IJCB 38B(5) 538

Indian Journal of Chcmistry
Vol. 38 B, May 1999, pp. 538-541
Microbial conversion of isoeugenol to vanillin by Rhodococcus rhodochrous
T Chatteljee, B K De & D K Bhattacharyya*
Departmcnt of Chcmical Tcchnology, Uni vc rsity Collcgcs of Science and Technology, Uni vcrsity of Calcu;ta,
92, Acharya Prafull a Chandra Road, Calcutta 700009, India
Received 31 Jllly 1998; accepted (re vised) I Febrtlw )' 1999
Microbial biotransformali on of isocugcnol to van illin by thc strain RhodococclIs rhodochrolls MTCC 289 has Decn stud icd .
Optimum conditions for thi s biotransformation such as tClllpcrature, pH, substrate conccntration and time course are also
establ ished. The strain RhodoCUCCIIS rllOdochrOlls MTCC 289 is shake-cultured for 3 days in a mcdium containing beef extract,
ycast cxtract. pcptonc and NaCl al 30ne. Thc bioconversion is carricd ou t with single substrate add ition, so that thc -ubstratc
concentration in the mediulll do not excccd 1.5% (w/v). The structurc of thc biotransformation product is cstablished by analytical
melhods, such as, FfIR, IH NMR, 1JC NMR and GC-MS spcctromctry. Undcr optimal conditions the yicld of vanill in is /bund to
be 58% (molar), dcmonstrating the cfficicncy of RhodococClis r/lOdochrolls for this biotransformation.
The role of aromatic aldehydes as flavor base
compound in food industries is well-estab li shed.
These aldehydes impart fruity smell in fo ods and
beverages I. Vanillin (4-hydroxy-3-methoxybenzaldehyde) is one such commerciall y ex tremely
important aromati c alde hyde, wh ich is responsible for
a vanilla-type flavor in many food products2.3.
Presentl y global demand for va nillin is met by
syn thet ic chemical method from eugenol, guaiacol or
lignin 4 •s. However, in recent years, th ere is a trend for
growing demand for biotlavors because sole microbial
' I route can prod
noi ogtca
uce" natura I" fl av or67. .
b·lotec h
Due to these inherent benefits of th e bioprocess and
some inev itable demerits of the chemical syntheti c
pathway, like stereochem ically impure prod uct
formati on, poor organoleptic property etc. the
microbial transformation process for th e sy nthesis of
vanillin has been explored during the past few yearss.
Several in vestigators have exam ined the microbial
production of vanillin from feruli c ac id using different
types of microorganisms9. 10 . Plant cell culture can also
produce vani llin but there are some technical
processing problems and the yield is also lowtl . The
li terature reveals th at the gen us Rhodococcus are able
to degrade short and long chain hydrocarbo ns apd
numerous aromatic compounds. Many authors also
by
report
that
biotransformations catalyzed
Rhodococci include enanti oselective synthesis which
make them excellent candidates for biomediatio n
treatments l2.
The present study has therefore aimed at the
syn th esis of va nillin startin g from an easi ly available
compound isoeugenol (2- methoxy-4-pr penylphenol)
by means of mi crobial oxidation catalyzed by
Rhodococcus rhodochrous MTCC 289 (Scheme I).
Our purpose was also to determine the best
experimental conditions to ac hi eve the desired
bioconversion.
Materials and Methods
Microorganism, maintenance and growth
The strain Rhodococcus rhodochrous MTCC 289
was purchased from the Institute of Microbi al
Technology (Cha ndi garh, India) . Stock cultures of
Rhodococcus rhodochrous MTCC 289 were grown at
30°C on agar plates for 2 to 3 days and were stored at
5°C. For growth in liquid medium, th e stock culture
was used to inoculate twenty 250 mL of Erlenmeyer
flask s, each contai nin g 100 mL of ste ril e growth
medium. The liquid medi um contained 1.0 g of beef
extract, 0.7 g of yeast extract, 1.7 g of peptone and 1.7
g of NaCI per litre. The pH of the mediulll was
maintained at 5.8 for storage and maintenance, and
R rhodococcus
30
"c,
3 day s
•
CHO
2
Scheme I
CHATTERJEE el al.: MICROBIAL CONVERSION OF ISOEUGENOL TO VANILLIN
5.0 to 6.0 for treatments. The flasks containing the
inoculated broth were incubated at 30°C with shaking
for 72 hr.
Fermentation procedure
..,..
The resultin g culture broths (contained in twenty
250 mL Erlenmeyer flasks) were divided into five sets
of four flasks. To each set 0.1, 0.2, 0.5, 1.0 and l.5 %
(w/v) of isoeugenol 1 was added to find out the
optimum concentration of isoeugenol , and was
incubated for five days under the same co ndition as
mentioned above. Sampl es fro m the treated cultures
were collected fro m each set after 2, 3, 4 and 5 days
respectively, and analyzed by thin layer chromatography and FflR spectra. Simultaneously, a control
experi ment was carried o ut without mi croo rgani sm by
adding substrate directly into the steri le broth.
Extraction and purification of products
..,.
After cultivation, the cells were removed by
centrifugation (6000 rpm, 10 min ) and th e supernatant
was ex tracted thrice wi th et hyl acetate (30 mL each
ti me). The combined extract was washed with
di stilled water (3x IS mL) and the organic phase was
dried over anh yd. MgS0 4 • The so lvent was th en
removed under red uced press ure (30 mm Hg at 35°C)
to ob tain crude reaction products. The crude reacti on
products were subjected to co lumn chromatography
using glass column (20 mm) o n silicic ac id (60-120
mesh). Column was packed with a slurry of silicic
acid (lOg) in n-hexane and eluted with 120 mL of /1hexane-diethyl ether (80:20 v/v) for iso lation of
bioconversion product. Rechro matography on fresh
si li cic acid column yielded pure reactio n product 2.
Analytical methods
)I.
The reaction product was analyzed by TLC o n
glass plates (20x20 cm), usi ng a 0.2 mm layer of
silica gel G. The plate was developed in 100 mL of
n-hexane-diethyl ether (40:60 v/v) and the ::.pot was
identified by iodine absorption (R r 0.42) . No
convers ion of isoeugenol was observed in control sets
as ev idenced from TLC (R r 0.76).
Chemical structure of the bioconversion product
was identified by FfIR spectroscopy, N~IlR and GCMS. FfIR spectra was obtained by a Perkin-Elmer
1600 Fourier transform spectrometer on KBr disk for
solid bioconversion product and NaCI plate for
isoeugenol. Proton and '3C-NMR spectra in CDCI 3
were obtained by the use of a Bruker AM-300L
539
spectrometer operating at the frequency of 300 MHz.
TMS was used as reference compound. GC-MS
spectra of the compound was obtained from a Hewlett
Packard Model HP 5890 series II GC-MSD apparatus,
equipped with fl ame ionization detector and a HP-I
capillary column . The carrier gas used was heliu m
with a flow rate of 30 mLimin.
Results and Discussion
Identification of isoeugenol bioconversion product
The bioconversion product of isoeugenol, which
was obtained as off-white crystal s after crystallization
in chloroform (0.54 g, 58 %, m.p. 79-80°C at 76 cm of
Hg) , was characterised by FfIR , 'H-NMR, I3C-NMR
and GC-MS spectroscopy (Table I). FflR spectru m
indicated the presence of a hydroxy (OH , 3320 cm")
and aldehyde carbonyl (CH=O, 1675 cm-') groups.
The 'H-NMR spectrum suggested the presence of
aldehyde group (CH=O, 09.80 ppm, s), aromati c
hydroxy (OH, 06.4 1 ppm, s), ':!lethoxy (OCH 3, 03 .94
ppm, s) groups. The aromatic protons, ortho to the
aldehyde functional group, were found to resonate at
07.40 ppm (s, IH) and 7.41 ppm (d, IH) whereas th e
meta proton resonated at 0 7.02 ppm (d, I H, J= 8.0
Hz) . The '3C-NMR spectrum also showed the
formation of aldehyde functional group (0 190.93
ppm). All the spectral data were iden ti cal with
4-hydroxy-3-methoxybenzaldehyde (vanillin), co mpared with authentic sa mple to co nfirm the identity.
Gro wth of Rhodococcus rhodochrous on isoeugeno!
The
growth
medium
inoC'Jlated
wi th
R. rhodochrous MTCC 289 resulted in a visible
increase in biomass with time, and a grow th curve
was constructed by measuring the dry weight of the
biomass formed at intervals (Figure 1). Sharp decline
in the amount of isoeugenol from the medium in the
first three days with the growth of microorganism, as
measured by complete extraction of the substrateproduct mixture followed by column chromatograph ic
Table I-Spectral data of isoeugenol bioconversion product by
the strain Rhodococcus rhodochrous MTCC 289
FT1R
IH NMR :
13C NMR
GC-MS
vmax
(ppm)
:0 (ppm)
m/z
190.93(C H=O), lSI (M3320,2926, 9.80(s,1 H,CHO),
151.68(C),
1,100),
2871,1675, 7.4I(d,IH ,l=6.4),
1590,1300, 7.40(s, IH),
147. II (C),
123(16),
1260,1030 7.02(d,IH , )=8.4), 129.74(C H),
109(20)
6.41 (br,s,1 H)
127.50(CH),
81 (6),
114.35(CH),
51(18)
3.94(s,3H ,
CH 30)
108.72(C),
56.03(CH 3)
o
540
INDIAN J. CHEM. SEC.B, MAY 1999
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Figure I--Growth of Rhodococcus rhodochrolls MTCC 289 on
isocugenol (e) and disappearance of growth substrate from the
medium (<1».
isolation of the substrate to determine its
consumption, was indicative of the ability of
R. rflOdochrous MTCC 289 to metabolize isoeugenol.
After three days, although there was an increase in the
quantity of biomass with time (measured upto five
days) , the bioconversion activity of the microorganism falls as measured by decline in its
consumption, indicating the maximum activity of the
microorganism after three days of inoculation.
Influence of substrate concentration
The growth of R. rhodochrous MTCC 289 in the
medium was stud ied with varying concentration of
isoeugenol between 0.1 to 1.5 gIL in the medium
(Figure 2). It was found that growth of the
microorganism increased steadily with the increasing
concentration of isoeugenol, and reached maximum at
the concentration of 1.0 giL. However, still higher
isoeugenol concentration suppressed the growth of
thi microorganism and use of l.5% (w/v) of growth
substrate lead to poor growth of R.rhodochrous,
which may be due to toxicity of isoeugenol on the
growth of the said biomass.
Effect of pH on bioconversiol1
It is important to know the optimal p H for the
acti vity of a pm1icular microorganism for a specific
bioconversion , since different microorganisms have
different pH optima. The effect of pH on the molar
percentage conversion of isoeugenol by the strai n
R. rhodochrolls MTCC 2g9 is shown in Figure 3. The
°o~.o~----~------~~------L---~
1'0
( gIL)
1-5
Conc~ntrQtion
Figure 2--Effect of concentration of isoeugenol on the activity of
Rhodococclls rhodochrous MTCC 289.
optimum pH was found to be 6.0, at which the molar
percentage yield of vanillin was maximum. Reactions
carried out at pH higher than 6.0, decreased the yield
as evident from Figure 3.
Time course of biotransformation of isoeugenol
To optimize the yield of production of vanillin the
time course of biotransformation of isoeugenol was
studied. The results obtained is shown in Figure 4.
The molar percentage conversion of isoeugenol to
vanillin was found to maximize (58%) in three days
of incubation (after the addi tion of the substrate) .
After three days the molar percentage yield of vanillin
was
found
to
decline,
indicating
no
further conversion of isoeugenol by the strain
R. rhodochrous to vanillin.
Optimum temperature for biotransformation
The effect of temperature for biotransformation of
isoeugenol is shown in Figure 5. It is evident from
the figure th at the optimum reaction temperature is
30°C. Increase of incubation temperature to 35 or
40°C found to reduce the yield of the
biotransfomlation product.
The strain Rhodococcus rhodochrous MTCC 289 is
therefore found to be capable of metabolizing
isoeugenol and thereby producing vanillin (4hydroxy-3-methoxybenzaldehyde) in acceptably good
yield. The product is valuable in creating food
fla vours and hence may be potentially important froln
the viewpoint of commercial production.
541
CHATIERJEE et al.: MICROBIAL CONVERSION OF ISOEUGENOL TO VANILLIN
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...&
-;;
II
>
u
-.:
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~~
5·0
6·0
5·5
7·0
100
pH .
120
Time,hr
Figure 3--Effect of pH on the bioconversion of isoeugenol by
RllOdococcus rhodochrous MTCC 289.
Figure 5-Temperature dependence of the biotransformation of
isoeugenol to vanillin by RhodococclIs rhodochrous MTCC 289.
80.----------------------------,
is funded in part by the
Commission, New Delhi, India.
6.0
Grants
References
I
c:
0
'iii
t>
University
40
2
c:
0
u
3
-.:
20
4
5
20
40
60
80
100
120
Time,hr
Figure 4-Time dependence of the biotransformation of isoeugenol.to vanillin by Rhodococcus rhodochrous MTCC 289 .
6
7
8
9
10
Acknowledgement
11
We would like to thank Ms . A Dan of the
Department of Polymer Science and Technology for
performing FTIR spectra of the compound. This work
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