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Biocatalytic Synthesis of Vanillin

Article in Applied and Environmental Microbiology · March 2000

DOI: 10.1128/AEM.66.2.684-687.2000 · Source: PubMed Central

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APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Feb. 2000, p. 684–687 Vol. 66, No. 2
0099-2240/00/$04.00⫹0
Copyright © 2000, American Society for Microbiology. All Rights Reserved.

Biocatalytic Synthesis of Vanillin

TAO LI AND JOHN P. N. ROSAZZA*
Division of Medicinal and Natural Products Chemistry, and Center for Biocatalysis and
Bioprocessing, College of Pharmacy, University of Iowa, Iowa City, Iowa 52242
Received 1 July 1999/Accepted 17 November 1999

The conversions of vanillic acid and O-benzylvanillic acid to vanillin were examined by using whole cells and
enzyme preparations of Nocardia sp. strain NRRL 5646. With growing cultures, vanillic acid was decarboxylat-
ed (69% yield) to guaiacol and reduced (11% yield) to vanillyl alcohol. In resting Nocardia cells in buffer, 4-O-
benzylvanillic acid was converted to the corresponding alcohol product without decarboxylation. Purified No-
cardia carboxylic acid reductase, an ATP and NADPH-dependent enzyme, quantitatively reduced vanillic acid to
vanillin. Structures of metabolites were established by 1H nuclear magnetic resonance and mass spectral analyses.

Vanilla is one of the most widely used flavors in food indus-
try. Natural vanilla extracted from the cured pods of the flow-
ers of Vanilla planifolia has an estimated net value of more
than $ 1 billion annually (18). Vanillin (3-methoxy-4-hydroxy-
benzaldehyde) is the most important organoleptic component
in vanilla. More than 12,000 tons of synthetic vanillin are pro-
duced each year from petrochemical and wood pulping indus-
tries (7). Strong market demand for natural and environmen-
tally friendly products has spawned efforts to produce vanillin
by microbial transformation from natural substrates, including
phenolic stibenes (27), eugenol (19, 26), and ferulic acid (14,
17).
Ferulic acid [3-(4-hydroxy-3-methoxyphenyl)-propenoic
acid] is an extremely abundant plant product available from
corn kernel hulls obtained from wet milling (21). Vanillic acid
is the major product obtained by ␤-oxidation of ferulic acid by
species of Bacillus (6), Pseudomonas (11, 23), Polyporus (9),
Rhodotorula (8), and Streptomyces (17, 22). Thus, vanillic acid
is an abundant, readily available precursor for the biocatalytic
synthesis of vanillin.
Microbial reductions of carboxylic acids are widely observed
in bacteria and fungi (1, 2, 4, 5, 10, 12, 13, 24, 25). One com-
mon mechanism of enzymatic carboxylic acid reduction in-
volves activation of carboxyl groups with ATP to highly reac-
tive carbonyl-AMP intermediates which are readily reduced by
NADPH to aldehydes (5, 13, 16) (see Fig. 1). Both the adeny-
lation and carbonyl-AMP reduction steps are catalyzed by a
single enzyme (5, 13, 16). A separate alcohol oxidoreductase
reduces the resulting aldehydes to their corresponding alcohol
products (16). Although carboxylic acid reductases have been
purified from two species of Nocardia (13, 16), properties of
the reported enzymes are significantly different.
It is possible to envision an enzymatic synthesis of vanillin
beginning either with ferulic acid (8, 17, 21) or with vanillic
acid as the starting materials. Such approaches are attractive
because the natural carboxylic acid starting materials are abun-
dant and inexpensive and are soluble in aqueous media. Fur-
thermore, reductions of carboxylic acids to aldehydes are dif-
ficult to achieve by chemical means, and the reduction of
vanillic acid to vanillin has not been widely reported.
* Corresponding author. Mailing address: Division of Medicinal and
Natural Products and Center for Biocatalysis and Bioprocessing, Col-
lege of Pharmacy, University of Iowa, Iowa City, IA 52242. Phone:
(319) 335-8842. Fax: (319) 335-8766. E-mail: john-rosazza@uiowa
.edu.
In this report, we describe biotransformations of vanillic acid
by growing cells of Nocardia sp. strain NRRL 5646 and a pure
aryl aldehyde oxidoreductase (carboxylic acid reductase) from
this organism. We also show that a complicating vanillic acid
decarboxylation reaction can be overcome by use of a vanillic
acid derivative as a substrate in whole-cell bioconversions.
MATERIALS AND METHODS
Biocatalysts. Nocardia species strain NRRL 5646 is maintained in the Uni-
versity of Iowa College of Pharmacy culture collection and is grown and main-
tained on slants of Sabouraud-dextrose agar or sporulating agar (ATCC no. 5
medium). The carboxylic acid reductase used in enzymatic reductions of vanillic
acid was isolated from Nocardia sp. strain NRRL 5646 by our procedure (16).
The enzyme used in this work was pure, based on sodium dodecyl sulfate-
polyacrylamide gel electrophoresis analysis (15).
Chemicals. Vanillic acid (4-hydroxy-3-methoxybenzoic acid) (compound 1a
[Fig. 1]), vanillin (4-hydroxy-3-methoxybenzaldehyde) (compound 3a), vanillyl
alcohol (4-hydroxy-3-methoxybenzyl alcohol) (compound 4a), guaiacol (2-me-
thoxyphenol) (compound 2), and benzyl bromide were purchased from Aldrich
Chemical Co. (Milwaukee, Wis.). Other chemicals were purchased from Sigma
Chemical Co. (St. Louis, Mo.) and Aldrich unless otherwise indicated.
4-O-Benzylation of vanillic acid was carried out by adding 11 ml of 1 N NaOH
to 900 mg of vanillic acid (5.4 mmol) in 16 ml of ethanol, followed by dropwise
addition of 1 g (5.8 mmol) of benzyl bromide in 2.5 ml of ethanol over 60 min.
The reaction mixture was refluxed for 2 h before being poured into 150 ml of
water and acidified to pH 2 with 6 N HCl. The precipitate was collected by
filtration and crystallized in xylene to afford 802 mg of compound 1b (3.1 mmol,
56% yield). The structure of compound 1b was confirmed by low-resolution
electron impact mass spectrometry (EIMS) with the following parameters: m/z
258 (8%, M⫹) and 91 (100%, benzonium ion); 1H nuclear magnetic resonance
(NMR) at 360 MHz (d6-acetone), ␦H 3.83 (3H, s, OCH3), 5.21 (2H, s, C-7⬘), 7.12,
7.14 (1H, d, J ⫽ 8.3 Hz, H-5), 7.33 to 7.52 (5H, m, H-2⬘, -3⬘, -4⬘, -5⬘, and -6⬘), 7.58,
7.57 (1H, d, J ⫽ 2.0 Hz, H-2), and 7.63 to 7.65 (1H, dd, J ⫽ 8.3, 2.0 Hz, H-6).
4-O-Benzylvanillin (compound 3b) was prepared by refluxing 32 g of vanillin
(210 mmol) with 26 ml (213 mmol) of benzyl bromide, and 30 g of potassium
bicarbonate in 200 ml of ethanol for 5 h. After cooling, the filtrate was crystal-
lized at 4°C overnight. Recrystallization in ethanol gave 45.3 g (187 mmol, 85%
yield) of compound 3b confirmed by EIMS: m/z 242 (4%, M⫹), 91 (100%,
benzonium ion); 1H NMR at 360 MHz (CDCl3), ␦H 3.95 (3H, s, OCH3), 5.25
(2H, s, C-7⬘), 7.00, 6.98 (1H, d, J ⫽ 8.3 Hz, H-5), 7.34 to 7.43 (7H, m, H-2, -6, -2⬘,
-3⬘, -4⬘, -5⬘, and -6⬘), and 9.84 (1H, s, H-7).
4-O-Benzylvanillyl alcohol (compound 4b) was prepared by refluxing a mixture
of 15 g of potassium bicarbonate, 16 g (104 mmol) of vanillyl alcohol, and 18.3 g
of benzyl bromide (107 mmol) in 115 ml of ethanol for 3 h. The reaction workup
was like that for 4-O-benzylvanillic acid to give pure product. Recrystallization
from cyclohexane gave 17 g of compound 4b (64% yield). The low-resolution,
fast atom bombardment (FAB) mass spectrum gave m/z 244 (17%, M⫹), 227
(9%, M⫹ ⫺ OH), and 91 (100%, benzonium ion); 1H NMR at 360 MHz
(CDCl3), ␦H 3.89 (3H, s, OCH3), 4.58 (2H, s, C-7), 5.16 (2H, s, H-7⬘), 6.76 to 6.79
(1H, dd, J ⫽ 8.3, 1.9 Hz, H-6), 6.82, 6.84 (1H, d, J ⫽ 8.2 Hz, H-5), 6.91, 6.92 (1H,
d, J ⫽ 1.7 Hz, H-2), and 7.26 to 7.43 (5H, m, H-2⬘, -3⬘, -4⬘, -5⬘, and -6⬘).
Chromatography. Thin-layer chromatography (TLC) was carried out on silica
gel GF254 plates (E. Merck, Darmstadt, Germany). Developed chromatograms
were directly visualized under 254-nm UV light to observe fluorescence quench-
ing. Phenolic compounds were also visualized with Pauly’s reagent, which con-
sisted of solution A (0.5% sulfanilic acid in 2 N HCl), solution B (0.5% sodium

684
VOL. 66, 2000
FIG. 1. Whole-cell and enzyme transformation pathways for vanillic acid and O-benzyl vanillic acid by Nocardia sp. strain NRRL 5646.
nitrite in water), and solution C (1 N potassium hydroxide in 50% ethanol-
water). Developed plates were first sprayed with a freshly prepared equal-volume
mixture of solutions A and B and then sprayed with solution C before being
heated with a heat gun to develop colors from yellow to orange. Aldehydes such
as vanillin were also detected with 2,4,-dinitrophenylhydrazine (0.4% in 2 N HCl
[wt/vol]) spray. With dichloromethane-acetonitrile-formic acid (75:25:1 [vol/vol/
vol]), Rf values for vanillic acid, vanillin, vanillyl alcohol, and guaiacol were 0.5,
0.4, 0.8, and 0.9, respectively. With dichloromethane-acetic acid-benzene (100:
2:2 [vol/vol/vol]), Rf values for 4-O-benzylvanillic acid (compound 1b), 4-O-
benzylvanillin (compound 3b), and 4-O-benzylvanillyl alcohol (compound 4b)
were 0.7, 0.8, and 0.2, respectively.
High-performance liquid chromatography (HPLC) was performed with a Shi-
madzu liquid chromatograph equipped either with a LC-10AD system controller,
four FCV-10AL pumps, and a photodiode array UV-Vis detector (SPD-M6A) or
with a SCL 6-B system controller, two LC-6A pumps, and a variable-wavelength
UV detector. Eluted peaks were detected at 273 to 276 nm or at 290 nm and
identified by comparison with authentic compounds. Separations carried out
under isocratic conditions over a Versapack C18 column (250 by 4.6 mm, 10-␮m
particle size; Alltech, Deerfield, Ill.) with acetonitrile-water-formic acid (20:80:1)
at a flow rate of 0.9 ml/min gave retention volumes (Rv) of 5.0 ml for vanillyl
alcohol, 8.1 ml for vanillic acid, 13.2 ml for vanillin, 16.1 ml for o-anisic acid, and
18.7 ml for guaiacol. Standard curves for vanillic acid (compound 1a), vanillin
(compound 3a), vanillyl alcohol (compound 4a), and guaiacol (compound 2)
were established over the range of 0.3 to 8 ␮g. For quantitation of 4-O-benzyl-
vanillic acid biotransformations, separations were carried out under isocratic
conditions over a 10 ␮m C18 Versapack column (300 by 4.1 mm; Alltech) with
acetonitrile-water-formic acid (30:70:1) at a flow rate of 2 ml/min. Rv values were
8.1 ml for p-anisic acid, 21.9 ml for 4-O-benzylvanillyl alcohol (compound 4b),
33.4 ml for 4-O-benzylvanillic acid (compound 1b), and 61.1 ml for 4-O-benzyl-
vanillin (compound 3b). Standard curves for these compounds were established
over the range of 0.3 to 3 ␮g.
Spectroscopy. Mass spectra were obtained by using a Trio-1 MS linked with a
5890 Hewlett-Packard gas chromatograph. EIMS was performed at an ionization
voltage of 70 eV. For direct inlet probe analysis, the probe temperature was set
at 30°C for 1 min, raised to 300°C at 150°C/min, and held at 300°C for 10 min for
analysis. For gas chromatography-mass spectrometry, separations were carried
out on an OV-1 capillary column (10 m by 0.25 mm; 1-␮m film thickness) with
helium as carrier gas at a flow rate of 20 ml/min. The column oven temperature
was held at 50°C for 1 min, raised to 250°C at a rate of 15°C/min, and held at that
temperature for 10 min. Injector and detector temperatures were 220 and 270°C,
respectively. FAB mass analyses were performed by using a ZAB-HF mass
spectrometer (VG Analytical, Inc.). Ionizing matrices were either 3-nitrobenzyl
alcohol or thioglycerol.
NMR spectra were obtained with a Bruker WM 360-MHz high-field spectrom-
eter equipped with an IBM Aspect-2000 processor. Tetramethylsilane was used
as the internal standard. Chemical-shift values are reported in parts per million
(ppm), and coupling constants (J values) are given in hertz. Abbreviations for
NMR are as follows: s, singlet; d, doublet; t, triplet; dd, doublet of doublets; and
m, multiplet.
Biotransformation of vanillic acid by Nocardia isolates. A preparative scale
incubation was grown by our standard two-stage incubation protocol (3) in
200-ml volumes of sterile soybean flour-glucose medium held in stainless-steel-
capped, 1-liter, DeLong culture flasks. The medium contained 20 g of glucose,
5 g of yeast extract, 5 g of soybean flour, 5 g of NaCl, and 5 g of K2HPO4 per liter
BIOCATALYTIC SYNTHESIS OF VANILLIN 685
in distilled water and was adjusted to pH 7.2 with 6 N HCl before being auto-
claved at 121°C for 20 min. Cultures were incubated by shaking at 250 rpm at
28°C on G25 Gyrotory shakers (New Brunswick Scientific Co., Edison, N.J.). A
10% inoculum derived from a 72-h-old first-stage culture was used to initiate
second-stage cultures, which were incubated as described above. After 24 h of
incubation in the second stage, 160 mg of vanillic acid (in 2 ml of dimethyl
sulfoxide) was added to each of three flasks. Control cultures included everything
except vanillic acid.
Samples were removed at various time intervals, adjusted to pH 2 with 6 N
HCl, extracted with 1 ml of ethyl acetate, and centrifuged for 1 min at 2,500 rpm.
Organic layers were removed, evaporated to dryness, and reconstituted in 0.5 ml
of methanol. The samples were spotted onto TLC plates for analysis.
Samples of 3 ml were also taken over the course of vanillic acid transformation
for HPLC analysis. To each sample, 200 ␮l of o-anisic acid solution (30 mg/ml in
acetonitrile) was added as an internal standard. Culture samples were then
acidified to pH 2 with 6 N HCl, 1 ml was loaded onto solid-phase extraction
cartridges (Chem Elut CE 1001, 1-ml aqueous solution capacity; Varian, Harbor
City, Calif.). After 5 min, cartridges were extracted twice with 3 ml each of
dichloromethane-acetonitrile (90:10). These organic extracts were combined and
used for HPLC analysis.
The microbial reaction was terminated 40 h after the addition of vanillic acid.
The culture was acidified to pH 2 with 6 N HCl. After centrifugation to remove
cells and other solids, the supernatant from 200 ml of culture was loaded onto a
solid-phase extraction cartridge (Chem Elut 1200, 200-ml aqueous solution ca-
pacity). After 5 min of equilibration, the cartridge was washed three times with
200 ml of hexane and then three times with 200 ml of dichloromethane. The
organic extracts were separately concentrated by rotary evaporation to give 45
mg of oil (from hexane) and 90 mg of residue (from dichloromethane). The
dichloromethane extracts were streaked onto a 1-mm-thick preparative TLC
plate (20 by 20 cm), which was developed in dichloromethane-acetonitrile-formic
acid (75:25:1 [vol/vol]). The separated bands after development were scraped
from the plate, and compounds were eluted from silica gel with a mixture of
dichloromethane and acetonitrile (70:30 [vol/vol]). The band extracts were
checked for purity by TLC, and concentrated for mass spectrometry and 1H
NMR spectroscopy.
Reduction of vanillic acid to vanillin by Nocardia carboxylic acid reductase.
The reduction was carried out in a reaction mixture of 200 ml of 50 mM Tris-HCl
buffer (pH 7.5) containing 34 mg of vanillic acid, 59 mg of NADPH, 110 mg of
ATP, and 100 ␮g of purified carboxylic acid reductase (0.6 U) (16). The reaction
mixture was incubated at 30°C with gentle shaking (50 rpm) for 24 h after which
the entire reaction mixture was loaded onto a solid-phase extraction cartridge
(Chem Elut 1200, 200-ml aqueous solution capacity). After 5 min of equilibra-
tion, the cartridge was washed twice with 100 ml of hexane, followed by two
washes 100 ml of dichloromethane. The dichloromethane solution was concen-
trated to less than 2 ml. The solution was transferred to a vial, and the solvent
was removed with a stream of nitrogen to give pure vanillin as determined by
TLC, EIMS, and 1H NMR analyses.
Nocardia resting cell conversions of 4-O-benzylvanillic acid. Nocardia sp. was
grown by using the standard two-stage incubation protocol. After 24 h in the
second-stage culture, 5 mg of benzoic acid per ml was added as an inducer for
carboxylic acid reductase (16). The cells were harvested by centrifugation at
8,000 ⫻ g for 20 min at 24 h after the benzoate addition. Cells were washed twice
with 0.9% saline solution and pelleted before use.
For preparative scale biotransformations, 2.8 g of cells (wet weight) were
686 LI AND ROSAZZA APPL. ENVIRON. MICROBIOL.

M⫹ ⫺ CH3 ⫺ H2O), and 81 (100%, M⫹ ⫺ CH3 ⫺ CO), and

FIG. 2. Time course of growing cell biotransformations of vanillic acid by
Nocardia sp. strain NRRL 5646. Triplicate sample averages for vanillic acid (F),
vanillyl alcohol (■), vanillin (䊐), and guaiacol (E) had variances of not more
than ⫾5%.
suspended in 400 ml of 50 mM Tris HCl (pH 7.4) containing 1% glucose, 10 mM
MgCl2, and 5 mM phosphate. 4-O-Benzylvanillic acid (0.7 g in 2 ml of dimethyl
sulfoxide) was added to the cell suspension, which was incubated with shaking
at 150 rpm at 30°C for 15 min. The reaction mixture was acidified to pH 2,
centrifuged to remove cells and other unwanted solids, and loaded onto two
solid-phase extraction cartridges (Chem Elut 1200, 200 ml on each). After 5 min
of equilibration, each cartridge was washed three times with 200 ml of ethyl
acetate. The ethyl acetate solution was concentrated by rotary evaporation to
give 670 mg of crude product, which was reconstituted to 2.0 ml with dichlo-
romethane and loaded onto a silica gel column (0.9 by 20 cm). 4-O-Benzylvanillin
(170 mg, 24% yield) was eluted with dichloromethane, while 4-O-benzylvanillyl
alcohol (210 mg, 30% yield) was eluted with dichloromethane-benzene-acetic
acid (100:4:6 [vol/vol/vol]). These compounds were spectrally (EIMS and NMR)
and chromatographically identical to synthetic standards described earlier.
4-O-Benzylvanillin obtained from the preparative-scale incubation was dis-
solved in 4 ml of ethanol, 5 ml of concentrated HCl was added, and the mixture
was refluxed for 2 h. The solvent was removed by rotary evaporation, and the
residue dissolved in 0.5 ml was purified by silica gel column chromatography (0.9
by 5 cm) eluted with dichloromethane to give 65 mg of pure vanillin as deter-
mined by TLC, EIMS, and 1H NMR.
Analytical scale biotransformations were conducted in the same medium as for
preparative biotransformations. Suspensions of cells (0.05 g [wet weight]/ml)
containing 0.6 mg of 4-O-benzylvanillic acid per ml were incubated at 30°C.
Samples (1 ml) taken during the course of incubation received 50 ␮l of p-anisic
acid (4 mg/ml in methanol) as an internal standard. Samples were acidified to pH
2 with 6 N HCl and loaded onto solid-phase extraction cartridges (Chem Elut CE
1001), which were equilibrated for 5 min and then eluted twice with 3 ml of ethyl
acetate. Pooled eluates were analyzed by HPLC.
an 1H NMR spectrum at 360 MHz (CDCl3), ␦H 3.76 (3H, s,
OCH3), 6.80, 6.85 (3H, m, H-3, H-4, H-6), and 6.90 to 6.93
(1H, m, H-5). These properties were identical to those for an
authentic sample of guaiacol.
The dichloromethane extract gave two compounds (4 mg
each) by preparative TLC. The compound at Rf 0.77 was spec-
trally identical to authentic vanillin (compound 3a): low-reso-
lution EIMS, m/z 152 (88%, M⫹), 151 (100%, M⫹ ⫺ H), 137
(6%, M⫹ ⫺ CH3), 123 (13%, M⫹ ⫺ CHO); 1H NMR at 360
MHz (CDCl3), ␦H 3.96 (3H, s, OCH3), 6.22 (1H, s, OH), 7.03,
7.06 (1H, d, J ⫽ 8.5, H-5), 7.26 to 7.44 (2H, m, H-2, H-6), and
9.83 (1H, s, CHO). The compound at Rf ⫽ 0.35 was identical
to vanillyl alcohol (compound 4a): low-resolution EIMS, m/z
154 (28%, M⫹), 136 (50%, M⫹ ⫺ H2O); 1H NMR at 360 MHz
(CDCl3), ␦H 3.90 (3H, s, OCH3), 4.61 (2H, s, CH2OH), and
6.82 to 6.92 (3H, m, H-2, H-5, H-6).
Reduction of vanillic acid by purified Nocardia carboxylic
acid reductase. The enzymatic reaction gave 8 mg (53 ␮mol) of
vanillin by TLC, EIMS, and 1H NMR analyses, all of which
were identical to an authentic sample of vanillin. Based upon
the estimated amount of NADPH consumed (51 ␮mol,
[change in optical density at 340 nm]) during the course of the
reaction, the yield of vanillin was essentially quantitative. The
remainder of the vanillic acid substrate was unreacted.
Transformation of 4-O-benzylvanillic acid with resting cells
of Nocardia sp. By HPLC, benzoate-induced cells of Nocardia
sp. rapidly transformed 4-O-benzylvanillic acid to metabolites
within 40 min (Fig. 3). In the first 15 min, the aldehyde 3b
accumulated in transient fashion to a maximum 30% yield (175
␮g/ml) after which compound 3b was further quantitatively
reduced to the corresponding alcohol 4b.
In the preparative scale incubation reaction, TLC analysis
indicated that benzoate-induced Nocardia cells produced two
major metabolites corresponding to 4-O-benzylvanillin (com-
pound 3b, 170 mg, 24% yield) and 4-O-benzylvanillyl alcohol
(compound 4b, 210 mg, 30% yield). These products were spec-

RESULTS
Biotransformation of vanillic acid with growing cells of No-
cardia sp. HPLC analyses of vanillic acid biotransformation by
Nocardia sp. (Fig. 2) showed that almost all vanillic acid was
consumed within 48 h to give guaiacol (69% yield) by decar-
boxylation and vanillyl alcohol (11% yield) by carboxylic acid
reduction. Only traces of vanillin were detected in this analyt-
ical experiment. To properly characterize vanillic acid metab-
olites, a preparative scale incubation gave 45 mg of a sample
from the hexane extract of a 40-h biotransformation culture.
The product from the hexane extract gave a single spot by TLC
FIG. 3. Time course of resting cell biotransformations of O-benzylvanillic
(Rf ⫽ 0.89), was orange with Pauly’s phenolic spray reagent, acid by Nocardia sp. strain NRRL 5646. Triplicate sample averages for O-ben-
gave an EIMS spectrum data as follows: m/z (percent relative zylvanillic acid (F), O-benzylvanillyl alcohol (■), and O-benzylvanillin (䊐) had
abundance) 124 (78%, M⫹), 109 (92%, M⫹ ⫺ CH3), 91 (44%, variances of not more than ⫾5%.
 VOL. 66, 2000
trally and chromatographically comparable to synthetic com-
pounds 3b and 4b. Thus, 4-O-benzylvanillic acid was reduced
first to the aldehyde 3b and subsequently to the alcohol 4b
(Fig. 1). To confirm the identity of compound 3b, chemical
removal of the benzyl group from the microbial metabolite
gave 65 mg of vanillin identified by spectral and chromato-
graphic comparisons with authentic vanillin.
DISCUSSION
Many microbial transformation approaches have been used
to produce vanillin. In most cases, the yields of vanillin were
low, and times for biotransformation reactions are long (7, 14,
17, 19, 26, 28).
The synthesis of vanillin from ferulic acid has been reported
with several microorganisms (6, 9, 17, 22, 23). In P. acidivorans
(23), manometric studies suggested that vanillin was produced
by a two-step reaction including water addition to the cin-
namoyl side chain and subsequent retro-aldol condensation to
give vanillin. In general, yields were low because vanillin was
further oxidized to vanillic acid which was O-demethylated to
protocatechuic acid or decarboxylated to guaiacol. The addi-
tion of sulfhydryl compounds to ferulic acid transformation me-
dium increased vanillin accumulations by Pseudomonas putida
ATCC 55180 (14). However, the mechanism by which sulfhy-
dryl reagents improved vanillin yields was not clear. Mulheim
and Lerch recently described a relatively high yield direct con-
version of ferulic acid to vanillin by cultures of Streptomyces
setonii (17). With this organism, yields of vanillin were directly
proportional to ferulic acid concentrations in culture media.
In our laboratory, the mechanism of ferulic acid transforma-
tion to vanillic acid by R. rubra IFO 889 occurred by ␤-oxida-
tion (8). The ready availability of vanillic acid by this process,
and the discovery of a vanillic acid reduction pathway in No-
cardia sp. strain NRRL 5646 suggested the potentials for whole
cells or the pure carboxylic acid reductase from this microor-
ganism for vanillin synthesis.
With pure Nocardia carboxylic acid reductase, the ATP- and
NADPH-dependent reduction of vanillic acid was quantitative,
yielding only vanillin and no complicating byproducts. In whole-
cell Nocardia biotransformation reactions, vanillic acid decar-
boxylation to guaiacol was the major complicating pathway.
The identification of guaiacol and vanillyl alcohol as metabo-
lites confirmed that Nocardia sp. strain NRRL 5646 possesses
two different metabolic pathways for the biotransformation of
vanillic acid. These pathways are (i) decarboxylation to guaia-
col and (ii) reduction to vanillin and further reduction to va-
nillyl alcohol (Fig. 1).
We also observed vanillic acid decarboxylation in Rhodo-
torula rubra (8) and in Bacillus pumilis (21). Deuterium isotope
incorporation studies revealed that the decarboxylation reac-
tion involved the enzymatic tautomerization of vanillic acid
through the para-hydroxyl group to a quinoid intermediate be-
fore decarboxylation. Blocking the p-phenolic group was con-
sidered as a possible means of preventing the enzymatic tau-
tomerization reaction and subsequent decarboxylation. To test
this hypothesis, we employed the benzylic functional group,
which is readily removed by simple acid treatment. However,
other alternative blocking groups, such as esters, could func-
tion equally well. 4-O-Benzylvanillic acid was not decarboxy-
lated and was quantitatively reduced to 4-O-benzylvanillin and
4-O-benzylvanillyl alcohol. We know that Nocardia sp. also
contains another reductase that converts aldehydes to alcohols
View publication stats
BIOCATALYTIC SYNTHESIS OF VANILLIN 687
(unpublished data). We are now exploring the possibility of clon-
ing the carboxylic acid reductase in a suitable biocatalytic host.
ACKNOWLEDGMENTS
Tao Li acknowledges support through a Center for Biocatalysis and
Bioprocessing Fellowship, and we are grateful for financial support
from USDA through the Byproducts for Biotechnology Consortium.
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