Purification and partial characterization of acetic acid esterase from malted finger millet (Eleusine coracana, indaf-15)

Article abstract of DOI:10.1021/jf0618527

Acetic acid esterase (EC 3.1.1.6) cleaves the acetyl groups substituted at O-2/O-3 of the xylan backbone of arabinoxylans and is known to modulate their functional properties. To date, this enzyme from cereals has not received much attention. In the present study, acetic acid esterase from 72 h ragi malt was isolated and purified to apparent homogeneity by a four-step purification, i.e., ammonium sulfate precipitation, DEAE-cellulose, Sephacryl S-200, and phenyl-Sepharose column chromatography, with a recovery of 0.36% and a fold purification of 34. The products liberated from a-NA and PNPA by the action of purified ragi acetic acid esterase were authenticated by ESI-MS and ¹H NMR. The pH and temperature optima of the enzyme were found to be 7.5 and 45 °C, respectively. The enzyme is stable in the pH range of 6.0-9.0 and temperature range of 30-40 °C. The activation energy of the enzymatic reaction was found to be 7.29 kJ mor^-1. The apparent K_m and V_max of the purified acetic acid esterase for a-NA were 0.04 μM and 0.175 μM min^-1 mL^-1, respectively. The molecular weight of the native enzyme was found to be 79.4 kDa by GPC whereas the denatured enzyme was found to be 19.7 kDa on SDS, indicating it to be a tetramer. EDTA, citric acid, and metal ions such as Fe⁺³ and Cu ⁺² increased the activity while Ni⁺², Ca⁺², Co⁺², Ba⁺², Mg⁺², Mn⁺², Zn ⁺², and AI ⁺³ reduced the activity. Group-specific reagents such as eserine and PCMB at 25 mM concentration completely inhibited the enzyme while iodoacetamide did not have any effect. Eserine was found to be a competitive inhibitor.

Full text of DOI:10.1021/jf0618527

J. Agric. Food Chem. 2007, 55, 895−902 895
Purification and Partial Characterization of Acetic Acid Esterase
from Malted Finger Millet (Eleusine coracana, Indaf-15)
G. MADHAVI LATHA AND G. MURALIKRISHNA*
Department of Biochemistry and Nutrition, Central Food Technological Research Institute,
Mysore 570020, Karnataka, India
Acetic acid esterase (EC 3.1.1.6) cleaves the acetyl groups substituted at O-2/O-3 of the xylan
backbone of arabinoxylans and is known to modulate their functional properties. To date, this enzyme
from cereals has not received much attention. In the present study, acetic acid esterase from 72 h
ragi malt was isolated and purified to apparent homogeneity by a four-step purification, i.e., ammonium
sulfate precipitation, DEAE-cellulose, Sephacryl S-200, and phenyl-Sepharose column chromatog-
raphy, with a recovery of 0.36% and a fold purification of 34. The products liberated from R-NA and
PNPA by the action of purified ragi acetic acid esterase were authenticated by ESI-MS and 1H NMR.
The pH and temperature optima of the enzyme were found to be 7.5 and 45 °C, respectively. The
enzyme is stable in the pH range of 6.0-9.0 and temperature range of 30-40 °C. The activation
energy of the enzymatic reaction was found to be 7.29 kJ mol-1. The apparent Km and Vmax of the
purified acetic acid esterase for R-NA were 0.04 µM and 0.175 µM min-1 mL-1, respectively. The
molecular weight of the native enzyme was found to be 79.4 kDa by GPC whereas the denatured
enzyme was found to be 19.7 kDa on SDS, indicating it to be a tetramer. EDTA, citric acid, and
metal ions such as Fe+3 and Cu+2 increased the activity while Ni+2, Ca+2, Co+2, Ba+2, Mg+2, Mn+2
,
Zn+2, and Al+3 reduced the activity. Group-specific reagents such as eserine and PCMB at 25 mM
concentration completely inhibited the enzyme while iodoacetamide did not have any effect. Eserine
was found to be a competitive inhibitor.
KEYWORDS: Acetic acid esterase; ragi; finger millet; purification; homogeneity
INTRODUCTION
properties (11). They are not degraded in the rumen by microbes
wherein the degree of acetylation of arabinoxylans is believed
to be one of the important factors (12, 13).
The complex nature of plant cell walls and the structure of
the individual polysaccharides have been the subject of many
investigations (1-3). Hemicellulose is the second most abundant
renewable polysaccharide in nature after cellulose (4). Hemi-
celluloses are composed of arabinoxylans, 1, 3/1, 4 â-D-glucans
and glucomannans, etc. Cereal arabinoxylan is a â-1,4-linked
D-xylose polymer which is generally substituted either at O-2/
O-3 with arabinose and also sometimes with 4-O-methylgluc-
uronic acid (5) in the side chains. Cinnamic acid derivatives
such as ferulic acid and coumaric acids are esterified to the 5I-
OH group of arabinose side chains (6-7) whereas acetic acid
is found to be esterified to the free hydroxyl groups present at
the C-2 and C-3 positions of xylose residues present in the
backbone (8). According to one school of thought, the presence
of occasional acetyl groups in the xylan backbone contributes
substantially to its solubility, while deacetylation leads to the
formation of xylan aggregates (9). However, recent literature
suggests that extensive acetylation does contribute to the
insolubility of arabinoxylans (10), indicating the bifunctional
nature of acetyl groups. Acetylated xylans have poor gelling
Enzymatic hydrolysis of cereal arabinoxylans requires the
participation of several hydrolytic enzymes. These are classified
in to two groups based on the nature of the linkages that they
cleave (14). The first group of enzymes is hydrolases involved
in the hydrolysis of the glycosidic bonds of xylan. These include
endoxylanases (EC 3.2.1.8), â-D-xylosidase (EC 3.2.1.37), R-L-
arabinofuranosidase (EC 3.2.1.55) and R-D-glucuronidase (EC
3.2.1.1). The second group includes enzymes that cleave the
ester linkages which include acetic acid esterases (EC 3.1.1.6)
and cinnamic acid esterases (EC 3.1.1.73).
Acetic acid esterases (EC 3.1.1.6) hydrolyze the ester linkages
of the acetyl groups present in the xylan side chains. Acetic
acid esterases obtained from plant and animal sources differ
from microbial acetyl xylan esterases (EC 3.1.1.72) such as
Trichoderma reesei, Aspergillus niger, and Schizophyllum
commune (15) with respect to their substrate specificity. Partial
characterization of acetic acid esterase from barley malt was
recently reported (16).
Finger millet (Eleusine coracana), also known as ragi, is an
indigenous minor millet, rich in calcium and dietary fiber. It is
extensively consumed by the south Indian rural population and
* Corresponding author: E-mail:
krishnagm2002@yahoo.com.
Fax: + 91- 821- 2517233. Tel. + 91-821- 2514876.
10.1021/jf0618527 CCC: $37.00
© 2007 American Chemical Society
Published on Web 01/06/2007

896 J. Agric. Food Chem., Vol. 55, No. 3, 2007
Latha and Muralikrishna
80 fractions. Ammonium sulfate fractions of 40-60, 60-80 were
pooled (80% activity) and loaded onto a DEAE-cellulose column (1.5
× 18 cm), pre-equilibrated with Tris-HCl buffer (5 × 28 mL, 20 mM,
pH 8.5) at a flow rate of 18 mL h-1 and washed with the same buffer
to remove unbound proteins. A linear NaCl gradient (0-0.6 M) in
equilibrating buffer was used to elute the bound proteins, which were
collected (1.5 mL each) and monitored for protein (280 nm) as well as
acetic acid esterase activity. The active fractions from the anion
exchange chromatography were concentrated and loaded on a Sephacryl
S-200 column (0.6 × 90 cm) which was pre-equilibrated with 10 mM
Tris-HCl buffer (pH 7.0), and fractions (1.5 mL) were collected and
monitored for protein and acetic acid esterase activity. The pooled and
dialyzed active fractions obtained from Sephacryl S-200 were concen-
trated and further fractionated on a phenyl Sepharose CL-4B column
which was pre-equilibrated with sodium phosphate buffer (5 mM, pH
6.5) containing 1 M (NH4)2SO4 which was used to remove unbound
proteins. A linear (NH4)2SO4 gradient (1 M to 0 M) followed by 50%
ethylene glycol in sodium phosphate buffer (5 mM, pH 6.5) was used
to elute the bound proteins (2.5 mL) individually. Active fractions were
monitored for protein and acetic acid esterase activity.
is used both in the native and processed (malted) forms (17).
Studies were reported from our group regarding (a) isolation,
purification, and characterization of ragi amylases (18), (b)
structure and functional relationship of alkali soluble arabin-
oxylans (19), and also (c) water extractable feruloyl polysac-
charides and their antioxidant properties (20).
Both alkali soluble arabinoxylans (hemicelluloses) and water
soluble feruloyl polysaccharides were proved to have an effect
on the functionality of cereal nonstarch polysaccharides with
respect to (a) foam stabilization, (b) gelling, and (c) viscosity
(21). However, to date the effect of O-acetyl groups on the
functionality of cereal water soluble nonstarch polysaccharides
was not studied. Hence, a study was undertaken to purify and
characterize O-acetic acid esterase from malted finger millet
and explore its potential in modulating the functionality of
various nonstarch polysaccharides which is one of the most
important aspects in food science and technology. Very few
attempts were made to purify and characterize this novel enzyme
from plant sources, hence the present study.
Purity Criteria. Polyacrylamide Gel Electrophoresis (PAGE).
PAGE (12.5%) under native conditions was carried out to evaluate the
purity of acetic acid esterase. Duplicate samples were run for
simultaneous protein (silver staining) (26) and activity staining (27).
MATERIALS AND METHODS
Materials. An authenticated variety of finger millet (Eleusine
coracana, ragi, Indaf-15) was procured from the V.C. farm of the
University of Agricultural Sciences, Bangalore, located at Mandya,
Karnataka, India. Acrylamide, bis-acrylamide, ammonium persulfate
(APS), N,N,N1-tetramethylenediamine (TEMED), glycine, Tris-HCl, and
reduced glutathione were obtained from Sisco Research Laboratories,
Mumbai, India. Sephacryl S-200, phenyl Sepharose, BSA (bovine serum
albumin), protein molecular mass standards, Comassie brilliant blue,
R-NA (R-naphthyl acetate), R- naphthol, fast blue RR salt, polyvi-
nylpolypyrrolidone (PVPP), Triton X-100, PNPA (p-nitrophenyl ace-
tate), EDTA (ethylenediamine tetraacetate), esereine, PCMB (p-
chloromercuric benzoate), and iodoacetamide were obtained from Sigma
Chemical Company, St. Louis, MO. Protein molecular mass markers
for SDS (sodium dodecyl sulfate) obtained from Genei, Bangalore,
India. DEAE-cellulose was obtained from Pharmacia Fine Chemicals,
Uppsala, Sweden.
Malting. Malting was carried as reported earlier (22). Ragi seeds
(50 g) were cleaned and steeped for 16 h and germinated under
controlled conditions on moist cloth at 25 °C in a B.O.D. incubator up
to 72 h. Germinated seeds were taken and dried at 50 °C in an air
oven for 12 h, and vegetative growth portions were removed (deveg-
etated) by gentle manual brushing. Devegetated seeds were weighed
powdered and used for the extraction of acetic acid esterase.
Enzyme Extraction. Malted ragi flour (72 h; 50 g) was extracted
with 75 mM Tris-HCl buffer (1:7, pH 9.0, 350 mL) containing 25 mM
reduced glutathione, 1% Triton X-100 (w/v), and 1% PVPP for 2 h at
4 °C, and supernatant was collected by centrifugation (7000g, 4 °C for
20 min) using a refrigerated centrifuge and dialyzed against the
extraction buffer and used for further experiments.
Enzyme Assay/Activity. Acetic acid esterase activity was measured
as described by Poutanen and Sundberg (23) using 1 mM R-NA
dissolved in ethanediol as substrate. The reaction mixture containing
acetic acid esterase and R-NA (made to a final assay volume of 1.0
mL with 75 mM Tris-HCl buffer, pH 9.0) was carried out for 30 min
at 30 °C, and the reaction was stopped by adding 100 µL of 0.33 M
H2SO4. One unit of acetic acid esterase activity is defined as the amount
of enzyme required to liberate 1 µmol of R-naphthol min-1. The reaction
was monitored spectrophotometrically at 235 nm for the release of
R-naphthol. The activity with PNPA was determined by monitoring
photometrically the release of PNP at a wavelength of 400 nm (24),
making use of PNP standard graph.
Protein Determination. The presence of protein was monitored in
the column fractions measuring absorbance at 280 nm and quantified
by Bradford method at 590 nm using BSA as standard (25).
Enzyme Purification. Crude enzyme preparation of acetic acid
esterase from malted ragi was subjected to ammonium sulfate precipita-
tion which resulted in 4 fractions, i.e., 0-20, 20-40, 40-60, and 60-
Acetic Acid Esterase ActiVity Staining. The gel after electrophoresis
was incubated in activity staining solution (50 mM sodium phosphate
buffer, 100 mL pH 7.2; R-NA dissolved in 1 mL of acetone, 10 mg;
fast blue RR salt, 50 mg) in the the dark at 37 °C until dark gray or
until black bands appeared. The stained gel was washed with water
and fixed in 3% acetic acid (28).
Estimation of Molecular Weight (Mr). GPC. This was performed
on a column (0.6 × 90 cm) of Sephacryl S-200 HR calibrated by using
standard protein markers such as papain (12 kDa), carbonic anhydrase
(29 kDa), BSA (66 kDa), alcohol dehydrogenase (1.50 kDa), and
â-amylase (2.00 kDa). The molecular mass was calculated from a plot
of Ve/Vo against the log of molecular weight.
SDS-PAGE. Mr values were estimated by SDS-PAGE by the method
of Laemmli (1970) (29) using a 12% w/v acrylamide gel. Proteins were
detected by silver staining. Mr values were estimated from a plot of
log Mr versus mobility using the following protein standards such as
lysozyme (14.3 kDa), soybean trypsin inhibitor (20.1kDa), carbonic
anhydrase (29 kDa), egg albumin (43 kDa), BSA (66 kDa), phosphor-
ylase (97.4 kDa), and myosin (2.05 kDa).
Effect of pH. Acetic acid esterase activity was determined at various
pH values using different buffers such as sodium acetate (pH 4.0-
6.0), sodium phosphate (pH 6.0-8.0), and Tris-HCl (pH 7.0-9.0) at
75 mM concentrations. The maximum activity was taken as 100% and
relative activity plotted against different pH values.
pH Stability. Stability of purified acetic acid esterase was carried
out by preincubating the enzyme in different buffers such as glycine-
HCl (pH 2.0-3.0), sodium acetate (pH 4.0-6.0), sodium phosphate
(pH 6.0-8.0), and Tris-HCl (pH 7.0-9.0), followed by determining
the residual activities at different time intervals. The original activity
was taken as 100%, and relative activity was plotted against different
time intervals.
Temperature Optima. Freshly purified enzyme (50 µL) was
incubated with 1 mM R-NA in Tris-HCl buffer (pH 7.5, 75 mM) in a
temperature range of 30-60 °C (with an interval of 5 °C) using a
thermostatically controlled incubator. The optimum activity was taken
as 100%, and relative activities were plotted against different temper-
atures.
Thermal Stability. Purified acetic acid esterase was preincubated
in a temperature range of 30-80 °C for 15 min. The residual activity
was estimated taking original activity as control (100%), and relative
activity was plotted against different temperatures.
Measurement of Activation Energy. To determine the temperature
dependence of acetic acid esterase activity, reaction rates/activities at
a series of temperatures (30-60 °C) were determined. An Arrhenius
plot was drawn taking the natural log of activity on y-axis and 1/T in
K on x-axis. The activation energy was determined from the slope of

Acetic Acid Esterase from Malted Finger Millet
J. Agric. Food Chem., Vol. 55, No. 3, 2007 897
the plot using the Arrhenius equation:
slope ) -Ea/R
where R is universal gas constant whose value is 8.314 J mol-1
.
Effect of Substrate Concentration. Different concentrations of
R-NA (2-10 µg) in Tris-HCl buffer (75 mM, pH 7.5) was incubated
with purified acetic acid esterase for 30 min at 45 °C, and activities
were measured at every 5 min. Initial velocities (Vo) were calculated
for all substrate concentrations and the Km and Vmax values were
calculated from double reciprocal plot (30).
Effect of Metal Ions. Purified acetic acid esterase was incubated
with 5 mM solution of salts of metal ions (chlorides of as Fe+3, Cu+2
,
Ni+2, Ca+2, CO+2, Ba+2, Mg+2, Mn+2, Zn+2, Al+3, etc.) at 45 °C for 15
min, and residual activities was measured. The enzyme activities without
metal ion were taken as control (100%), and relative activities were
calculated.
Figure 1. Elution profile of ragi acetic acid esterase (40−80% ammonium
sulfate fraction) on a DEAE-cellulose column.
Effect of Group Specific Reagents. Purified acetic acid esterase
was incubated with 25 mM of PCMB, iodoacetamide, and eserine in
Tris-HCl buffer (pH 7.5) at 45 °C for 15 min, and the residual activities
were estimated. The enzyme activities without these chemicals were
taken as 100%, and relative activities were calculated.
the disulfide bonds and Triton X-100 to extract membrane bound
enzymes, if any.
Acetic acid esterase and acetyl xylan esterase activities have
been detected in a variety of microorganisms, particularly fungi
such as Penicillium purpurogenum (32), Trichoderma reesei
(33), Ruminococcus flaVefaciens (34), Streptomyces liVidans
(35), and Bacillus pumilus (36), etc. Recently an acetic acid
esterase from Barley malt (16) was isolated, purified, and
partially characterized.
Purification of Acetic Acid Esterases. Crude Tris buffer
extract (containing 1% PVPP, 1% Triton X-100, and 25 mM
reduced glutathione) from 72 h ragi malt was subjected to
ammonium sulfate precipitation and separated into four fractions
(0-20, 20-40, 40-60, and 60-80%). Several chromatographic
techniques, i.e., anion exchange, gel filtration, and hydrophobic
interaction, were previously used to purify the acetic acid
esterases from microbes. Since more than 80% of the activity
was present in the 40-80% ammonium sulfate fraction, it was
subjected to further purification on DEAE-cellulose column
(Figure 1). This step was successful in removing the colored
material, large amounts of unbound and contaminating proteins;
in addition, it has also reduced the viscosity of the 40-80%
fraction. The bound proteins were eluted with a linear gradient
of NaCl (0-0.6 M). Acetic acid esterase was eluted as two
peaks, one major (P-1) and one minor (P-2), at 0.27 and
0.43 M NaCl concentrations, respectively.
The DEAE-cellulose purified acetic acid esterase major peak
(P-1) was further purified on a Sephacryl S-200 column (figure
not shown). Acetic acid esterase was eluted as a single peak
with a fold purification and recovery of 25.18 and 4.96%,
respectively. However, on a native PAGE a large amount of
carbohydrate streaking was observed along with minor con-
taminating protein bands (figure not shown). Most of the
hydrophobically associated carbohydrate as well as contaminat-
ing minor proteins was removed by phenyl Sepharose column
chromatography with a decreasing linear gradient of 1 M to
0 M (NH4)2SO4. Acetic acid esterase was eluted with 50%
ethylene glycol in sodium phosphate buffer (5 mM, pH 6.5)
(figure not shown). The purified acetic acid esterase was
obtained in 0.36% yield with fold purification of 34.
Recovery of the enzyme was significantly decreased after
DEAE-cellulose column separation as well as after hydrophobic
interaction chromatography, which might be due to (a) enzyme
inactivation, and (b) removal of the carbohydrate which might
be hydrophobically associated with the acetic acid esterase by
stabilizing it. The overall scheme employed in the purification
of acetic acid esterase from ragi malt is summarized in
Table 1.
Enzymic Deesterification of Acetyl Substrates. Water soluble
portions of larch wood xylan, gum karaya, and water extractable
polysaccharides (0.5%, 1 mL) isolated from ragi and wheat were taken
in Tris buffer (75 mM, pH 7.5) and incubated with purified ragi acetic
acid esterase (0.1 mL). The reaction was allowed to proceed at 45 °C
for 2 h and was stopped by boiling for 10 min and centrifuged at
15 000g for 30 min to separate the supernatant from the residue. The
supernatant was analyzed by HPLC for acetic acid using Supelco-
C610H ion exchange column at room temperature at 210 nm using
orthophosphoric acid (0.05%) as the eluent with a flow rate of 0.5 mL
min-1. The retention time for acetate under these conditions was 19.865
min. The specific activities of purified ragi acetic acid esterase using
various water soluble polysaccharide preparations were compared with
small molecular weight synthetic substrates such as R-NA and PNPA.
IR Analysis of Water Soluble Polysaccharides. Freeze-dried water
soluble polysaccharides of ragi, wheat, larch wood xylan, and gum
karaya (2 mg each) were taken in KBr pellet discs (10 mg) and analyzed
by infrared spectroscopy for the presence of acetyl groups.
ESI-MS Analysis. A 1 mg amount of R-NA and PNPA was
dissolved in ethanediol and DMSO (0.5 mL), respectively, and
incubated with purified acetic acid esterase at 45 °C for different time
intervals (0-8 h). The reaction was stopped with methanol (0.3 mL)
and centrifuged. The methanol layer which consists of products was
analyzed on an ESI-MS Alliance Waters 2695 mass spectrometer using
negative mode electrospray ionization. The capillary voltage was
3.5 kV, core voltage 100 V, source temperature 80 °C, dissolvation
temperature 150 °C, core gas (argon) 35 L h-1, and dissolvation gas
(Nitrogen) 500 L h-1
.
1H NMR Spectral Analysis. A 2 mg amount of R-NA or PNPA in
solution was incubated with purified ragi acetic acid esterase for 8 h at
45 °C. Subsequently the reaction mixture was freeze-dried and dissolved
1
in DMSO-d6, and the products were analyzed by H NMR using a
Bruker 500 MHz spectrometer operating at 27 °C.
RESULTS AND DISCUSSION
Extraction of Acetic Acid Esterase. The 72 h malted ragi
was found to be the best (0.380 µmol min-1gm-1 malt)
compared to 24 (0.171), 48 (0.254), and 96 h (0.160) ragi malts
with respect to the acetic acid esterase activity. Maximum
enzyme activity was extracted with Tris-HCl buffer at pH 9.0
(10.21 µmol min-1gm-1 malt) compared to pH 7.0 (1.97), pH
7.5(1.31), pH 8.0 (1.8), and pH 8.5 (4.02). Various substances
such as PVPP, reduced glutathione, and Triton X-100 were
added to the extracting buffer to enhance the yield (31). PVPP
was included to minimize the coextraction of phenolic com-
pounds present in ragi malt and reduced glutathione to disrupt

898 J. Agric. Food Chem., Vol. 55, No. 3, 2007
Latha and Muralikrishna
Table 1. Summary of the Purification of Acetic Acid Esterase from
Ragi Malt
total
total
specific
fold
%
step
activityb proteinc activityd purification recovery
crudea
40-80%
0.725
0.59
266.05
15
0.0027
0.04
1
14.8
100
81.2
(NH4)2So4 fraction
DEAE-cellulo se
Sephacryl S-200
0.04
0.036
0.0016
0.83
0.526
0.028
0.045
0.068
0.093
16.8
25.2
34.37
5.24
4.96
0.36
phenyl-Sepha rose 4B
a 50 g scale (values are average of three independent experiments). b One unit
-1
is equivalent to 1
µ
mol of
R
-naphthol released min
mol of
.
c Total protein is expressed
R-naphthol released min
-1
in mg. d Specific activity is expressed in 1
µ
-
1
mg of protein.
Figure 2. PAGE of purified ragi acetic acid esterase. (a) Protein staining.
(b) Activity staining.
Criteria of Purity. The purity of acetic acid esterase was
confirmed by SDS and native PAGE. The activity and protein
bands coincide on native PAGE (Figure 2a,b). By SDS-PAGE
a single subunit protein band with an estimated Mr of 19.7 kDa
was observed (Figure 3a). The apparent molecular mass,
determined under denaturing conditions, is comparable to the
ones reported from the acetyl xylan esterases isolated from
Pencillium purpurogenum (32), Bacillus pumilus (36) and acetic
acid esterase from Barley malt (16). The estimated Mr of acetic
acid esterase under nondenaturing conditions using Sephacryl
S-200 was found to be 79.4 kDa (Figure 3b), indicating it to
be a homotetramer similar to the one reported from acetyl xylan
esterase of Bacillus pumilus (36).
transition state, represents the half-way point where the bonds
of the substrate are distorted sufficiently so that the conversion
to products becomes possible. The activation energy of the
reaction was calculated at the optimum pH of the enzyme (7.5),
using R-NA as substrate. The energy of activation as calculated
from the Arrhenius plot was found to be 7.29 kJ mol-1
(Figure 6).
Effect of Substrate Concentration. Effect of different
substrate concentrations on the initial velocity was calculated,
and the kinetic constants Km and Vmax were calculated from the
double reciprocal plots (LB plot, Lineweaver and Burk, 1934
(Figure 7). The Michaeli constant Km Value of acetic acid
esterase from ragi was found to be 0.40 µM for R-NA, and
pH Optima and Stability. Acetic acid esterase was found
to have pH optima of 7.5 and 80% activity was retained between
pH 6.0-9.0 (Figure 4a). The activity of acetic acid esterase in
alkaline pH was more than the one observed in acidic pH. The
activity has decreased both in acetate and phosphate buffers at
lower pH compared to Tris-HCl buffer. However, the drop in
activity in acetate buffer was much more pronounced in
phosphate buffer. Most of the activity was retained in Tris-
HCl buffer, indicating better stability of acetic acid esterase in
Tris-HCl buffer. The enzyme activity has decreased drastically
in the pH range 4.0-5.5 in acetate buffer and 6.0-7.0 in
phosphate buffer, indicating the labile nature of the enzyme in
acidic pH. The optimal pH of 7.5 is similar to the pH values of
8.0 reported for Bacillus pumilus (36), 7.7 for Schizophyllum
commune (37), and 7.5 for Streptomyces liVidans (35). The
enzyme was stable over a broad pH range from 5.5 to 9.0,
retaining about 80-90% activity after 2 h of incubation.
(Figure 4b).
V
max was found to be 0.175 µM min-1 mL-1. The Km value of
0.40 µM determined for purified finger millet esterase is lower
than the values reported for B. pumilus (1.54 mM) (36) and
F. succinogenes (2.7 mM) (39), indicating a higher specificity
for R-NA. Km values for two acetyl xylan esterases from
Thermoanaerobacterium sp. were determined using 4-methyl-
umbelliferyl acetate at 0.45 and 0.52 mM, respectively (38). A
Km value of 25 mM for barley malt acetic acid esterase was
reported using diacetin as the substrate (16). No information is
available with respect to the detailed kinetics and substrate
specificity of cereal/millet acetic acid esterases.
Effect of Metal Ions. A range of metal ions such as Fe+3
,
Cu+2, Ni+2, Ca+2, Co+2, Ba+2, Mg+2, Mn+2, Zn+2, and Al+3 at
5 mM concentration were tested for acetic acid esterase
activation/inhibition effects, and the results are given in Table
2. Metal ions such as Ni+2, Ca+2, Co+2, and Ba+2 reduced the
activity by 60-70%, while Mg+2, Mn+2, Zn+2, Al+3 reduced
the activity by 30-40% (Table 2). Similar concentrations of
Fe+3, Cu+2, Cu+1, and citric acid enhanced the enzyme activity.
Oxalic acid did not show any significant effect on the enzyme
activity. The inhibitory effect of Ca+2, Co+2, Ba+2, Mg+2, and
Zn+2 was in accordance with the results reported for acetic acid
esterase from Schizophyllum commune (37) and Bacillus pumilus
(36) and with ferulic acid esterase from Aspergillus awamori
(40). Fe+3 and Cu+2 increased the enzyme activity in accordance
with a 10% increase in activity of feruloyl esterase of Pencillium
expansum by Fe+3 ions (41).
Temperature Optima and Stability. To determine the
temperature optima of ragi acetic acid esterase, activities were
determined at a temperature range of 30-80 °C (Figure 5).
The optimum temperature was found to be 45 °C, and this was
comparatively lower than the temperatures reported for micro-
organisms such as Bacillus pumilus (55 °C), Streptomyces
liVidans (70 °C), and Thermoaerobacterium species (80 °C) (35,
36, 38). The purified enzyme was thermally active at 30 °C.
The stability of the enzyme decreased gradually, and at 60 °C
about 40% of the activity was retained. Less than 5% of the
activity was retained at 80 °C (Figure 5). Increase in the three-
dimensional structure of the enzyme, which is maintained by a
number of forces, mainly hydrophobic interactions and hydrogen
bonds, will be disrupted which results in the denaturation of
the protein and leads to inactivation of the enzyme.
The inhibitory and stimulatory effects of these ions may be
important factors in the commercial exploitation of this enzyme
where enzyme stability and activity are paramount.
Activation Energy. Activation energy, defined as the mini-
mum energy required by the reactants in order to pass into a

Acetic Acid Esterase from Malted Finger Millet
J. Agric. Food Chem., Vol. 55, No. 3, 2007 899
Figure 3. Molecular weight determination of purified ragi acetic acid esterase: (a) On SDS-PAGE: (A) Molecular weight markers: 205 kDa myosin; 97.4
kDa phosphorylase; 66.0 kDa bovine serum albumin; 43.0 kDa ovalbumin; 29 kDa carbonic anhydrase; 20.1 kDa soybean trypsin inhibitor; 14.0 kDa
lysozyme. (B) Purified acetic acid esterase. (b) On Sephacryl S-200: a,
â-amylase, 2.00 kDa; b, alcohol dehydrogenase, 1.50 kDa; c, bovine serum
album, 66 kDa; d, carbonic anhydrase, 29 kDa; e, papain, 12 kDa. Purified ragi acetic acid esterase, 79.4 kDa.
Figure 4. (a) pH optima and (b) pH stability of purified ragi acetic acid esterase.
statement, as the ionization values of these amino acids fall in
this range. PCMB is known to form complexes with cysteine
residues in the active site region of the enzymes. The effect of
eserine on the enzyme kinetics of purified ragi acetic acid
esterase showed that it is a competitive inhibitor (Figure 7).
Eserine is a structural anaolgue of the amino acid serine which
is known to be present in the active site of several of the
esterases (42).
Enzymic Deesterification of Acetylated Substrates. The
amount of acetylation achieved is determined by measurement
of enzymically released acetic acid from water soluble polysac-
charides and synthetic substrates. The specific activities of
purified ragi acetic acid esterase using various water soluble
polysaccharides and synthetic substrates is as follows: ragi,
0.027 U mg-1; wheat, 4.44 U mg-1; larch wood xylan, 0.061
Figure 5. Temperature optima and thermal stability of purified ragi acetic
acid esterase.
U mg-1; gum karaya, 0.282 U mg-1; R-NA, 3.38 U mg-1
;
Effect of Specific Reagents on Acetic Acid Esterase
Activity. Acetic acid esterase activity was determined in the
chemicals such as PCMB, iodoacetamide, and eserine at 45 °C
and 25 mM concentration. The acetic acid esterase was found
to be completely inactivated by PCMB as well as eserine
whereas iodoacetamide had showed almost negligible effect on
the activity (data not shown). The inhibition of PCMB and
eserine suggests the possible presence of amino acids such as
cysteine or serine residues at the active site pocket. The pH
optima (7.5) value of the acetic acid esterase also supports this
PNPA, 5.39 U mg-1 (U is µM min-1). The specific activity of
purified ragi acetic acid esterase with respect to water soluble
wheat preparation is higher than the other polysaccharide
substrates. Acetic acid esterase from ragi was active both on
small molecular weight substrates as well as polysaccharides
as indicated by the present study.
IR Analysis of Water Soluble Polysaccharides. The IR
spectra of water soluble preparations of polysaccharides such
as ragi, wheat, larch wood xylan, and gum karaya showed

900 J. Agric. Food Chem., Vol. 55, No. 3, 2007
Latha and Muralikrishna
Table 2. Effect of Metal Ions, EDTA, and Citric Acid on Purified Ragi
Acetic Acid Esterase
metal ions
control
relative activity (%)
100
+2
Cu
1130
1000
728.2
489.1
293.4
141.3
82.6
79.3
77.1
69.5
50.0
44.5
36.9
36.5
+
3
Fe
Cu
Hg
+
+
2
EDTA
citric acid
+
2
Mg
Zn
+2
+2
Mn
+
3
Al
+2
Co
Ba
Ni
Ca
+2
+
+2
not shown). The chemical shifts at 8.20-8.22 ppm were
assigned to H-3 and H-5 protons, while those at 7.34-7.36 ppm
were assigned to H-2 and H-6 protons in the aromatic ring of
PNPA. The chemical shift values at 2.235 were assigned to
acetyl protons of PNPA (figure not shown). The chemical shift
values for these protons were shifted after enzymatic deacetyl-
lation of PNPA to PNP (8.022-8053 and 6.83-6.86 ppm). The
unreacted PNPA showed a slight chemical shift value at 7.3
and 8.2 ppm for aromatic protons and at 2.2 for acetyl protons
(figure not shown). In the case of R-NA the chemical shift values
at 7.22-7.91 ppm were assigned for the aromatic protons of
naphthol and 2.35 ppm was assigned for the protons of the acetyl
group (43) (figure not shown). After enzymatic deacetylation,
there is a change in chemical shift values in the nucleus of
naphthol ring. The complete absence of acetyl protons and the
presence of a carbonyl proton at 10.0 ppm indicates the complete
deacetylation of R-NA to R-naphthol (figure not shown). The
complete hydrolytic cleavage of acetyl group of R-NA and
PNPA by ragi acetic acid esterase was found to be quite slow.
The practical use of acetic acid esterases particularly from
plant sources is limited because of the lack of sufficient
knowledge regarding their isolation, purification, and kinetic
properties. This particular study is an attempt in that direction
for possible exploitation of malt enzymes in modulating the
functional properties of cereal nonstarch polysaccharides in
various food applications.
Figure 6. Arrhenius plot for the temperature dependence of acetic acid
esterase using
R-naphthyl acetate as substrate.
Figure 7. Determination of Km and Vmax of ragi acetic acid esterase by
Lineweaver Burk plot with and without inhibitor (substrate used: -naph-
thyl acetate).
−
R
Conclusions. Ragi malt of 72 h gave maximum acetic acid
esterase activity compared to other malts. The enzyme was
purified to apparent homogeneity from 72 h malt by four-step
purification with a recovery of 0.36% and a fold purification
of 34. It was found to be a homotetramer with a molecular
weight of 79.4 kDa. The pH and temperature optima of the
enzyme were found to be 7.5 and 45 °C, respectively. The
activation energy of the enzymatic reaction was found to be
7.29 KJ mol-1. The apparent Km and Vmax of the purified acetic
medium-intensity absorption at 1200 cm-1, indicating the
presence of acetyl groups (figure not shown).
Electro Spray Ion-Mass Spectrometry (ESI-MS). Analysis
by ESI-MS confirmed the gradual deacetylation of R-NA and
PNPA to R-naphthol and PNP, respectively, by purified ragi
acetic acid esterase. The formation of PNP by the enzymatic
deacetylation of PNPA using ragi acetic acid esterase increased
with increase in incubation period (0-8 h). The ratio of signals
at m/z for PNP and PNPA (a. 0.6:1.0, b.1.33:1.0, c. 5.0:1.0, d.
100%) showed the complete deacetylation of PNPA to PNP
after 8 h of reaction (figure not shown). The m/z values of PNPA
and PNP are 193.70 and 137.15, respectively. Similar results
were observed with R-NA to R-naphthol. The m/z value of
R-naphthol is 142.70 and showed complete deacetylation of
R-NA (183.89, m/z negative mode). The amount of R-naphthol
has increased concurrently with increase in the incubation time
(figure not shown).
acid esterase were 0.04 µM and 0.175 µM min-1 mL-1
,
respectively, with respect to R-NA. Acetic acid esterase from
ragi malt is specific both for natural polysaccharides as well as
synthetic substrates. Eserine was found to be a competitive
inhibitor. The products liberated from R-NA and PNPA by the
action of purified ragi acetic acid esterase were identified by
1
ESI-MS and H NMR.
ABBREVIATIONS USED
1H NMR. The enzymatic deacetylation of R-NA and PNPA
PVPP, polyvinylpolypyrrolidone; PCMB, p-chloromercuric
benzoate; R-NA, R-naphthyl acetate; PNPA, p-nitrophenyl
1
was confirmed by carrying out H NMR experiments (figure

Acetic Acid Esterase from Malted Finger Millet
J. Agric. Food Chem., Vol. 55, No. 3, 2007 901
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ACKNOWLEDGMENT
We thank Dr. V. Prakash F.R.SC, Director CFTRI for his keen
interest in the work and encouragement.
Supporting Information Available: Elution profile of ragi
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JF0618527