L. plantarum Coumaric Acid Decarboxylase
J. Agric. Food Chem., Vol. 56, No. 9, 2008 3069
T
Figure 1. Comparison of protein sequence of L. plantarum PDC from strains WCFS1 (WCF) (accession no. CAD65735), CECT 748 (748), and LPCHL2
(LPC) (accession no. AAC45282). Asterisks, amino acid identity; dashes, gaps introduced to maximize similarities.
MATERIALS AND METHODS
(pH 6.5) at different temperatures (4-90 °C) for 20 min using
p-coumaric acid (4 mM) as substrate.
T
Materials. L. plantarum CECT 748 (ATCC 14917, DSMZ 20174)
was purchased from the Spanish Type Culture Collection. The 19
phenolic acids analyzed in this study were 7 cinnamic acids, 9 benzoic
acids, and 3 other food phenolic acids, such as phloretic acid (Aldrich
H524006), chlorogenic acid (Sigma C3878), and ellagic acid (Sigma
E2250). The cinnamic acids were p-coumaric acid (Sigma C-9008),
o-coumaric acid (Fluka 28170), m-coumaric acid (Aldrich H23007),
cinnamic acid (Aldrich C8, 085-7), caffeic acid (Sigma C0625), ferulic
acid (Sigma F3500), and sinapic acid (Sigma D7927). The benzoic
acids assayed were benzoic acid (Merck 6391513), syringic acid (Fluka
The temperature stability was assayed by preincubating the purified
PDC in 25 mM phosphate buffer (pH 6.5) at different temperatures
during 1, 2, 3, 5, 12, 24, and 48 h at 17, 22, 30, 37, or 52 °C without
the substrate, and then 4 mM p-coumaric acid was added as substrate
for residual activity assay.
To assay the effects of metals and other additives on L. plantarum
PDC activity, the enzyme (90 ng of protein) was incubated with a 1
mM concentration of different metals and inhibitors and 4 mM
p-coumaric acid in 1 mL of 25 mM phosphate buffer (pH 6.5) at 30 °C
for 20 min. The activity was calculated as relative to the sample
containing no additives.
To validate the method the MINITAB Student test was used. Three
replicate determinations were carried out for each experiment. Relative
standard deviations were e5%.
HPLC Analysis of the Degradation Products from Phenolic
Acids. PDC in phosphate buffer (25 mM, pH 6.5) was incubated during
8
6230), gallic acid (Fluka 48630), salicylic acid (Merck 631), gentisic
acid (Aldrich 149357), veratric acid (Fluka 94872), p-hydroxybenzoic
acid (Fluka 54630), protocatechuic acid (Sigma P5630), and vanillic
acid (Fluka 94770).
The phenolic acid derivatives 4-vinylphenol (Lancaster L10902) and
4
-vinylguaiacol (Lancaster A13194) were used as standards for the
identification of the decarboxylated compounds from p-coumaric and
ferulic acids.
Expression and Purification of the p-Coumaric Acid Decarboxy-
lase. The cloning and overexpression of the pdc gene from L. plantarum
have been previously described (7). Briefly, the pdc gene (coding for
p-coumaric acid decarboxylase) from L. plantarum CECT 748 was
PCR-amplified and inserted into the pURI3 vector by using a restriction
enzyme- and ligation-independent cloning strategy (9).
Protein Assay. Protein concentration was measured according to
the method of Bradford using a protein assay kit purchased from Bio-
Rad Laboratories (Germany) with bovine serum albumin as
standard.
Enzyme Activity Assay. PDC activity was assayed by measuring
the amount of 4-vinylphenol produced from p-coumaric acid. The
standard reaction was performed by adding 30 µL of purified PDC
enzyme (3 µg/mL) into 1 mL of reaction solution containing substrate
at 4 mM in 25 mM phosphate buffer (pH 6.5) and incubating at 30 °C
for 20 min. The assay time was under the linear range of enzyme
reaction. The reaction was terminated by extracting twice with ethyl
acetate. One unit of enzyme activity was defined as the amount of
enzyme that catalyzes the formation of 1 µmol of 4-vinylphenol per
minute. Substrate and enzyme blanks were also prepared in which the
enzyme or substrate was incubated with the buffer.
4
h at 30 °C in the presence of each phenolic acid at 1 mM final
concentration. As control, phosphate buffer containing the phenolic acid
was incubated in the same conditions. The reaction products were
extracted twice with ethyl acetate (Laboratory-Scan, Dublin, Ireland)
and analyzed hy HPLC-DAD.
T
A Thermo (Thermo Electron Corp., Waltham, MA) chromatograph
equipped with a P400 SpectraSystem pump, an AS3000 autosampler,
and a UV6000LP photodiode array detector was used. A gradient of
solvent A (water/acetic acid, 98:2, v/v) and solvent B (water/acetonitrile/
acetic acid, 78:20:2, v/v/v) was applied to a reversed-phase Nova-Pak
C
18 (25 cm × 4.0 mm i.d.) 4.6 µm particle size, cartridge at room
temperature as follows: 0-55 min, 80% B linear, 1.1 mL/min; 55-57
min, 90% B linear, 1.2 mL/min; 57-70 min, 90% B isocratic, 1.2 mL/
min; 70-80 min, 95% B linear, 1.2 mL/min; 80-90 min, 100% linear,
1
.2 mL/min; 100-120 min, washing 1.0 mL/min, and reequilibration
of the column under initial gradient conditions. Detection was performed
by scanning from 220 to 380 nm (10). Samples were injected in
duplicate onto the cartridge after being filtered through a 0.45 µm PVDF
filter (Teknokroma, Spain).
The identification of degradation compounds was carried out by
comparing the retention times and spectral data of each peak with those
of standards from commercial suppliers or by LC-DAD/ESI-MS.
Kinetic analysis was performed under conditions of pH 6.5 and 30 °C
for 20 min in 25 mM phosphate buffer containing substrate (p-coumaric,
caffeic, or ferulic acid) at different concentrations ranging from 0.125
RESULTS AND DISCUSSION
Enzymatic Activity of L. plantarum PDC. L. plantarum
to 48 mM. Values of K
M
were calculated by fitting the initial rates as
T
CECT 748 isolated from pickled cabbage has been shown to
a function of substrate concentration to the Michaelis-Menten equation.
To determine the optimal pH of the PDC, the purified enzyme was
incubated within different pH values (3-10) at 30 °C for 20 min using
p-coumaric acid (4 mM) as the substrate. Citric acid-sodium citrate
buffer (100 mM) was used for pH 3-5, phosphate buffer (100 mM)
for pH 6-7, Tris-HCl buffer (100 mM) for pH 7-8, and 100 mM
glycine-KOH buffer for pH 9 and 10. The optimal temperature was
assayed by incubating the purified PDC in 25 mM phosphate buffer
produce a p-coumarate decarboxylase identical to that produced
by L. plantarum WCFS1 strain, isolated from saliva (7).
Strikingly, the purification of another PDC from L. plantarum
LPCHL2 strain, with a different amino acid sequence, has been
previously reported. Figure 1 shows an alignment of the
available L. plantarum PDC protein sequences. PDCs from L.
T
plantarum WCFS1 and CECT 748 were identical and showed