the Lac tobacillus plantarum p-coumarate decarboxylase gene (pdc) (J.-F. Cavin , L. Barthel-mebs, and C, Divies, App't, Environ, Microbiol. 63:1939-1 944, 1997), a DNA probe of about 300 nucleotides for the L, plantarum pdc gene was used to screen a B. subtilis genomic library in order to clone the corresponding gene in this bacterium, One clone was detected with this heterologous probe, and this clone exhibited phenolic acid decarboxylase (PAD) activity. The corresponding 5-kb insertion was par tially sequenced and was found to contain a 528-bp open reading frame coding for a 162-amino-acid protein exhibiting 71 and 84% identity wit h the pdc- and fdc-encoded enzymes, respectively. The PAD gene (pad) i s transcriptionally regulated by p-coumaric, ferulic, or caffeic acid; these three acids are the three substrates of PAD. The pad gene was o verexpressed constitutively in Escherichia coli, and the stable purifi ed enzyme was characterized, The difference in substrate specificity b etween this PAD and other PADs seems to be related to a few difference s in the amino acid sequence, Therefore, this novel enzyme should faci litate identification of regions involved in catalysis and substrate s pecificity.