Molecular characterization of an inducible p-coumaric acid decarboxylase from Lactobacillus plantarum: gene cloning, transcriptional analysis, overexpression in Escherichia coli, purification, and characterization.
By using degenerate primers designed from the first 19 N-terminal amino acids of Lactobacillus plantarum p-coumaric acid decarboxylase (PDC), a 56-bp fragment was amplified from L. plantarum in PCRs and used as a probe for screening an L. plantarum genomic bank. Of the 2,880 clones in the genomic bank, one was isolated by colony hybridization and contained a 519-bp open reading frame (pdc gene) followed by a putative terminator structure. The pdc gene is expressed on a monocistronic transcriptional unit, which is transcribed from promoter sequences homologous to Lactococcus promoter sequences. No mRNA from pdc and no PDC activity were detected in uninduced cell extracts, indicating that the expression is transcriptionally regulated by p-coumaric acid, which corresponds to an activation factor up to 6,000. The pdc gene was overexpressed constitutively in Escherichia coli, and the recombinant enzyme was purified and characterized.
Cavin
J F
JF
Laboratoire de Microbiologie, US, INRA, ENSBANA, Université de Bourgogne, Dijon, France. cavinjf@u-bourgogne.fr
Barthelmebs
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L
Diviès
C
C
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D015139:Blotting, Southern; D002262:Carboxy-Lyases / Q000096:biosynthesis* / Q000235:genetics* / Q000302:isolation & purification; D002874:Chromosome Mapping; D003001:Cloning, Molecular; D001330:Electronic Data Processing; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D004926:Escherichia coli / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D015723:Gene Library; D007778:Lactobacillus / Q000201:enzymology* / Q000235:genetics*; D017045:Lactococcus / Q000235:genetics; D008969:Molecular Sequence Data; D009693:Nucleic Acid Hybridization; D016366:Open Reading Frames; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D012329:RNA, Bacterial / Q000032:analysis; D011994:Recombinant Proteins / Q000302:isolation & purification; D011995:Recombination, Genetic; D014158:Transcription, Genetic
D012329:RNA, Bacterial; D011994:Recombinant Proteins; D002262:Carboxy-Lyases; C087194:phenylacrylic acid decarboxylase