Ferulic acid metabolism was studied in cultures of two micromycetes ... Pestalotia palmarum catabolized ferulic acid via 4-vinylguaiacol, vanillin, vanillyl alcohol, ...
Vol. 55, No. 9
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Sept. 1989, p. 2391-2398
0099-2240/89/092391-08$02.00/0 Copyright C) 1989, American Society for Microbiology
Metabolism of Ferulic Acid by Paecilomyces variotii and Pestalotia palmarum REGINE STEIMAN,1 KARL-ERIK ERIKSSON2 Laboratoire de Botanique, Cryptogamie, Biologie Cellulaire et Genetique, 38243 Meylan, France,' and Department of Biochemistry, The University of Georgia, Athens, Georgia 306022
MOHAMMED RAHOUTI,1
FRANQOISE SEIGLE-MURANDI,1* AND
Received 21 February 1989/Accepted 14 June 1989
Ferulic acid metabolism was studied in cultures of two micromycetes producing different amounts of phenol oxidases. In cultures of the low phenol oxidase producer Paecilomyces variotii, ferulic acid was decarboxylated to 4-vinylguaiacol, which was converted to vanillin and then either oxidized to vanillic acid or reduced to vanillyl alcohol. Vanillic acid underwent simultaneously an oxidative decarboxylation to methoxyhydroquinone and a nonoxidative decarboxylation to guaiacol. Methoxyhydroquinone and guaiacol were demethylated to yield hydroxyquinol and catechol, respectively. Catechol was hydroxylated to pyrogallol. Degradation of ferulic acid by Paecilomyces variotii proceeded mainly via methoxyhydroquinone. The high phenol oxidase producer Pestalotia palmarum catabolized ferulic acid via 4-vinylguaiacol, vanillin, vanillyl alcohol, vanillic acid, and methoxyhydroquinone. However, the main reactions observed with this fungus involved polymerization reactions.
and benzidine were obtained from Fluka (Buchs, Switzerland). Guaiacol, o-anisidine, and bis(trimethylsilyl)trifluoroacetamide were from E. Merck AG, Darmstadt, Federal Republic of Germany, and syringaldazine was from Sigma Chemical Co., St. Louis, Mo. 1,2,4-Trihydroxybenzene(hydroxyquinol) was from Aldrich Chemical (Strasbourg, France), and vanillin, catechol, pyrogallol, a-naphthol, pcresol, tyrosine, and tetramethylsilane were from Prolabo (Paris, France). 4-Vinylguaiacol was purified from cultures of Pestalotia palmarum grown on ferulic acid. Phenol oxidase production. Phenol oxidases were produced by growth of Paecilomyces variotii and Pestalotia palmarum on solid malt extract medium (1.5%) by the methods of Kaarik (21), Harkin and Obst (15), and Harkin et al. (14). Culture conditions. The synthetic medium of Galzy and
Ferulic acid is a precursor in lignin biosynthesis (11) and has also been shown to be a product of lignin degradation (18). It has earlier been used as a model in lignin biodegradation studies (8, 13). The metabolism of ferulic acid by bacteria generally leads to the production of vanillic acid and vanillin (22, 31, 32). Similar results were reported by Henderson (17) and Ishikawa et al. (19), using fungi. Other authors found high levels of reduced products, including coniferyl alcohol (25). In the same way, Gupta et al. (13), using Sporotrichum pulverulentum, showed that only small amounts of vanillic acid and methoxyhydroquinone were obtained while >80% of the metabolites were accounted for as coniferyl aldehyde, dihydroferulic acid, and dihydroconiferyl alcohol. The functions of the reduced products remain unclear. The purpose of this investigation was to study along which metabolic pathways two species of Fungi Imperfecti with different phenol oxidase activities degrade ferulic acid. This ability was first established for the two species. A scheme for the metabolism of ferulic acid by Paecilomyces variotii is proposed.
TABLE 1. Production of phenol oxidases by Paecilomyces variotii and Pestalotia palmarum on solid malt extract medium Specificity
MATERIALS AND METHODS Microorganisms. Two strains belonging to the mycological collection of the Laboratory of Cryptogamy (Collection Mycology Pharmacy Grenoble [CMPG]) were used in this study: Paecilomyces variotii Bainier (CMPG 116), isolated from green walnut (30); and Pestalotia palmarum Cooke (CMPG 222), obtained from the Museum Mycotheque, Paris, France. Stock cultures of both organisms were maintained on solid malt extract medium (1.5%) at 4°C. Paecilomyces variotii and Pestalotia palmarum were selected from among 826 micromycetes, belonging to the CMPG, for their ability to degrade ferulic and syringic acids (submitted for publication). Chemicals. Ferulic acid, vanillic acid, and gallic acid, vanillyl alcohol and veratryl alcohol, methoxyhydroquinone, *
Tyrosinase
Reagents
p-Cresol Tyrosine
Color reactiona P. variotii
P. palmarum
0 0
0 0
Phenol oxidases Gallic acid Pyrogallol
0
++ ++
Benzidine a-Naphthol
0 +
+++ +++
Guaiacol o-Anisidine
0 0
+++ +
Laccase
Syringaldazine
0
++
Peroxidaseb
Syringaldazine + H202
0
c
Laccase or
peroxidase
+
0, No reaction; +, + +, + + +, intensity of color reactions. b If no reaction was obtained without H202, H202 was added to test for peroxidase activity. -, Peroxidase activity was detected only when laccase activity was negative.
Corresponding author. 2391
APPL. ENVIRON. MICROBIOL. RAHOUTI ET AL.APLENRN.Mcoo.
2392
IN~~~~~~~~
acid
~anicobo
Iday7
Iday
2days
incub-tioo incubation
incubation
32
24 28
4 8 1 16 20 32 36 ~44 .A2
2428 Al
t~10
hydro#om 2 days
3 days incubation
i day incubation
incubation
6
5~~~~~~~~1
3 V,
~ ~
~ ~ ~~8? ~~~~~~~7
7 4
112
8
A4
-4
8'w12 AS
8 12
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8
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lday
incubation
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A$
~~~~~4 Iday
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hdoa i day incubation
:,.
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10
K~~~~~~~~~~~~~1
12~~ ~~~~~~1 13
50 7 90 1012 12 11830 RI
4
8 52
12
4
8 53
214
4 8 12 8 12 8~~~~~~~~~~~4 ~ a5
Retention time( ) FIG. 1. HPLC chromatograms of cultures of (A) Paecilomyces variotii and (B) Pestalotia palmarum incubated with different substrates (500 mg/liter): ferulic acid, Al and Bi; 4-vinylguaiacol, A2; vanillin, A3 and B2; vanillic acid, A4 and B3; vanillyl alcohol, AS and B4; methoxyhydroquinone, A6 and B5; guaiacol, A7; catechol, A8. Metabolites for A and B panels were as follows: hydroxyquinol, 1; pyrogallol, 2; methoxyhydroquinone, 3; vanillyl alcohol, 4; catechol, 5; vanillic acid, 6; vanillin, 7; guaiacol, 8; cis-ferulic acid, 9; residual trans-ferulic acid, 10; 4-vinylguaiacol, 11; polymer, 12; transformation products of methoxyhydroquinone, 13; unknown compound,?9.
METABOLISM OF FERULIC ACID
VOL. 55, 1989
2393
x
HB
HA C C
HX
R
ABX system
7, 7
9
8
7
5
6 6
4
3
2
1
0
( pp m )
FIG. 2. NMR spectrum of 4-vinylguaiacol.
Slonimsky (12) was used. Glucose was added to a concentration of 1%. Cultures were grown in 250-ml or 1-liter Erlenmeyer flasks containing 100 or 300 ml of medium, respectively. After sterilization by autoclaving for 20 min at 121°C, the medium was inoculated with 5 x 105 spores per 100 ml of culture medium. Cultures were incubated with shaking (180 rpm) at 24°C. The phenolic compounds (2.5%) were dissolved in 96% ethanol, sterilized by filtration through a syringe filter (Millipore Corp., Bedford, Mass.) and added to 2-day-old cultures to a final concentration of 0.05%. The incubation times, given in the text and figures, indicate the culture time after addition of phenolic substrates to the culture. Extraction of metabolites. The culture medium was filtered off, and the aqueous phase was acidified to pH 2.0 with 6 M HCl and extracted with ethyl acetate. Combined extracts were dried over anhydrous Na2SO4 and evaporated to dryness at 40°C under vacuum. The residue was dissolved in a small amount of methanol for high-performance liquid chromatography (HPLC) or in ethyl acetate for thin-layer chromatography (TLC) analysis. TLC. TLC was carried out in solvent systems I (chloroform-acetic acid, 20:1) and II (benzene-dioxane-acetic acid,
90:25:4). Silica plates from Merck were used: Kieselgel 60 PF254 (Art. 7747) for preparative work and Kieselgel 60 F254 (Art. 5562) for quantitative determinations. Aromatic compounds were detected by UV light at 254 and 360 nm. Aromatic aldehydes were detected by spraying with 2,4dinitrophenylhydrazine (0.1% in perchloric acid, 30%). Phenolic compounds were detected after spraying with diazotized sulfanilic acid (29). For nuclear magnetic resonance (NMR) spectrometry, unsprayed bands were scraped from TLC plates and extracted with ethyl acetate. HPLC. HPLC was performed with a liquid chromatograph (Waters Associates, Inc., Milford, Mass.) equipped with a pump (model 510), an injector (model U6K), and a UV detector (Lambda-Max model 481) operating at 279 nm. The separation column (model H220; Societe Francaise de Chromato Colonne, Paris) was 4.6 mm in inside diameter by 250 mm in length, packed with ODS (Hypersil, 10 ,um). The mobile phase consisted of methanol, water, and acetic acid (30:70:1, vol/vol/vol). The flow rate was 1.4 mllmin. Chromatographic peaks were identified by comparison with the reference. GC-MS. Gas chromatography (GC) was carried out with a Girdel instrument (model 32) equipped with a capillary
2394
APPL. ENVIRON. MICROBIOL.
RAHOUTI ET AL.
COOH
CH HC
CH 1u2 CH
CHO
COOH
OCH3
OH
OH
rrans-ferulic acid
-OH
OH
Iproducts
OH
Vanillin
4-Vinylgusiacol
cleavage
OH3
OCH
OH
Ring-
Hydroxyquinol
Methoxyhydroqui noms
Vanillic acid
OCH3
CH20H
OH Gusiacol
OH
HO
OH
OH
OH
Pyrogallol
Catechol
OCH3 OH
Vanillyl alcohol
FIG. 3. Proposed pathway for degradation of trans-ferulic acid by Paecilomyces variotii. Dashed lines indicate tion.
column (Chrompack CP Sil 5 CB, 25
m
by 0.32 mm). The
temperature of injection was 250°C. The column was heated at 100°C for 1 min and then programmed to rise 10°C/min to 280°C. The carrier gas was He at a flow rate of 1.5 ml/min. Mass spectrometry (MS) was performed with a Nermag (model R10-10C) mass spectrometer.
The compounds, dissolved in dry pyridine, were silylated with bis(trimethylsilyl)trifluoroacetamide for 2 h at room temperature. Identification of products was based on comparison with authentic compounds or by interpretation of their mass spectra. Spectral analysis. 1H-NMR spectra were recorded on a Bruker 100-MHz instrument with Fourier transform, using deuterated chloroform with tetramethylsilane as internal standard. Infrared spectra were obtained with a Pye Unicam SP3-100 spectrophotometer. UV spectra were measured with a Beckman Acta CII spectrophotometer. COOH
I
tC-H (3)
(2)
H-I
a
postulated transforma-
To determine whether the substrate, ferulic acid, was in the cis or trans configuration, it was analyzed by NMR and UV spectrometry. It was shown that the acid was in the trans form. RESULTS Phenol oxidase production. Production of phenol oxidases by Paecilomyces variotii and Pestalotia palmarum was examined from 14-day-old cultures grown on solid malt extract medium. Color formation was examined 1 day after addition of substrates to the solid medium. In the case of syringaldazine, the time of reaction was only 15 min. Results are shown in Table 1. No reaction was observed with tyrosine-specific substrates, which indicates that these fungi do not produce tyrosinase. However, syringaldazine gave a positive reaction in Pestalotia palmarum cultures, indicating that this fungus produces laccase or peroxidase. The extent and intensity of the color reactions indicate that Paecilomyces variotii and Pestalotia palmarum have weak and strong phenol oxidase activity, respectively. Ferulic acid metabolism by Paecilomyces variotii. The initial pH value of the Galzy and Slonimsky medium was 4.4. This medium became more acidic during the degradation of TABLE 2. 'H-NMR spectral data of trans-ferulic acid and the unidentified product B from Fig. lAla Compound
Proton(s)
trans-Ferulic acid H(2)
Type
Doublet
Doublet H(3) Singlet Methoxyl aromatics Multiplet
cis-Ferulic acid (product B) FIG. 4. Structures of trans-ferulic acid (top) and unidentified product B from Fig. lAl (bottom). See Table 2.
H(2)
H(3) Methoxyl
Doublet Doublet
Singlet
aromatics Multiplet a
Chmcl Chical shift (ppm)
Coupling constant (z
6.22 7.64 3.87 6.99
J2 3 J3-2
= 15.8
5.77 7.0 3.84 6.84
J2-3
= 12.9
= 17.2
Structures of trans-ferulic acid and product B are given in Fig. 4.
VOL. 55, 1989
METABOLISM OF FERULIC ACID
2395
TABLE 3. Appearance of metabolites in culture media of Paecilomyces variotii with trans-ferulic acid (500 mg/liter) Amt (mg/liter) after given period of incubation (h)
Phenolic compound
trans-Ferulic acid cis-Ferulic acida 4-Vinylguaiacola Vanillin Vanillyl alcohol Vanillic acid Methoxyhydroquinone Guaiacol
Catechol
14
18
22
24
400 ++++
290 ++++
130.4 +++
+
+++
++++
6 8 16 15.8 1.4 14
12.8 20 62 30.6 8.4 17.4
2.4 1.2 3.2 2.4 0.8 1.8
38
48
67
96
4.4 +++
1.6 ++
1.3 +
0.3 +
0 0
++++
++
++
++
++
+
+
15.2 7.4 144 44 15.6 16
6.8 4 3 50.6 20 10.4
5 3.4 2 53.5 25 4
0.7 2 1.5 35 17 2.2
0 1.4 0.4 15 6 2
0 0.9 0 3.7 2 1.2
0 0.7 0 1.6 0 1.1
120
150
168
0
0
0
0
0 0 0
0 0 0 1.4 0
0.7
a 4-Vinylguaiacol and cis-ferulic acid were not assayed.
ferulic acid by Paecilomyces variotii. After 7 days of incubation, the pH was 2.3. The yellow color of the culture medium became slightly darker. After 67 h of incubation of Paecilomyces variotii, the culture medium was analyzed by HPLC. Figure lAl reveals that trans-ferulic acid was completely transformed into other phenolic compounds. Methoxyhydroquinone (compound 3), guaiacol (compound 8), and an unidentified product, A (compound 11) (retention time, 33.7 min), were found to be the major metabolites. Other compounds were formed but in minor quantities: vanillyl alcohol (compound 4), catechol (compound 5), vanillin (compound 7), and an unidentified product, B (compound 9) (retention time, 13 min); two unidentified compounds (?) were present in trace amounts. The unidentified products A and B were further analyzed by TLC. They had retardation factors of 0.82 and 0.48, respectively, in system I (see Materials and Methods) and brown-yellow and violet colors after being sprayed with diazotized sulfanilic acid. The products were purified by preparative TLC. The solvents used for the preparative work were systems I and II for product A and system I for product B. MS analysis indicated molecular ions of mlz 150 and 194, respectively. Analysis by UV spectrometry showed a maximum UV absorbance at 258 nm in ethyl acetate for product A and one at 316 nm in ethanol for product B. The infrared absorption spectrum of product A showed the presence of an aromatic ring (1,500 and 3,050 cm-'), a vinyl double bond (925 and 1,600 cm-'), a hydroxyl group (3,500 cm-'), and a methoxyl group (2,820 cm-'). The NMR spectrum (Fig. 2) indicated one methoxyl group (83.84, s, 3H), one hydroxyl group (85.55, s, 1H), three aromatic protons (86.81,
m,
3H), and
a
vinyl group including HB
(85.05, dd, JB-X = 10.8 Hz), HA (85.51, dd, JA-X = 17.5 Hz), and Hx (86.57, q, JX-A = 17.5 Hz). This spectrum showed
the ABX system typical of RHC CH2 compounds. The NMR analysis of product B is presented in Table 2. The results obtained by infrared and NMR analyses allowed the identification of compounds A and B: product A is 4-vinylguaiacol, and product B is cis-ferulic acid. The rapid catabolism of trans-ferulic acid by Paecilomyces variotii is evidenced in Table 3. After 1 day of incubation, 99% of trans-ferulic acid was transformed. The cis and trans isomers of this acid were completely degraded after 3 days of incubation. cis-Ferulic acid, formed from the trans isomer, did not accumulate in the culture medium but was transformed at the same rate as trans-ferulic acid. 4-Vinylguaiacol, vanillin, vanillyl alcohol, vanillic acid, and catechol could be detected in culture medium after 14 h of incubation, but they reached peak levels only after 22 or 24 h. Later, these levels decreased and the compounds were no longer present after 3 or 7 days of incubation. Methoxyhydroquinone and guaiacol were detected also after 14 h of incubation, reaching a maximum after 2 days and then gradually diminishing. Metabolism of detected metabolites from ferulic acid by Paecilomyces variotii. With 4-vinylguaiacol as substrate (Fig. 1A2), vanillin (compound 7) was the main compound formed in the cultures after 33 h of incubation. Vanillyl alcohol (compound 4), vanillic acid (compound 6), and guaiacol (compound 8) were also detected, but in minor quantities. Vanillin was completely transformed after 24 h of incubation (Fig. 1A3 and Table 4). Methoxyhydroquinone (compound 3), vanillyl alcohol (compound 4), and vanillic acid (compound 6) were the main products of transformation. A small amount of guaiacol (compound 8) was also formed. Some 85% of vanillic acid was metabolized by this fungus after 1 day of culture (Fig. 1A4 and Table 4). The major products formed were methoxyhydroquinone (compound 3) =
TABLE 4. Metabolism of different phenolic compounds by Paecilomyces variotii Substrate (500 mg/liter)
Vanillin Vanillic acid Vanillyl alcohol Methoxyhydroquinone Guaiacol Catechol Pyrogallol a ND, Not determined.
Incubation time (days)
1 1 2 1 3 2 2
Vanillin
Vanillic Vaci acid
Vanillyl Vanlchl alcohol
1.8 0 2.2 0 0 0 0
48 73.8 16 0 0 0 0
168 6.8 138 0 0 0 0
Metabolites (mg/liter) MethoxyMethydroqui
hydroquinone 36.6 78 25.6 227 0 0 0
Guaiacol
Catechol
Pyrogallol
9.4 41.4 9 0 26 0 0
NDa
