Philippine Journal of Science
132 (2): 129-136, December 2003
ISSN 0031 - 7683
Partial Purification and Characterization
of Invertase from Flowers of
Madhuca longifolia (Mi)
M.K.B Weerasooriya and H.P. Yatawara
Department of Chemistry
University of Kelaniya, Sri Lanka
Enzyme Invertase (β-D-fructofuranosidase, EC 3.2.1.26), present in the flowers of
Madhuca longifolia plays an important role during the preparation of fermented Ayurvedic
drugs known as ‘Arishta’. This enzyme was partially purified with a yield of 11.6%, using
(NH4)2SO4 fractionation, followed by gel filtration through Sepharose 4B and DEAE cellulose
chromatography at pH 6.5 and 4.2. The molecular mass of the enzyme as determined by
elution through Sepharose 4B gel column was found to be about 210 kDa . The enzyme
showed a broad pH optimum between 4 -7 . Optimum assay temperature was 37ºC and
above 45ºC enzyme activity slowly declined and inactivated around 80ºC. Approximate
Km value for the enzyme for sucrose is 125 mM.
Keywords: purification, characterization, invertase, Madhuca longifolia
In ayurveda, fermentation processes have been used
for ages to modify the potency of drugs and to extract
and to preserve active constituents from medicinal
plants. Therapeutic application of these fermented
drugs are based on the properties of ingredients
and the method of preparation. In general, these
fermented ayurvedic preparations posses a moderate
potency and administered in the convalescent phase
of a disease (Namijoshi 1979, Caraka Samhita 1949).
One such fermented preparations known as ‘Arishtas’
are widely used in Sri Lanka as well as in other
South Asian countries. ‘Arishtas’ are weak alcoholic
preparations used for the treatment of a wide variety of
diseases and specially, prescribed for diseases which
require long term treatments. Since, Arishtas are not
perishable very often they are used as tonics (Kroes
et al. 1992, Kroes 1990, Ayurvedic Pharmacopoeia
1976 &1979).
In the preparation of many Arishtas, dried flowers
of Woodfordia fruticosa (L.) Kurz ( Malitta in Sinhalese,
Dataki in Sanskrit; Family, Lythraceae), or Madhuca
longifolia (Mi in Sinhalese; Family, Sapotaceae) are
used to enhance the sucrose hydrolysis which leads to
production of alcohol (Caraka Samhita 1949, Ayurvedic
Pharmacopoeia 1976 &1979, Hand Book of Domestic
Medicine 1978, Kroes 1990). Thus, produced alcohol
contributes preservation of drugs and also promotes
the extraction of biologically active components from
plant materials and absorption of active components
from gastro-intestinal tract (Kroes 1990, Ayurvedic
Pharmacopoeia 1976 &1979).
So far, little is known about these enzymes. Hence,
we aimed to purify and characterize the endogenous
invertases of the above two sources in view of
providing better insight to rationalize, standardize and
optimize the quality and preparation procedures of
Ayurvedic drugs used in Sri Lanka.
In a previous work we reported the purification and
properties of invertase from the flowers of Woodfordia
fruticosa (Weerasooriya and Yatawara 2002).
Here, we present the kinetic properties of partially
purified of invertase of Madhuca longifolia.
129
Weerasooriya & Yatawara
Materials and Methods
were then stored in the freezer until required for further
purification and other analysis.
Apparatus
Sepharose 4B gel filtration chromatography
Microbiological work was carried out under sterile
conditions in a Labcare microbiological cabinet.
Sterilization of the culture media, glass ware and other
consumables prior and subsequent to use was carried
out in an autoclave at 15 p.s.i for 30 minutes.
Enzyme fraction was further purified by passing
through a Sepharose-4B column (58x2cm) and eluted
with citrate buffer (pH 5.2, 50mM) containing 1mM
EDTA, 5mM 2-mercaptoethanol and 50mM NaCl at a
flow rate of 20ml/hr. Fractions (2 ml) were collected and
the enzyme activity was assayed.
Chemicals
All the bio-chemicals were purchased from Fluka
Chemical company, Sweden. All other chemicals used
were of analytical grade.
Plant materials
Flowers of Madhuca longifolia (Mi) were collected
from the garden of Pallkelle Ayurveda Hospital. They
were identified by Mr. D.B.M.U. Dhanasekera and
Mrs.R.W. Edirisinghe by comparing with authentic
samples in the National Herbarium at Peradeniya
Botanical Gardens, Kandy. The collected flowers were
cleaned and stored in the refrigerator.
Enzyme purification
All the purification steps were carried out at room
temperature unless otherwise specified.
Preparation of the crude extract
Microorganisms presence on the surface of the
Madhuca longifolia flowers were removed by washing
the flowers with sterile distilled water. In each washing,
the flowers were shaken for about 30 minutes and this
process was continued for nearly 55 times. The flowers
(120 g) were next immersed in citrate buffer (pH 5.2,
50 mM, 75.0 ml) containing 1 mM EDTA and 5 mM
2-mercaptoethanol , ground with mortar and pestle
and the crude extract thus obtained was centrifuged
at 15,000 g for 30 minutes at 1°C. The supernatant
solution was filtered through a sterile filter paper, (0.22
µm). A small aliquot which obtained from the filtrates
was cultured in nutrient Agar (NA) and potato dextrose
agar (PDA) and incubated for 2 days at 37°C to check
for the presence of any microflora.
Since, no microflora was observed, above filtrate
was used for the following ammonium sulphate
fractionation.
(NH4) 2SO4 fractionation
Invertase in the crude extract was precipitated
at 1°C between 50-95% saturation of (NH4) 2SO4,
centrifuged at 12,000g for 30 min and resuspended in
citrate buffer (pH 5, 50mM) containing 1mM EDTA and
5mM 2-mercaptoethanol. The enzyme preparations
130
DEAE Cellulose chromatography at pH 6.5
Active fractions eluted from sepharose-4B column
were pooled and purified on a DEAE Cellulose column
(12x 3cm) eluting with phosphate buffer (50mM, pH 6.5)
containing 1mM EDTA and 5mM 2-mercaptoethanol.
The enzyme was eluted with same buffer at a flow rate
of 20ml/hr. Fractions (1.5 ml) were collected and the
enzyme activity was assayed.
DEAE Cellulose chromatography at pH 4.2
Active fractions were pooled and chromatographed
on second DEAE column (9 x 2.2 cm) eluting at a flow
rate of 18ml/hr, with a linear gradient of 0-1M NaCl added
to citrate buffer (50mM citrate, pH 4.2) containing 1mM
EDTA and 5mM 2-mercaptoethanol. Fractions (1.5 ml)
were collected and the enzyme activity was assayed.
The active fractions were pooled and concentrated by
osmosis against solid polyethylene glycol.
Sepharose 4B gel filtration chromatography
(final step)
Concentrated enzyme fraction was further purified
by passing through a sepharose 4B column (12 x 1 cm)
eluted with citrate buffer (pH 5.2, 50mM) containing
1mM EDTA, 5mM 2-mercaptoethanol and 150mM
NaCl. Fractions (1.0 ml) were collected and the enzyme
activity was assayed.
Protein estimation
Quantitative estimation of protein after each step
of purification was done by Bradford method (Scopes
1982) using bovine serum albumin as the standard
protein.
Invertase Assay
Activity of invertase was assayed by Nelson’s
method as follows:
The reaction mixture in a total volume of 2.5ml
contained citrate buffer (100mM, pH 5, 1.3ml)
containing 1mM EDTA and 5mM 2-mercaptoethanol
and sucrose (5%, 1.00 ml) . The reaction carried out at
37 °C for 3 min, was initiated by the addition of enzyme
(200µl , 1 nkat). Then Nelson’s reagent (1.00ml) was
Partial Purification and Characterization of Invertase of M. longifolia flowers
added to the reaction mixture, boiled for 20 minutes
and cooled to room temperature. Arseno-molybdate
reagent (1.00 ml) was added and the mixture was
diluted with water up to 10ml. Then absorbance was
measured at 510 nm.
The standard curve was constructed for known
concentration of product glucose against the absorbance
and the amount of glucose was determined. The
enzyme activity is expressed as as nmole of sucrose
reacted min-1 under specified conditions.
Determination of the molecular weight
The molecular mass of the purified enzyme
was estimated by gel filtration through a column of
Sepharose 4B, which had previously been calibrated
with Urease (340 kDa), Alcohol Dehydrogenase (141
kDa), Bovine Serum Albumin (66 kDa), Egg albumin (45
kDa), Pepsin (34 kDa) and Trypsin (24 kDa).
For the characterisation of the purified enzyme,
following experiments were carried out under the
standard assay conditions (1 nkat in assay). All the
experiments were performed three times in duplicates
under the same conditions and mean standard deviation
were calculated.
PH optima
The optimum pH was determined by assaying the
enzyme activity at different pH ranging from 2.6 to
9.49 using the buffers glycine-HCl (pH 2.6), Citric acidNa2HPO4 (pH 3-7.5) and Tris-HCl (pH 8-8.9). Blank
determinations were done simultaneously at all pH’s
without the enzyme.
Temperature optima
The optimum temperature was studied by assaying
the enzyme at different incubation temperatures
ranging from 25°C to 65°C. Blank determinations were
done simultaneously at all temperatures without the
enzyme.
Thermal denaturation
Thermal stability of the purified enzyme was studied
by pre-incubating 1nkat of the enzyme at different
temperatures ranging from 40 °C-100 °C for 30 min.
Samples were cooled immediately in ice-water and the
residual enzyme activity was determined.
Determination of Km
Km value was obtained by assaying the invertase
activity of purified preparation with sucrose as a
substrate at concentrations ranging from 10mM to
250 mM.
Results and Discussion
Preparation of the crude enzyme extract
The flowers were ground merely to disrupt only
the plant tissues without harming the cellulolic walls
of micro-organisms, if any present in the flowers. This
step would avoid the release of microbial invertase into
the extracting solution.
Subsequent filtering the above enzyme extracts
through 0.22µm sterile filter under sterile conditions was
aimed at eliminating any micro-organisms, conidia or
spores which could be present in the extract.
Purification
The enzyme was partially purified about 51.6
fold by ammonium sulphate fractionation followed by
gel filtration and anion exchange chromatography.
The results of the enzyme purification are given in
Table 1.
During the purification by DEAE column at pH 6.5
the enzyme didn’t bind to the column and was eluted
before applying the gradient. However, at this DEAE
step enzyme showed purification of 15.95 fold with
54.2% recovery. As the pH was changed to pH to 4.2.
Table 1. Purification of invertase from Madhuca longifolia
Step
Total Protein (mg)
Specific activity
(nkat mg-1 protein)
Recovery
%
Purfication fold
(NH4)2SO2
Step
585.2
0.42
100
1
107.6
1.95
85.3
4.64
19.9
6.7
54.2
15.95
5.48
11.95
26.6
22.6
1.32
21.7
11.6
51.6
Gel filtration
(1st step)
Ion exchange
At pH 6.5
Ion exchange
At pH 4. 2
Gel filtration
(2st step)
131
Weerasooriya & Yatawara
Molecular weight
Table 2. Properties of Invertase from Madhuca longifolia
Native molecular mass of the enzyme as determined
by elution through Sepharose 4B gel filtration ~210kDa
(Fig. 1). Interestingly this value falls in the molecular
mass range that reported invertases from Woodfordia
fruticosa, another invertase source used for the
preparation of arishta (Weerasooriya and Yatawara
2002), banana fruit (Sum et al. 1980) , Iraqui date fruit
(Mahrouf & Zerki 1982), rice grains (Chang et al. 1994)
and sprouting soya bean (Chen & Black 1992) .
Property
Molecular mass (kDa)
210
Optimum pH
4- 7
Optimum temperature
37°C
Km (37°C, pH 5)
125 mM
Thermal stability
Enzyme is stable up to 45°C.
Beyond this point activity is slowly
declined and inactivated around
80° C
Optimum pH
The purified invertase of Madhuca was quite stable
and showed broad pH optimum between 4.0 - 7.0
(Fig. 2) which was quite comparable to that reported
for invertases from Woodfordia (Weerasooriya and
Yatawara 2002), Iraqui date fruit (Mahrouf & Zerki
1982), Allocasia leaves (Nakasone & Yasui 1982),
Ricinus communis (Prado et al. 1985 ) and sugarcane
leaf (Minouru 1942).
the enzyme retained in DEAE column. Upon gradient
elution, the enzyme showed activity peak at 0.42M
NaCl.
In the final purification step, reloading the active
enzyme fractions (obtained from above DEAE step)
on Sepharose 4B gave a slightly broader peak.
Native PAGE of this enzyme fraction (obtained
after fractionation through final Sepharose 4B
column) showed that enzyme has been partially
purified.
Optimum temperature
Enzyme showed a higher enzyme activity in the
range of 30ºC to 45ºC with a maximum of 37ºC (Fig.3)
which was very close to that reported for invertases
5.8
5.6
Log Molecular Weight
5.4
5.2
5
4.8
4.6
4.4
4.2
4
0
4
8
12
16
20
24
28
32
36
40
44
48
Elution volume (ml)
Figure 1. Determination of the molecular weight of Invertase of Madhuca longifolia (Urease 340kDa, Alcohol dehydrogenase
141kDa, Bovine serum albumin 66kDa, Egg albumin 45kDa, pepsin 34kDa and Trypsin 24kDa were used as the standards).
132
Partial Purification and Characterization of Invertase of M. longifolia flowers
40
Invertase activity (nmole/min)
35
30
25
20
15
10
5
0
2.07
3.07
4.03
5.11
6.12
7.04
8.06
8.97
pH value
Figure 2. Invertase activity of Madhuca longifolia as a function of pH (pH 2.6 glycine HCl 100mM; pH 3-7.5 Citric acid-Na2HPO4
100mM; pH 8-8.9 Tris-HCl 100mM; 37°C, 3min, Mean and standard deviation were determined from six replicates.)
35
Invertase activity (nmole/min)
30
25
20
15
10
5
0
20
30
35
37
40
50
55
60
65
70
75
0
Incubation temperature C
Figure 3. Invertase activity of Madhuca longifolia as a function of incubation temperature 0.1M citrate pH 5, temeperature
range 25-65°C, 3 min, Mean and standard deviation were determined from six replicates).
133
Weerasooriya & Yatawara
of Woodfordia (Weerasooriya and Yatawara 2002),
Allocasia leaves (Nakasone & Yusai 1982), sugarcane
leaf (Minouru 1942) and sugar beet (Bohuslavska et
al. 1984 ) .
Thermal inactivation
Beyond 45ºC, invertase activity of Madhuca
slowly declined and became almost nil by 80ºC (Fig.4)
similar to that reported for invertases of Woodfordia
(Weerasooriya and Yatawara 2002), Allocasia leaves
(Chen & Black 1992) and potato tubers (Isla et al. 1998)
. However, invertase of Madhuca seems to be more
heat sensitive than invertases of muscat bailey A grapes
(Porntaveewat et al. 1994), semillon grapes (Nakanishi
et al. 1991) and yeast particularly, Saccharomycese
cerevisae (Raheja et al. 1998) and Saccharomycese
fragilis (Kulikora 1986) which were reported to be stable
up to 80ºC.
Kinetic constants (km)
The Km values of purified Madhuca invertase for
sucrose was found to be ~ 125 mM which was very close
to the values that reported for invertases of Woodfordia,
(Weerasooriya and Yatawara 2002). invertases from
Solanum tuberosum tubers (Isla et al. 1999) and higher
than Km values that reported for invertases of rice
grain (Km ~ 70mM) (Chang et al. 1994), and potato
tubers (Km ~ 100mM) (Isla et al. 1998).
In a summary, Invertase from Madhuca longifolia
fits many characteristics described for invertase
isolated from Woodfordia fruticosa (another invertase
source which used for the preparation of Arishta)
such as optimum pH, optimum temeperature, thermal
stability, molecular mass and Km value. Km of
Woodfordia invertase for sucrose is reported to be
~160 mM whereas that of Madhuca invertase found
to be ~125mM. This indicates possibly substrate
sucrose shows higher affinity towards Madhuca
invertase rather than that of Woodfordia. All arishta
preparations in Sri Lanka are carried out at room
temperature which is around 35-37ºC . This is in
agreement with the temperature 37ºC which shows
the optimum activity of the enzyme. pH of the solution
is also very importance during the preparation of
Arishta. As described in Kroes 1990, all arishta
preparations favour the weak acidic condition.
Alkaline conditions are unfavourable for the reaction.
This is in agreement with optimum pH of the enzyme.
As reported by Kroes 1992, high content of gallic
acid, (a weak acid present in preparation medium)
presumably maintain the pH of the solution around 4-
Invertase activity (nmole/min)
60
50
40
30
20
10
0
50
60
70
80
90
100
0
Temperature C
Figure 4. Thermal stability of invertase of Madhuca longifolia (Incubation of 1nkat of enzyme in 0.1M citrate buffer pH 5 for
30 min at different temperature 40-100°C and then measured for activity under standard conditions; 0.1M citrate pH 5, 37°C,
3min, Mean and standard deviation were determined from six replicates).
134
Partial Purification and Characterization of Invertase of M. longifolia flowers
-2
1/rate x 10 (nmole/min)
-1
20
15
10
5
0
-25
-5
-5
15
35
55
1/[Substrate](M)
75
95
115
-1
Figure 5. Determination of Km of Madhuca invertase - (Line-Weaver Burk plot; 0.1M citrate pH 5, 37°C, 3min, substrate
concentration ranging from 10-250mM; Mean and standard deviation were determined from six replicates.)
7 in order to provide optimum activity of the enzyme.
Apart from these data further information particularly
on the inhibitors of two enzymes are to be obtained.
All these data will enable to upgrade the quality and
preparation procedures of all arishtas in Sri Lanka
as well as in India.
Acknowledgements
National Science Foundation, Sri Lanka is
acknowledged for the financial support.
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