Biotransformation of isoeugenol catalyzed by growing cells of Pseudomonas putida

Article abstract of DOI:10.3109/10242422.2011.589898

Growing cells of Pseudomonas putida transformed isoeugenol after 5 days of incubation to give mainly vanillin, eugenol, 4-(E)-(3-hydroxyprop-1-enyl)-2- methoxyphenol and the dimeric molecule (+)-4-[2,3-dihydro-7-methoxy-3-methyl-5- (E)-(1-propenyl)-2-benzofuranyl]-2-methoxyphenol (licarin A). The formation of the latter compound from isoeugenol by biotransformation with P. putida is reported here for the first time.

Full text of DOI:10.3109/10242422.2011.589898

Biocatalysis and Biotransformation, July–August 2011; 29(4): 147–150
SHORT COMMUNICATION
Biotransformation of isoeugenol catalyzed by growing
cells of Pseudomonas putida
SANGEETHA VASUDEVAN & SUJATA V. BHAT
Laboratory for Advanced Research in Natural and Synthetic Chemistry,V. G.Vaze College, Mumbai University,
Mumbai, India
Abstract
Growing cells of Pseudomonas putida transformed isoeugenol after 5 days of incubation to give mainly vanillin, eugenol,
4
-(E)-(3-hydroxyprop-1-enyl)-2-methoxyphenol and the dimeric molecule (ϩ)-4-[2,3-dihydro-7-methoxy-3-methyl-5-(E)-
(1-propenyl)-2-benzofuranyl]-2-methoxyphenol (licarin A). The formation of the latter compound from isoeugenol by
biotransformation with P. putida is reported here for the first time.
Keywords: Biotransformation, isoeugenol, vanillin, Peudomonas putida, growing cells
Introduction
The aim of the present study was to examine the
ability of growing cells of P. putida NCIM 2176 to
transform isoeugenol and to study various products
other than vanillin and vanillic acid, which have been
found in previous reports. Transformation products
were identified using GC and GC–MS analyses.
Thus, we report herein the biotransformation of
isoeugenol treated with growing cells of P. putida
NCIM 2176, which yielded vanillin and appreciable
amounts of the dimer (ϩ)-4-[2,3-dihydro-7-methoxy-
Vanillin is the primary component derived from the
vanilla bean. Synthetic vanillin has been widely used
as a flavoring agent in foods, beverages and pharma-
ceuticals and as an intermediate in the production
of pharmaceuticals and other fine chemicals. Syn-
thetic and biotechnological production of vanillin
and vanillic acid has been achieved starting from
isoeugenol. Various microorganisms such as
Pseudomonas putida (Furukawa et al. 2003; Yamada
et al. 2007), Bacillus fusiformis (Zhai et al. 2005; Zhao
et al. 2006) and Nocardia iowensis (Sheshadri et al.
3
-methyl-5-(1-propenyl)-2-benzofuranyl]-2-meth-
oxyphenol (licarin A). The latter compound, which
has been found to display antimicrobial and platelet
aggregation inhibition activities, occurs naturally in
Aristolochia maxima and Myristica fragrans (Aiba &
Gottlieb 1975; Li & Yang 2007). The formation of
cis-dihydrodiols was not observed.
2008) have been reported to degrade isoeugenol to
vanillin and vanillic acid.The cultured cells of Euca-
lyptus perriniana (Orihara et al. 1992) and Capsicum
frutescens root (Suresh et al. 2003) have been found
to transform isoeugenol to give vanillin.
Several aromatic hydrocarbons have been trans-
formed by P. putida to cis-dihydrodiols, which find
wide applications in the synthesis of natural products
Experimental
Chemicals
(Hudlicky et al. 1993;Bui et al. 2000, 2002;Hudlicky
2
005; Reed & Hudlicky 2009). Pseudomonas oleo-
Methylene dichloride (MDC), isoeugenol and chem-
icals required for fermentation were obtained from
commercial suppliers. P. putida (NCIM 2176) was
obtained from the National Collection of Industrial
vorans and P. putida have also been used in the pro-
duction of alcohols from the aliphatic hydrocarbons
(Bosetti et al. 1992).
Correspondence: S.V. Bhat, Laboratory for Advanced Research in Natural and Synthetic Chemistry,V. G.Vaze College, Mumbai University, Mithagar Road,
Mulund (East), Mumbai 400 081, India. Tel:ϩ91-022-21631421 ext 113. Fax:ϩ91-022-2163-4262. E-mail: sujata8b@gmail.com
(
Received 8 April 2011; accepted 17 May 2011)
ISSN 1024-2422 print/ISSN 1029-2446 online © 2011 Informa UK, Ltd.
DOI: 10.3109/10242422.2011.589898

148
S.Vasudevan & S.V. Bhat
Microorganisms, National Chemical Laboratory,
Pune, India.
Analyses of the products
The compounds were identified by comparison of
GC–MS spectra with those in the NIST library and
of standards.The compounds were separated on a 5%
polysiloxane column using aVarian Star 2400 (Varian,
California, USA) CX gas chromatograph coupled to
a Saturn 2 mass spectrometer with a 5% phenylmeth-
ylsiloxanecolumn(dimensions30mϫ0.25μmϫ0.25
Fermentation conditions
P. putida (NCIM 2176) was grown on a nutrient agar
slant. A small portion of the 1- day-old growth was
transferred into two 100-mL flasks each containing 25
mL of nutrient broth.When the solution became tur-
bid both fractions were transferred into a growth cul-
ture medium containing glucose (3.6 g in 10 mL),
potassium dihydrogen phosphate and sodium hydrox-
ide (6.8. g + 1.85 g respectively in 50 mL), ammonium
sulfate (1.2 g in 50 mL) and a trace elements solution
–1
μm) and helium flow rate of 1 mL min . The tem-
perature program was 70°C (1 min), ramp at 3°C
–1
min to 250°C (60 min) and 250°C (4 min). The
quantitative data shown in Scheme 1 are the relative
percentages based on the GC integrated area. NMR
spectra were recorded on a Varian 300 MHz instru-
ment with TMS as internal standard.
(10 mL).The latter contained iron(ii) sulfate (0.04 g),
magnesium sulfate (0.025 g), zinc sulfate (0.002 g),
copper(ii) sulfate (0.004 g), cobalt sulfate (0.0005 g),
citric acid (0.5 g) and boric acid (0.0004 g). Distilled
water (1 L) was added to broth. The culture was
distributed and grown in twelve 250 mL conical flasks,
each containing ~100 mL of medium for 8 h at 25°C
prior to the addition of the substrate.
Products data
Vanillin (2), white solid, mp 81–83°C. EIMS m/z
151 (100), 137 (6.34), 123 (14.28), 109 (15.8), 91
(
3.2), 81 (20.6), 65 (6.34), 53 (9.5); EIMS m/z
1
1
4
64 (100), 149 (35), 131 (20), 121 (12.5),
03 (22.5), 91 (20), 77 (22.5), 65 (7.5), 55 (12.5),
1 (2.5); Coniferyl alcohol: EIMS m/z 180 (24), 137
Incubation of isoeugenol with P. putida
(
100), 122 (16), 94 (8), 77 (4), 65 (4), 51 (4), 43
Isoeugenol (1) (50 mg) was added to the bottom of the
flasks every 12 h with a 1-mL pipette for a period of 4
days.The flasks were removed on the sixth day and fil-
tered to remove the bacterial mass, which was repeatedly
washed five times with 25 mL of water and MDC.The
washings and filtrate were combined and continuously
extracted using MDC.The extract was dried (anhydrous
Na SO ) and the solvent evaporated to give a residue
(8); Licarin A: EIMS m/z 326 (100), 311 (10.8), 297
(2.7), 283 (4.0), 269 (1.3), 251 (1.3), 235 (.2), 221
(.2), 189 (4.0), 176 (2.7), 163 (5.4), 151 (6.7), 137
(8.1), 115 (5.4), 103 (4.0), 91 (5.4), 77 (4.0), 65
(2.7), 51 (1.3).
ϩ
Eugenol (3), liquid. EIMS, m/z: 164 [M ], 149,
131, 121, 103, 91, 77, 65, 55, 41.
4-(E)-(3-Hydroxyprop-1-enyl)-2-methoxyphe-
2
4
ϩ
which was subjected to chromatography on silica.
nol (4), liquid. EIMS, m/z: 180 [M ], 164, 146, 137
OH
OH
OH
OH
OCH3
OCH3
OCH3
5
OCH3
2
P. putida
Growing cells
H
6
α
€
O
γ
OH
1
2 (24%)
3 (3%)
4 (10%)
γ
4
α
€
3
6'
6
5'
1'
2
O
OMe
HO
MeO
2'
5
(16%)
Scheme1. Products obtained from isoeugenol by treatment with P. putida (NCIM 2176).

Biotransformation of isoeugenol by Pseudomonas putida 149
1
(
100), 122 (13), 94 (3), 77 (1), 66 (1), 43 (3). H
Conclusions
NMR (CDCl ), δ (ppm): 3.78 (1H, bs, OH); 3.85
3
The biotransformation of isoeugenol by treatment
with growing cells of P. putida NCIM 2176 yielded
vanillin and an appreciable amount of the dimer (ϩ)-
(
3H, s, –OMe); 4.14 (2H, td, Jϭ5.5 and 1.5 Hz,
Hγ); 6.22 (1H, dt, Jϭ15.9 and 5.5 Hz, Hβ); 6.49
(
1H, dt, Jϭ15.9 and 1.5 Hz, Hα); 6.76 (1H, d,
4
2
-[2,3-dihydro-7-methoxy-3-methyl-5-(1-propenyl)-
-benzofuranyl]-2-methoxyphenol (licarin A).
The formation of (ϩ)-licarin A from isoeugenol
Jϭ8.5 Hz, H ); 6.84 (1H, dd, Jϭ8.1 and 1.9 Hz,
5
H ); 7.04 (1H, d, Jϭ1.9 Hz, H ); 7.64 (1H, s,
6
2
Ar-OH).
by the treatment with growing P. putida cells has not
been previously reported.
(
ϩ)-Licarin A (dehydrodiisoeugenol) (5), white
24
solid. [a ] ϭϩ17.8° (cϭ1.0, CHCl ); reported,
D
3
2
D
0
[
a ] ϭϩ18° (cϭ1.0, CHCl ) (Li & Yang, 2007).
3
Acknowledgements
ϩ
EIMS, m/z: 326 [M ], 311 (11), 202 (6), 151 (6),
1
1
37 (7). H NMR (CDCl ), δ (ppm): 1.37 (3H, d,
Jϭ6.8 Hz, C -Me); 1.86 (3H, dd, Jϭ6.5 and 1.5 Hz,
The authors are thankful to the Sophisticated
Analytical Instrumentation Facility, Indian Institute
of Technology, Bombay, for NMR spectral data.
3
3
Meγ); 3.44 (1H, m, H ); 3.88 (6H, s, –OMeϫ2); 5.10
3
(
1H, d, Jϭ9.0 Hz, H ); 5.65 (1H, s, OH); 6.11 (1H,
2
dq, Jϭ15.5 and 6.5 Hz, Hβ); 6.33 (1H, dd, Jϭ15.5
Declaration of interest: The support from the
Kelkar Education Trust is gratefully acknowledged.
The authors report no conflicts of interest. The
authors alone are responsible for the content and
writing of the paper.
and 1.5 Hz, Hα); 6.76, 6.70 (1H each, s, Ar-H )’
4,6
6
.88 (1H, d, Jϭ8.0 Hz, H ); 6.91 (1H, dd, Jϭ8.0
5'
and 1.5 Hz, H ); 6.98 (1H, d, Jϭ1.5 Hz, H ).
6'
2'
Results and discussion
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24
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D
3
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150
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