CHAPTEU
3
MATEnIALS AND METHODS
MATERIALS AND METHODS
3.1
CHEMICALS
Eugenol,
isoeugenol,
eugenol
isoeugenol
dehydrodiisoeugenol,
Ferul ic
ac id
was
Shinshu
University,
acetate,
ferulic
protocatechuic
and
acetate,
acid
obta i ned
as
Japan
bisdehydro
acid,
were
vanillin,
used
a
and
bisdehydro
as
gift
vanillin
isoeugenol,
vanillic
the
from
eugenol,
substrates.
Dr. Koh j i
was
acid
Tadasa,
purchased
from
Sisco Research Laboratories, Bombay.
Isolation of eugenol from clove oil
3.1.1
Eugenol
01.
cove
1
1
by
was
.
treat lng
well
cleaned
250 cc
was
introduced.
extracted
Wl. t h
round
To
hydroxide
solution
5 minutes.
Then
it
bottom flask,
75 ml
added
was
contents
funnel
aqueous
and
of
the
layer
the
and
heated
flask
were
non-phenolic
was
f i 1 tered
a
Into a
of clove
1 N
shaken
on
potassium
thoroughly
water
oil
bath
for
with
The flask was cooled and
poured
portion
through
57
10 ml
aqueous
occasional shaking for 10 minutes.
the
commercially available
potassl. urn h y d rOXl. d e. 140
this
was
from
a
was
into
a
separatory
separated.
f i 1 ter
paper
and
The
the
58
filtered
acid
was
solution
until
The
sulpha te,
8.5 g
acidified
mixture
t rans fer red
ether.
get
the
was
to
a
ether
was
with
dilute
strongly acid
separa tory
extract
was
funnel
dried
to
and
with
hydrochloric
litmus.
This
ext rac t ed
wi t h
anhydrous
f i 1 tered and evaporated under reduced
of
eugenol
as
a
pale
yellow
liquid,
pressure to
boiling
at
141
255°C.
3.1.2
Preparation of eugenol acetate
Into
a
dry
50 ml
RB
flask
1.5 ml
eugenol,
pyridine and 3 ml acetic anhydride were placed.
for
sodium
6
hours,
poured
into
extracted
shaking
intermittently.
ice-cold water,
with
ether
and
stirring
the
4 ml
It was kept
The mixture was
continuously.
ether
extract
was
It
then
was
washed
several times first with 1 N hydrochloric acid and then with
5% sodium bicarbonate solution.
It was dried with anhydrous
sodium sulphate and evaporated under vacuum to get 2.14 g of
eugenol
acetate.
Eugenol
acetate
is
a
pale
yellow
liquid
142
freezing at 30°C.
3.1.3
Preparation of isoeugenol
Isoeugenol was prepared by the reaction of eugenol
.
wlth
.
potasslum
h y d rOXl. d e. 143
In
a
250 ml
RB
flask
10 g
59
eugenol was mixed with 4.5 g KOH
was
then
removed at
in 10 ml of water.
Water
reduced pressure.
Three
80-90°C under
gram diethylene glycol and 1 g triethanolamine were added to
it and the mixture was heated for about 5 minutes at 160°C.
The
me1 t
dilute
the
was
sulphuric
run
acid.
into water and
It
was
extracted
neut ra1ised
with
with
benzene
and
benzene extract was distilled under reduced pressure to
yield
9 g
at 266°C.
3.1.4
isoeugenol
ethanol
placed
as
a
pale
yellow
liquid,
boiling
141
Preparation of bisdehydroeugeno1
In
and
and
precipitate
was
cool ed
a
250 ml
6 g
ferric
shaken
was
RB
4 ml
chloride
in
well.
filtered
recrystallised
flask,
from
The
and
30 ml
mixture
washed
ethanol
eugenol
to
was
with
give
in
of
20 ml
water
cooled
40%
and
ethanol.
1.62 g
of
were
the
It
bisdehydro-
eugenol having a melting point of 106°C.
3.1.5
Preparation of isoeugeno1 acetate
Into a dry 50 ml
RB flask 1.5 ml
isoeugeno1,
pyridine and 3 ml acetic anhydride were placed.
for
6
hours
shaking
occasionally.
The
mixture
into ice-cold water, stirring continuously.
4 ml
It was kept
was
poured
It was filtered
60
and
the
prec i pi ta t e
followed
was
by 5% NaHC0
wi t h
1 N hydroch lor i c
ac id
solution to give 2.31 g of isoeugenol
3
142
acetate melting at 79°C.
3.1.6
washed
Preparation of bisdehydroisoeugeno1 and
dehydrodiisoeugeno1
In a
ethanol
and
250 ml
7 g
RB
ferric
flask,
5 g
chloride
in
placed and shaken well for 5 minutes.
off
under
reduced
was
transferred
The
ether.
sulpha te ,
It
was
melting
to
ether
a
separatory
extract
and
sUbjected
to
under
at
of
water
and
132°C were obtained.
was
were
Ethanol was distilled
funnel
dried
of
and
wi th
cooled.
extracted
anhydrous
It
wi th
sodi urn
con cent ra t ed under reduced pressure.
column
section
158°C
30 ml
in 30 ml
pressure and the mixture was
f i 1 t ered
described
isoeugenol
3.5.3.
1.48 g
chromatography
as
will
be
0.70 g bisdehydroisoeugenol
dehydrodiisoeugenol
melting
at
Both products were recrystallised from
ethanol.
3.1.7
Preparation of vanil1ic acid
Vanillic acid was prepared by fusing vanillin with
potassi urn
hydroxide
conditions. 144
In
and
a
sodi urn
hydroxide
stainless
steel
under
beaker
controlled
of
250 ml
61
capacity equipped
hot
plate,
with
1.8 g
a
of
stirrer
sodium
and
heated
hydroxide
by
an
electric
pellets,
1.8 g
potassium hydroxide pellets and 1 ml water were placed.
mixture was stirred well and heated to 160°C.
was
turned
off
and
1.5 g
of vanillin was
of
The
The hot plate
added
in
portions
at a rate sufficient to maintain the reaction temperature at
about 190°C.
hot
plate
was
stirring.
about
removed
When
150°C,
mixture
with
Stirring was continued for
was
15 ml
and
the mixture
5 more minutes,
allowed
10 ml
then
The
cool
with
the temperature of the mixture came down to
water
cooled
was
to
added
room
and
stirred
temperature
6 N hydrochloric acid keeping
stirring.
to
the
ppt.
formed
was
f i 1 t ered,
well.
and
The
acidified
in an ice bath and
washed
wi t h
wat er
It was recrystallised from water-acetone mixture
and dried.
which yielded, 1.42 g vanillic acid melting at 210°C.
3.1.8
Preparation of protocatechuic acid
Protocatechuic
vanillin
In
a
sodium
stainless
with
a
3.5 g
was
with
stirrer
KOH
pellets
stirred
and
hydroxide
steel
and
was
acid
beaker
heated
and
heated
1 ml
to
and
of
by a
prepared
.
potasslum
250 ml
hot
plate
1 g
Then
1.6 g
fusing
.
145
hydroxlde.
capacity,
water were placed.
160°C.
by
NaOH
equipped
pellets,
The mixture
vanillin
was
62
added
in
about
190°C,
until
the
at
portions
250°C
10 ml
15 ml
ice
to
rate
stirring
to
was
reached
to
the
for
2
hot
temperature at
Heat
was
applied
Temperature was
plate was
stirring.
dissolve
acid.
hours,
mixture
the
the
removed
At
kept
and
about
mixture.
the
150°C,
It
was
room temperature and acidified with
The mixture was
filtered
and
the
cooled
in an
precipitate
was
It was dried and recrystallised from
washed with ice water.
water-acetone
250°C.
with
to
6 N hydro chI or i c
bath
the
cool
added
cool
to maintain
simultaneously.
5 minutes,
allowed
water
allowed
a
temperature
for
mixture
at
to
yield
0.95 g
protocatechuic
acid
melting at 200°C.
3.2
SOURCE OF MICROORGANISMS
As the stock cultures in our laboratory were found
to
be
inactive
on
eugenol,
microorganisms
that
transform
eugenol were isolated from the local soil using the elective
culture
organisms
technique.
used
in
As
this
mentioned
study,
previously,
Pseudomonas
obtained as a gift from Dr.Kohji Tadasa.
one
of
the
aeruginosa
was
63
3.2.1
Screening of soil samples
The
screening
of
the soil samples was done
in the
following mineral salts medium.
NH N0
4
3
1 g
KH 2 P 04
0.9 g
Na HP0
2
4
0.55 g
MgS0 7H O
4
2
0.2 g
CaC1 2H O
2 2
0.1 g
FeS0 7H O
4
2
0.1 g
Tap water
1000 ml
pH
7.2
For
preparing
isoeugenol was added as
To get a
specified).
mineral
the
broth
0.1%
carbon source
solid medium,
2%
(v/v)
eugenol/
(unless otherwise
(w/v)
agar was added
to the above broth.
About
habi ta t
of
clove
tap water
and
and
of
10 ml
2 g
trees
shaken
the
of
soil,
was
collected
suspended
in
It
was
thoroughly.
supernatent
was
from
the
natural
of
st er i le
100 ml
allowed
transferred
to
to
settle
100 ml
of
64
the mineral medium containing eugenol/isoeugenol as the sole
carbon
source.
This was
rotary shaker.
One ml
another
of
100 ml
incubated at
mineral
medium
isoeugenol and incubated at 30°C.
mineral
agar
the
eugenol/
in sterile petri dishes with eugenol-
(EMA)/isoeugenol-mineral
on
containing
After 2 days 1 ml of the
The plat es were i ncubat ed at 30 ° C for
developed
2 days on a
of the broth was then transferred to
the
broth was pour-plated
30°C for
plates
were
agar
(IMA)
2 days.
isolated
medium.
The col oni es
and
purified
streaking repeatedly over fresh EMA/IMA plates.
by
Two strains
of bacteria, one growing on EMA and the other growing on IMA
slants,
were
CUAC 20
and
obtained.
CUAC 30
The
pure
respectively,
strains
were
designated
transferred
to
as
EMA
and IMA slants respectively.
3.2.2
Isolation of the strain CUAC 10
strain
The
contaminant
glucose.
fying
it
in
The
to
a
CUAC 10
mineral
broth was
pH 2.
The
was
medium
extracted
ether
isolated
as
containing
with
extract
ether,
was
a
chance
eugenol
and
after acidi-
concentrated
and
subjected to thin layer chromatography studies, which showed
the
presence
of
one
more
spot
other
than
that
of
eugenol.
65
This
isoeugenol
strain
in
a
CUAC 10
mineral
was
medium
also
able
containing
to
transform
isoeugenol
and
glucose.
But this strain was not able to grow on EMA or IMA
plates.
So
nutrient
it
was
purified
by
streaking
repeatedly
over
agar plates and the pure strain was transferred
to
nutrient agar slants.
3.2.3
Maintenance and preservation of the cultures
The
agar
s 1 ants
CUAC 20
culture
an d
and
CUAC 10
·
nutrl.ent
was
un d
er '
Ol. 1 • 146
agar
Pseudomonas
maintained
aeruginosa
were
on
nutrient
The
cultures
maintained
slants and CUAC 30 was maintained on IMA slants.
were
sterilised
for 15 minutes.
slants
and
by
autoclaving
15 Ibs
EMA
The media
pressure
(l210C)
The cultures were transferred to appropriate
incubated
refrigerator.
at
on
for
They were
one
day
before
transferred
to
storing
fresh
them
slants
in
every
month to keep them viable.
3.3
IDENTIFICATION OF THE MICROORGANISMS
The
gener i c
1 evel
isolated
organisms
based
thei r
characters. 147 ,148
on
were
identified
morpho log i cal
and
to
the
bi ochemi cal
66
3.3.1
Morphological characters
These
tests
spore
staining,
included
staining,
the
cell
morphology,
chromogenesis
and
gram
motility.
Chromogenesis was tested by growing the cultures on nutrient
agar.
3.3.2
Motility
was
tested
by
the
hanging
drop
method.
Biochemical characters
Biochemical
production
nitrate,
of
indole,
hydrogen
glucose,
characterisation
utilisation
sulphide
of
production,
oxidation-fermentation
included
citrate,
gas
test,
tests
for
reduction
production
cytochrome
of
from
oxidase
activity, catalase activity and ability to produce hydrolytic
·
.
149
enzymes name 1 y ge 1 atlnase,
amy 1 ase an d caselnase.
3.4
GROWTH STUDIES OF THE ISOLATED MICROORGANISMS
The
medium
used
for
the
studies
with
the
strain
CUAC 10 was a mineral medium containing 1% (w/v) glucose and
0.1%
(v/v)
eugenol/isoeugenol
The medium used
and
Pseudomonas
0.1 % (v /v)
for
the
otherwise specified).
studies with the strains
aeruginosa
eugenol
(unless
and that
was
used
mineral
for
medium
(CUAC 20)
containing
CUAC 30 was a mi nera 1
67
medium containing 0.1%
the
mineral
3.4.1
(v/v)
medium was
that
isoeugenol.
The composition of
d€',cribed under section
3.2.1.
Preparation of inoculum
A loopful of the culture from the agar slants was
transferred
to
for 24 hours.
100 ml
mineral
broth
and
incubated
at
30°C
The turbidity of the culture was then observed
at 600 nm and the absorbance was adjusted with fresh mineral
broth to give a final
sion,
in
quantities
absorbance of 1.0.
of
2 ml/lOO ml
This cell suspen-
broth,
was
used
as
the
inoculum.
3.4.2
Incubation procedure
To
10 ml
quantity
of
mineral
broth
in
each
test
tube, which was autoclaved at 15 lbs pressure for 15 minutes,
0.2 ml
of
the
incubat ed
at
inoculum
30 ° C
for
was
a
per iod
shaker
(unless
otherwise
tained
for
experiments.
in
all
These
of
24
specified).
All
test
hours
tubes
on
Controls
a
were
were
rot ary
mal.n-
the experiments were done
duplicate.
3.4.3
Measurement of growth
The
The
added.
growth
turbidi ty formed
was
measured
in the
in
ino(~ulated
terms
of
turbidity.
tubes due to the growth
68
of
the
bacteria
was
spectrophotometer
measured
(Hitachi
at
600 nm
Model
in
200-20)
24 hours (unless otherwise stated).
a
at
UV-visible
the
end
of
Growth was expressed as
optical density (OD) values.
3.4.4
Effect of concentration of eugenol/isoeugenol
The
effect
of
concentration of
eugenol/isoeugenol
on the growth of the bacteria was tested by inoculating the
test strain of bacteria in mineral broth adjusted to various
concentrations
0.25%
(v/v).
eugenol/isoeugenol,
of
viz.,
The dispensed medium was inoculated,
0.1
to
incubated
and the growth was measured.
3.4.5
Effect of pH
The
tested
in
substrate
effect
mineral
at
its
of
broth
optimal
pH
on
the
prepared
after
of
bacteria
was
incorporating
the
concentration and adjusting
incubat ing
inoculating
3.4.6
Effect of concentration of sodium chloride
chloride
from
and
for
after
By
broth
incorporat i ng var ious
0
to
10%
(w/v),
it
to
The growth was measured
various levels of pH from 5 to 10.
the
growth
10
concent ra t ions
ml
aliquots
24
hours.
of
sodi urn
of
mineral
69
broth
were
prepared.
The
growth
was
measured
after
inoculating the test strains and incubating it.
3.4.7
Effect of different nitrogen sources
Ammonium nitrate in the mineral broth was replaced
wi th
ammonium
nitrate
and
nitrogen
prepared.
chloride,
sodium
content
ammonium
nitrate
in
the
in
sulphate,
urea,
concentrations
medium
and
10 ml
pot ass i urn
giving
equal
aliquots
were
Cultivation was carried out with the cultures and
the gr~wth was measured after incubation for 24 hours.
3.4.8
Effect of temperature
Ten
optimal
ml
aliquots
conditions
test strains.
were
of the mineral
prepared
These were
and
broth adjusted to
inoculated
with
the
incubated at various temperatures
viz., 15° to 60°C and the growth was measured.
3.5
3.5.1
ANALYTICAL METHODS
Extraction procedure
After
fermentation,
6000 rpm for 15 minutes.
the
broth
was
centri fuged
at
The supernatent was acidified with
70
2 N sulphuric acid
solvent
ether.
anhydrous
The
sodium
pressure.
to pH 2 and exhaustively extracted with
extracts
sulphate
The residue was
and
were
pooled,
concentrated
with
dried
under
reduced
subjected to chromatographic
and
spectrophotometric studies.
3.5.2
Thin layer chromatography
Thin
with
silica
glass
layer
gel
plates
chromatography
plates
wi th
a
of
(TLC)
0.25 mm
spreader.
The
was
thickness
plates
carried
prepared
were
out
on
developed
with the following solvent systems.
i)
Chloroform
ii)
Ethyl acetate: Hexane - 3:7
iii) Benzene: Dioxane: Acetic Acid - 90:25:4
Spots were detected either by exposing the plates
to
iodine
reagents.
3.5.3
vapours
by
spraying
them
with
appropriate
150
Column chromatography
Column
mesh
or
silica
gel
chromatography
(unless
was
otherwise
performed
with
60-120
specified).
The
eluant
71
used was either chloroform : hexane system or ethylacetate :
hexane system,
depending
observed from TLC.
on
the
nature
of the substrate as
As elution progressed, fractions yielding
the same compound as shown by TLC were pooled and distilled
under reduced pressure to get the product.
3.5.4
High pressure liquid chromatography
High
used both for
pressure
440.
chromatography
(HPLC)
was
the detection and estimation of the substrate
and metabolites.
Model
liquid
The instrument used was a Waters Associates
The column used was an analytical reverse phase
The eluant was methanol.
C1S-p-Bondapack.
The UV detector
and refractive index detector were used.
Standard
were prepared and
and
eluted
with
solutions
10 pl
of
each was
methanol.
The
the
substrate
injected
was
observed
drawn
computed.
from
in
the
progress
which
chromatogram,
concentration
of
a
methanol
into HPLC system
of
monitored with the refractive index detector.
heights
in
elution
From the peak
calibration
the
was
curve
substrate
was
72
3.6
EFFECT OF PHYSICOCHEMICAL FACTORS ON TRANSFORMATION
The
temperature,
effect
of
different
physicochemical
nitrogen
factors
sources
and
namely
pH,
concentrations
of sodium chloride and glucose on the rate of transformation
of eugenol/isoeugenol
by the bacterial
For the
studies with
the
mineral
broth
1%
(w/v)
in 250 ml
otherwise
stated)
for
mineral
the
broth
mineral
medium
section 3.2.1.
studies
the
0.1%
used
wi th
strain
(v/v)
was
0.1%
(v/v)
CUAC 30,
isoeugenol
the
same
as
the
aliquots
eugenol
mineral
were
that
(unless
with
50 ml
Pseudomonas aeruginosa,
with
with
the
of
supplemented with
eugenol/ i soeugenol
For
supplemented
studies
supplemented
used.
studied.
50 ml aliquots
Erlenmeyer flasks
were
strains CUAC 20 and
of
strain CUAC 10,
0.1 % (v/v)
gl ucoseand
strains was
and
broth
used.
The
described
under
The medium was autoclaved at 15 Ibs pressure
for 15 minutes before cultivation and preparation of inoculum
was the same as described under section 3.4.1. The inoculated
flasks
were
6,
18,
All
12,
at
30°C
hours
on
incubated
24
experiments
and
36
were
done
kept for all experiments.
in
for
a
specific
rotary
duplicate
shaker
and
periods
of
(220 rpm).
controls
were
73
After
incubation
for
specific
intervals
of
time,
the broth was centrifuged and the supernatent was acidified.
It
was
extracted
with
ether
evaporated to dryness.
The
and
the
residue
ether
was
extract
was
then estimated for
residual eugenol/isoeugenol.
3.6.1
Estimation of eugenol/isoeugenol
Standard
methanol
were
281/260 nm
curves
From
the
prepared
were
were
solutions
measured
then
drawn
absorbance
l!nd
in
a
each
values
of
eugenol/isoeugenol
in
the
absorbance
at
values
spectrophotometer.
for
of
eugenol
the
test
and
Standard
isoeugenol.
solut ions
the
con-
centrations of eugenol/isoeugenol were then estimated.
3.6.2
Effect of concentration of sodium chloride
The effect of concentration of NaCl on transforma-
tion
having
1%
of
eugenol/isoeugenol
various
(w/v).
specific
chloride.
sodium
After
was
chloride
tested
mineral
concentrations,
inoculation the
intervals of time
in
flasks were
viz.,
broths
0
to
incubated for
for each concentration of sodium
The contents of the flasks were then analysed for
residual eugenol/isoeugenol.
74
3.6.3
Effect of concentration of glucose
Fifty ml
aliquots
of
mineral
broth were
prepared
by incorporating various glucose concentrations from 0.2 to
2%
(w/v)
in the case of studies with the strain CUAC 10 and
from 0 to 1%
CUAC 20 and
inoculated
(w/v)
in the case of studies with the strains
CUAC 30.
with
the
The
test
flasks
containing the broth were
strains,
incubated
for
specific
intervals of time and analysed.
3.6.4
Effect of pH
Fi fty
ml
aliquots
of
mineral
and adjusted to pH values ranging
inoculated,
incubated
and
broth were
from 5 to 9.
analysed
for
prepared
These were
residual
eugenol/
isoeugenol.
3.6.5
Effect of temperature
The
flasks
containing
the
mineral
broths
were
inoculated and incubated at different temperatures from 15°C
After specific intervals of time,
the flasks were analysed.
the contents of
75
3.6.6
Effect of different nitrogen sources
Ammonium nitrate in the mineral broth was replaced
wi th
ammoni urn
nitrate
and
nitrogen
out.
chloride,
sodium
content
After
contents of
nitrate
in
the
incubation
the
ammoni urn
flasks
in
sulphate,
concentrations
medium and
for
were
urea,
pot ass i urn
giving
cultivation was
specific
analysed
intervals of
for
equal
carried
time,
the
unreacted eugenol/
isoeugenol.
3.7
GROWTH CURVE
By
i ncorpora t i ng
t he
opt imal
cond i t ions,
mi neral
broths were prepared for the three cultures CUAC 10, CUAC 20
and CUAC 30 and the broths were inoculated with the cultures.
They were incubated and the growth was measured
in terms of
optical
densi ty
A graph was
plot t ed
wi th
at
time
various
interval
intervals of
versus
time.
opt ical
dens i t Y for
each
culture, which formed the growth curve.
3.B
FERMENTATION CONDITIONS
The
fermentor
mineral
fermentation
(Eyela
broth
model
was
M-lOO).
supplemented wi th
carried
out
A volume
of
in
a
1.5
eugenol/i soeugenol
mini
jar
li tres
of
was
used
76
for
fermentation.
strain
CUAC 10,
The
medium.
In
1%
the
case
(w/v)
of
glucose
fermentor
fermentation
was
together
also
with
the
to
the
medium
was
added
the
sterilised at 15 Ibs pressure for 20 minutes.
with
The ferment a-
tion was conducted at pH 7 at 30 D C (unless otherwise stated).
Continuous
aeration at
a
flow
rate of
1 VVM was given with
an air pump and a speed of 220 rpm of the impeller was used.
A loopful
of
the
mineral
inoculated
in
of the culture was transferred to 10 ml
broth
a
and
incubated
100 ml
broth
for
one
contained
in
flask and incubated for one day in a shaker.
transferred
incubated
to
for
10000 rpm for
400 ml
broth
15 hours.
1000 ml
The broth was
20 minutes.
with physiological
in a
day.
an
This
was
Erlenmeyer
This was again
shaking
flask
then centrifuged
and
at
The cells were separated, washed
saline and this was used as the inoculum
for fermentation.
Since eugenol/isoeugenol was strongly antibacterial,
cultivations
which
the
were
done
substrate
by
was
the
added
concentration from 0.01% (v/v).
feeding
in
culture
stages
method
increasing
in
the
77
3.9
ISOLATION AND CHARACTERISATION OF TRANSFORMATION
PRODUCTS
After
6000
rpm
for
supernatent
This
was
ether
fermentation
15
was
minutes
and
acidified
exhaustively
extracts
were
the
broth
was
the
cells
were
with
centrifuged
removed.
2 N sulphuric
extracted
pooled,
with
dried
with
The
acid to
sol.vent
pH
et her.
anhydrous
at
2.
The
sodium
sulphate and filtered.
A small portion of the ether extract
was
thin
layer
which
gave
subjected
solvent
systems,
number of
the
products
The
pressure.
chromatography
an
idea
formed during
extract
ether
graphy.
to
was
then
residue
was
on
concentrated
subjected
separated.
The
pooled
concentrated
and
fractions
the
the
fermentation.
Chloroform-hexane or ethyl
as the eluant dependi ng
about
with
under
nature
and
The rest of
under
column
reduced
chromato-
acetate-hexane was used
nat ure
yielding
to
different
0
f
the
reduced
the mi xt ure
same
product
pressure.
to
be
were
The
separated components were purified by recrystallisation from
appropriate solvents.
The
pure
compounds
were
identified by determining
the physical constants and chromatographic and spectrometric
met hods.
These
incl uded
compar i ng
wi t h
au thent i c
samples
78
the
melting
systems
points,
and
Rf
IR,
UV,
values
NMR
(TLC)
and
in
mass
different
spectra.
solvent
Suitable
derivatives of these compounds were also prepared.
3.9.1
Preparation of acetates of the transformation products
Five
1.5 ml
1 ml
for
mg
of
the
compound
was
dissolved
in
pyridine in a 50 ml clean dry round bottom flask and
ac et i c
6
hundred
anhydr i de was added
hours
poured
shaking
into
filtered.
it.
i nt ermi t tent ly.
water
ice-cold
The flask was kept
The
stirring
mi xt ure
was
then
continuously
and
The precipitate was washed several times with 1 N
hydrochloric
solution.
to
acid
The
followed
product
was
by
then
5%
sodium
recrystallised
bicarbonate
from
dilute
ethanol and the melting point determined.
3.9.2
Preparation of methyl esters of acidic products
I n a 50 m1 r 0 u n d bot tom f 1 ask,
and
2
ml
sulphuric
A reflux
absolute
acid
was
condenser
refluxed for 3 hours.
and 10 ml
of
methanol
were
added
was
pl aced
cautiously
fitted
to
the
700 mg
and
with
flask
0
f
0.2
the a cid
ml
con.
shaking. 151
and
it
was
It was then cooled to room temperature
water was
added.
The
excess of
methanol was
79
di st i lled . of f
flask
was
on
a
water
cooled
and
separatory funnel.
and
washed
wi th
was
then
dried
bath under
the
reduced
contents
pressure.
were
poured
The lower layer of ester was
water
followed
with
by
5%
anhydrous
NaHC0
3
magnesium
The
into
a
separated
solution.
It
sulphate
and
products
were
filtered.
3.10
TRANSFORMATION OF FERMENTATION PRODUCTS
The
studied
by
t rans forma t ions
cultivating
supplemented
with
the
the
of
ferment at ion
cultures
fermentation
in
the
mineral
product.
The
broth
mineral
medium used was the same as that described under section
The
inoculated
30°C.
with
The
a
broth
was
a
to
find
out
rotary
the
After incubation for different
the broth was centrifuged,
extracted
on
shaker
at
turbidity of the broth was periodically observed
spectrophotometer
bacteria.
incubated
3.2.l.
wi th
ether.
growth
of
the
intervals of time,
the supernatent was acidified and
The
ether
extract
was
concentrated
under reduced pressure and subjected to analysis.
3.11
FERMENTATION
OF
ACETATES
OF
EUGENOL
AND
ISOEUGENOL
The acetate of eugenol/isoeugenol was fermented in
the
mineral
medium
described
under
section
3.2.1.
80
For
the
studies
supplemented
with
with
1%
acetate/isoeugenol
CUAC 20
and
mented
with
medium
with
rest
of
the
(w/v)
(v/v)
0.1%
the
CUAC 10
glucose
acetate.
Pseudomonas
0.1%
strain
For
and
eugenol
(v/v)
the
was
eugenol
with
the
strains
medium
was
supple-
for
acetate
procedures
medium
(w/v)
acetate and
isoeugenol
fermentat ion
0.1%
studies
aeruginosa
the
was
CUAC
was
the
30
the
used.
same
The
as
wi th
eugenol/isoeugenol.
3.12
MODE OF AROMATIC RING CLEAVAGE
The
the
procedure
det ermi nat ion
ring. 152
The
agar
slants
days
of
of
of
the
organism
at
mode
was
supplemented
incubation
Ottow
0
and
f
Zolg
cl ea vage
subcul tured
wi th
30°C,
a
was
of
2
Then
tube.
protocatechuate
shaken.
positive
was
A
were
green
ml
for
aroma tic
on
mineral
eugenol/ isoeugenol.
After
thick
culture
loopful
of
the
(pH 8.0)
2
in a
toluene and 2.5 ml of 4 mM sodium
added
colour
meta-cleavage.
to
it
within
If
by
shaking
for
prepared
aqueous
1%
incubated
freshly
0.5
the
times
was suspended in 2 ml 0.05 M phosphate buffer
test
adopted
no
18
and
3
it
was
minutes
vigorously
indicated
reaction
occurred,
hours
30°C,
sodium
at
the
and
nitroprusside
a
tube
1 g
of
solution
81
and
0.5 ml
colour
concentrated
wi thin
3
ammonia were
minutes
was
added.
regarded
as
A deep purple
a
positive
test
for the ortho-cleavage of the substrate.
3.13
IMMOBILISATION OF WHOLE CELLS
Immobilisation
entrapment
alginate
in
on
of
calcium
treatment
whole
cells
b ea d s.
alginate
with
(IWC)
calcium
was
done
153,154
chloride
by
Sodium
gave
calcium
alginate beads.
3.13.1
Entrapment in calcium alginate
Hundred ml
distilled water was warmed in a
flask and 4 g sodium alginate was added.
the
start
of
boiling
with
constant
avoid the formation of lumps.
heater
and
allowed
to
cool
It was heated till
stirring
in
order
It was then removed
to
form
250 ml
the
sodium
to
from the
alginate
slurry.
About
homogeneous
20
paste
g
of
with
wet
25
volume was made upto 100 ml.
ml
bacterial
distilled
cells was
water
and
made
then
to
a
the
82
Sodium
alginate
slurry
and
the
bacterial
cell
slurry were mixed in 1:1 ratio and stirred well to obtain a
uniform mix.
It was extruded through a syringe needle into
0.1 M calcium
chloride
were
cured
6 hours.
in
the
solution
same
with
solution
at
stirring.
room
The
beads
temperature
for
The gel beads were washed with distilled water and
stored in bu ffer con ta i ni ng 50 rPM calc i urn chI or ide.
3.13.2
Packing of the column
A glass column of 30 cm
was
used
for
the
packing
of
length and 2.5 cm diameter
the
i mmobil i sed
cell s.
The
column was packed in the order of glass wool at the bottom,
a
perforated
plate,
cell
immobilised
then again a perforated plate.
eugenol
was
continuously
aliginate
beads
and
The mineral broth containing
passed
into
the
column
and
the
eluant was collected periodically.
3.13.3
Activation of immobilised cell beads
About 30 g of immobilised cell beads was packed in
the column.
The mineral broth containing eugenol/isoeugenol
was
into
passed
24 hours.
The
the
broth
column
was
and
eluted
incubated
off
and
at
the
30°C
beads
for
were
83
washed
passing
by
continuously through
freshly
prepared
the column.
saline
solution
This column was then used
for further studies.
3.13.4
Effect of temperature and pH on transformation by IWC
Fifty ml
aliquots of the mineral
the substrate adjusted to different
and
passed
into
and
eluted
and
analysed
at
ml
aliquots
of
Similarly
the
50
optimum
These were
pH
the
were
col umn.
pH values were prepared
These were
different
the
prepared
incubated at different
incubated at
intervals
mineral
and
broth containing
broth
passed
into
of
30 ° C
time.
adjusted
the
to
column.
temperatures from 15°C to
60°C, eluted and analysed.
3.14
ENZYME STUDIES
Cell
free
extracts
(CFE)
of
the
cultures
were
prepared and were used as the source of enzymes.
3.14.1
Preparation of cell free extracts
The
inoculated
ln
organism
400
eugenol/isoeugenol.
ml
It
was
of
was
subcultured
the
two
mineral
incubated
for
times
broth
15-18
and
then
containing
hours
and
84
10,000
centrifuged
at
separated,
washed
(pH 7.0)
cell
for
and the
paste
15
thick
paste
alumina,
cell
was
centrifugation
supernatent
a
two
solution
weights
was
and
at
for
30
used
as
the
O"C.
buffer
The
pestle.
with
walls
rpm
cells were
phosphate
and
ml
cell
The
frozen
of alumina was ground
mortar
10
to
cells
15,000
at
frozen
chilled
diluted
unruptured
0.05 M
with
paste was
wi th
with
20 minutes.
for
twice
together
minutes
rpm
155
buffer
were
minutes
crude
and
removed
4"C.
at
enzyme
The
the
by
The
prepara-
tion.
3.14.2
Effect of pH and temperature on transformation by CFE
Five ml aliquots containing 0.05 ml substrate, 0.5 ml
CFE
4.45
and
different
then
pH
values
analysed.
optimum
pH
0.05
ml
were
M
incubated
S imi lar 1 y
were
phosphate
incubated
5
ml
at
for
buffer
adjusted
3 hours at
al iquots
different
adj ust ed
to
30"C and
to
the
temperatures
for
3 hours and then analysed.
3.14.3
Transformations with CFE
Five
ml
aliquots
containing
0.05
ml
substrate,
0.50 ml CFE and 4.45 ml of 0.05 M phosphate buffer adjusted
to optimum pH were
incubated at the optimum temperature and
periodically analysed by TLC and HPLC.
85
The
formation
of
1-carboxy-oC...-hydroxy-cis,
~-carboxy-cis,
aromatic
cis-muconic
ring
cleavage
acid
products
semialdehyde
and
cis-muconic acid were monitored spectrophoto-
metrically by measuring
.
156 157
respect1vely.
'
the
Thus
absorption at 410 nm and 290 nm
to
a
cuvette
containing
2 ml
of
0.05 M phosphate buffer, 0.1 ml eFE and 0.4 ml of 1 mM sodium
protoca t echua t e
sol ut i on
measured at 410/290 nm.
were
added
and
the
absorpt i on
was