616 Hua et al.
Asian J. Chem.
Ferulic acid was dissolved in 1 M NaOH solution (1:10,
w/v) and the pH value was adjusted to 8.5 with 6 M HCl.
Ferulic acid solution was directly added to the mature cultures
described above. Control experiments were performed in
phosphate buffer without the addition of cells (pH 7.0, 0.06
M). The biotransformation was conducted at 200 rpm, 30 ºC
and 50 mL medium in 500 mL flask. Triplicate experiments
were performed under the same condition. At regular time
intervals, samples were removed from flasks to determine the
concentrations of ferulic acid and products.
Isolation and identification of biotransformation
product: Biotransformation product was isolated by prepa-
rative TLC and identified by gas chromatography-high
4
resolution mass spectrometry (GC-HRMS) . The product was
further confirmed by nuclear magnetic resonance, which was
analyzed on an Avance 600 spectrometer (Bruker) operating
at 600 MHz.
8
7
6
5
4
3
1
Fig. 1. H NMR spectrum of 4-vinyl guaiacol
Analytical methods: Cell growth was measured spectro-
photometrically at 620 nm. Dry cell weight was calculated
from the optical density (OD620 nm) with a linear correlation
-1
factor (OD620 nm of 1 = 0.4 g L dry cell weight).
Concentrations of ferulic acid and 4-vinyl guaiacol were
estimated by HPLC (Agilent 1100 series, Hewlett-Packard).
Before HPLC analysis samples were centrifuged at 12,000
rpm for 10 min. The supernatant was diluted with distilled
water and filtered with aqueous phase filter film of 0.45 µm.
The HPLC system was equipped with a KR100-5 C18 column
(
150 mm × 4.6 mm × 5 µm, Kromasil, Sweden) and a UV
detector operating at 254 nm. The column was eluted with a
mixture of methanol and water (90:10, v/v) at a flow rate of
0
.5 mL min-1 and 30 ºC. Quantitative data were obtained by
comparing the peak areas of the query compounds with those
of standards of known concentrations.
Fig. 2. Biotransformation of ferulic acid to 4-vinyl guaiacol (4-VG) by
Bacillus pumilus S-1. The biotransformation medium was composed
RESULTS AND DISCUSSION
-
1
-1
of 20 g L glucose, 5 g L yeast extract, 10 g L peptone and 10 g
-1
-1
Four major pathways of ferulic acid metabolism have been
found in different strains, which are nonoxidative decarbo-
xylation, side chain reduction, coenzyme-A-independent
L NaCl (pH 7.2). The biotransformation was carried out on a rotary
shaker with 180 rpm at 30 ºC and 50 mL medium in 500 mL flask.
The values were means of three replicates, and the error bars
indicated standard deviations. Symbols represent: , ferulic acid;
2
,6
deacetylation and coenzyme-A-dependent deacetylation .
Biotransformation of ferulic acid by B. pumilus S-1 was investi-
gated and the main metabolite was identified by GC-HRMS
and NMR. The mass spectrum from GC-HRMS (m/z 150.0683,
,
4-vinyl guaiacol
Fig. 3 shows the metabolic pathways of ferulic acid in
different strains. Some strains (Streptomyces setonii, Pseudo-
monas fluorescens, Amycolatopsis sp.) convert ferulic acid
directly to vanillin and vanillic acid through deacetylation,
which was further metabolized through guaiacol or protocat-
9 10 2
calculated mass 150.0681), with a formula of C H O , was
consistent with that of 4-vinyl guaiacol according to the
standard library (WILEY275). 4-Vinyl guaiacol was further
1
approved by H NMR spectrum (Fig. 1). Therefore, ferulic
2,6
echuic acid . While in some other strains (Debaryomyces
acid was metabolized through 4-vinyl guaiacol in B. pumilus
S-1.
hansenii, Candida versatilis, Schizophyllum commune, Lacto-
bacillus plantarum, Enterobacter sp., Aspergillus niger, Sporot-
richum thermophile, B. coagulans), 4-vinyl guaiacol was found
Time course of ferulic acid biotransformation and 4-
vinyl guaiacol production is shown in Fig. 2. The highest
as an intermediate in the ferulic acid metabolism, which was
l5,6,9-13
-1
concentration of 4-vinyl guaiacol obtained was 0.72 g L and
the molar yield was 93.1 %. All of the ferulic acid added was
dissimulated within 3 h, which was faster than many of the
further converted to vanillin or 4-ethylguaiaco
. Though
the fate of 4-vinyl guaiacol was different, it was quickly trans-
formed to other vanilla flavour compounds. Therefore, most
of the produced 4-vinyl guaiacol was not accumulated.
Different from former reports, 4-vinyl guaiacol was not
further transformed and maintained unchanged in the following
10
reports to date. Before this study Karmakar et al. reported
the fastest degradation speed of ferulic acid by organisms, in
-1
which 0.95 g L ferulic acid was dissimilated in 7 h by a B.
coagulans strain. The result suggested that the biotransfor-
mation ability of B. pumilus S-1 was better than the report.
9 h during the biotransformation. The result showed that B.
pumilus S-1 had a weak biotransformation ability for 4-vinyl