Gene cloning, transcriptional analysis, purification, and characterization of phenolic acid decarboxylase from Bacillus subtilis

Jean François Cavin, Véronique Dartois, Charles Diviès

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Abstract

Bacillus subtilis displays a substrate-inducible decarboxylating activity with the following three phenolic acids: ferulic, p-coumaric, and caffeic acids. Based on DNA sequence homologies between the Bacillus pumilus ferulate decarboxylase gene (fdc) (A. Zago, G. Degrassi, and C. V. Bruschi, Appl. Environ. Microbiol. 61:4484-4486, 1995) and the Lactobacillus plantarum p-coumarate decarboxylase gene (pdc) (J.-F. Cavin, L. Barthelmebs, and C. Divies, Appl. Environ. Microbiol. 63:1939-1944, 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 partially sequenced and was found to contain a 528-bp open reading frame coding for a 161-amino-acid protein exhibiting 71 and 84% identity with the pdc- and fdc-encoded enzymes, respectively. The PAD gene (pad) is transcriptionally regulated by p-coumaric, ferulic, or caffeic acid; these three acids are the three substrates of PAD. The pad gene was overexpressed constitutively in Escherichia coli, and the stable purified enzyme was characterized. The difference in substrate specificity between this PAD and other PADs seems to be related to a few differences in the amino acid sequence. Therefore, this novel enzyme should facilitate identification of regions involved in catalysis and substrate specificity.

Original languageEnglish (US)
Pages (from-to)1466-1471
Number of pages6
JournalApplied and environmental microbiology
Volume64
Issue number4
DOIs
StatePublished - Apr 1998
Externally publishedYes

All Science Journal Classification (ASJC) codes

  • Biotechnology
  • Food Science
  • Applied Microbiology and Biotechnology
  • Ecology

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