MOLECULAR CHARACTERIZATION OF AN INDUCIBLE P-COUMARIC ACID DECARBOXYLASE FROM LACTOBACILLUS-PLANTARUM - GENE CLONING, TRANSCRIPTIONAL ANALYSIS, OVEREXPRESSION IN ESCHERICHIA-COLI, PURIFICATION, AND CHARACTERIZATION
Jf. Cavin et al., MOLECULAR CHARACTERIZATION OF AN INDUCIBLE P-COUMARIC ACID DECARBOXYLASE FROM LACTOBACILLUS-PLANTARUM - GENE CLONING, TRANSCRIPTIONAL ANALYSIS, OVEREXPRESSION IN ESCHERICHIA-COLI, PURIFICATION, AND CHARACTERIZATION, Applied and environmental microbiology, 63(5), 1997, pp. 1939-1944
By using degenerate primers designed from the first 19 N-terminal amin
o 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 clon
es in the genomic bank, one was isolated by colony hybridization and c
ontained a 519-bp open reading frame (pde gene) followed by a putative
terminator structure. The pdc gene is expressed on a monocistronic tr
anscriptional unit, which is transcribed from promoter sequences homol
ogous to Lactococcus promoter sequences, No mRNA from pdc and no PDC a
ctivity were detected in uninduced cell extracts, indicating that the
expression is transcriptionally regulated by p-coumaric acid, which co
rresponds to an activation factor up to 6,000, The pde gene was overex
pressed constitutively in Escherichia coli, and the recombinant enzyme
was purified and characterized.