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FTB-2803
original scientific paper
Studies of Extraction, Purification and Thermodynamic Characterization of
Almond (Amygadalus communis) -Galactosidase for the Preparation of
Delactosed Milk
Ajay Pal*
, Melita Lobo and Farhath Khanum
Biochemistry and Nutrition Discipline, Defence Food Research Laboratory,
Siddarthanagar, Mysore 570 011, India
Received: April 22, 2011
Accepted: September 24, 2012
Running Title Purification and properties of almond-galactosidase
SummaryThe conditions with respect to buffer (type, pH and ionic strength) and polyvinylpyrrolidone
(PVP) were optimized for efficient extraction of -galactosidase from almond seeds. Enzyme was
purified up to electrophoretic homogeneity employing (NH4)2SO4 (15-60 %) fractionation, size
exclusion (sephadex G-100) and ion-exchange chromatography (DEAE-cellulose). Molecular mass
of -galactosidase as estimated by gel filtration and SDS-PAGE was ~62 kDa confirming its
monomeric nature. The enzyme was optimally active at pH=5.5 while stable within pH range 5.0-
6.0. Various kinetic parameters of -galactosidase towards thermo-inactivation were calculated.
H, S and Gof thermal denaturation suggested that enzyme undergoes significant processes of
unfolding during denaturation. Using almond seed powder (10 g / 100 mL), lactose hydrolysis in
Corresponding author; Phone: ++91 821 2474 676; Fax: ++91 821 2473 468; E-mail:
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milk up to the extent of ~50 % was observed. The findings indicate the potential of almond seeds
for the production of low/de-lactosed milk for lactose intolerant population.
Key words: almond, extraction, -galactosidase, lactose intolerance, thermodynamics
Introduction
Lactose, the principal carbohydrate of milk occurring at a concentration of ~5 %, has
attracted the attention of researchers and dairy industry because of its associated nutritional (lactose
intolerance), technological (crystallization) and environmental (whey disposal) problems (1).
Lactose malabsorption / intolerance is common among approximately 70 % of the world's adult
population which is caused by the intestinal insufficiency of the enzyme -galactosidase/lactase,
EC. 3.2.1.23 (2,3). Deficiency of this enzyme causes the accumulation of undigested lactose in
small bowel, leading to increased influx of fluids inside the intestinal lumen. The unabsorbed
lactose is passed into the large intestine, which in addition to increasing fluid volume of gastro-
intestinal content, is acted upon by the colonic bacteria, resulting in the production of short chain
fatty acids, hydrogen gas and associated symptoms of lactose intolerance (4).
Besides lactose, milk and dairy products provide calcium, protein, potassium and riboflavin
in the human diet. Due to the gastrointestinal discomfort experienced after lactose consumption,
lactose intolerant individuals often avoid dairy products and thus eliminate a major source of
calcium and energy from their diet thereby inviting other complications like osteoporosis (3).
However, the problem can be solved by removing lactose from diet by the addition of exogenous -
galactosidase enzyme. The major products of hydrolysis, glucose and galactose, are sweeter, more
soluble and more readily absorbed into the mammalian intestine (5).
-Galactosidase is widely distributed in nature, being found in numerous microorganisms,
plant and animal tissues (1,6,7). But, before an enzyme preparation can be used in a food system, it
must be granted GRAS (generally recognized as safe) status (8). Although various -galactosidases
have been granted GRAS status and purified from many microorganisms for the preparation of de-
lactosed milk but, many of them have shown inhibition to Ca+2
, an intrinsic milk component
occurring at the concentration of 30 mM, indicating that lactose hydrolysis with these preparations
are not effective (1). Therefore, to improve commercial applications, other sources are being
explored in the search of a robust enzyme.
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Almond (Amygadalus communis) seeds have been traditionally consumed with milk with
almost unknown scientific reasons. While working on lactose intolerance, we found that almond
seeds are very rich source of -galactosidase. Although preparation of lactose free milk has been
tried using salt fractionated almond proteins (9) but to exploit an enzymes potential efficiently it is
necessary to absolutely purify and thoroughly characterize the enzyme in terms of its biochemical
properties. Hence, this study was aimed at extraction, purification and characterization of -
galactosidase from almond seeds and to explore the possibility of preparation of de-lactosed milk
for lactose intolerance population using the purified enzyme.
Materials and Methods
Materials
Almond seeds were procured from the local market while milk was obtained from the
Nandini Dairy, Mysore, Karnataka, India. Unless indicated otherwise, all the chemicals used in the
present investigation were of high quality analytical grade and were purchased from Sigma
Chemicals Co., USA, Sisco Research Laboratory, Himedia and E. Merck, Bombay.
Methods
Enzyme assay
The procedure as described in our earlier report (1) was used for the estimation of enzyme
activity. Hydrolytic activity of the -galactosidase was determined by measuring the release of o-
nitrophenol (ONP) from o-nitrophenyl--D-galactopyranoside (ONPG) and enzyme activity is
expressed as International Unit (IU) where IU is defined as the amount of enzyme that liberates 1.0
mol of ONP per minute under the assay conditions. All the experiments were done in triplicate and
the results are expressed as meanSD.
Optimization of extraction conditions for -galactosidase
Effect of buffer pH
-Galactosidase from almond seeds was extracted in buffers ofdifferent pHs such as sodium
acetate (pH=4.0-6.0), sodium phosphate (pH=6.5-7.5) and Tris-HCl (pH=8.0) at 50 mM ionic
strength.
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was first prepared and from the elution volume, the molecular mass of -galactosidase was deduced.
The molecular mass (Mr) standards used were alcohol dehydrogenase (150 kDa), bovine serum
albumin (66 kDa), ovalbumin (45 kDa) and carbonic anhydrase (29 kDa).
The molecular mass under denaturing condition was determined by sodium dodecyl sulfate-
polyacrylamide gel electrophoresis (SDS-PAGE) using a gel system (Bio-Rad Laboratories). The
SDS-PAGE was performed with a 5 % acrylamide stacking gel (pH=6.8) and 10% separating gel
(pH=8.8) (11). Proteins were visualized by staining with coomassie brilliant blue. Molecular mass
of the -galactosidase was calculated from the relative mobility of molecular mass markers run
simultaneously.
Determination of kinetic constants
To determine the maximum velocity (Vmax) and MichaelMenten constant (Km) of the -
galactosidase, initial reaction rates were measured by using ONPG substrate at different
concentrations (0-18 mM). Km and Vmaxvalues were calculated by means of the Lineweaver and
Burk (12).
Effect of pH on activity and stability of -galactosidase
The effect of pH on the activity of purified enzyme was determined in 50 mM HCl-KCl
buffer (pH=2.0), glycine-HCl buffer (pH=2.5-3.0), citrate buffer (pH=3.5-5.5) and phosphate buffer
(pH=6.0-8.0) using ONPG as the substrate. The optimum pH value obtained from these assays was
used in all the other experiments. Effect of pH on the stability was performed by pre-incubation of
the enzyme overnight at various pHs (pH=2.0-8.0).
Effect of temperature on activity and stability of -galactosidase
Optimal temperature for -galactosidase activity was determined by incubating the enzyme
solution with substrate in 100 mM acetate buffer (pH=5.5) for 30 min at various temperatures. The
activation energy (Ea) of catalysis of -galactosidase was determined from the slope of a plot
between ln v and T-1
(K) using the Arrhenius equation-
aESlope
R
= /1/
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The thermal stability of -galactosidase was investigated at seven different temperatures
between 40 and 60 C for varying periods of time in a temperature controlled water bath. The
enzyme solution was placed in a pre-warmed tube at the specified temperature and aliquots were
withdrawn at 30 min time intervals, cooled and residual activity assayed. The stability of the
enzyme was expressed as percent residual activity (%RA). The incubation was carried out in sealed
vials to prevent change of volume of the sample and hence, the enzyme concentration due to
evaporation. The data obtained from the thermal stability profile were used to analyze
thermodynamic parameters related to the -galactosidase activity. The experimental points were
plotted according to the equation given below-
0
ln .dA
k tA
= /2/
Where A0 is the initial activity, A is the residual activity after heat treatment, kd is thermalinactivation rate constant (min
-1) and t is the exposure time (min).
The half-life of the -galactosidase (t1/2, min-1
) was determined from the relationship-
1 / 2
ln 2
d
tk
= /3/
The D-values (decimal reduction time or time required to pre-incubate the enzyme at a given
temperature to maintain 10 % residual activity) was calculated from the following relationship-
ln10
d
D valuek
=
/4/
The z-value (temperature rise necessary to reduce D-value by one logarithmic cycle) was
calculated from the slope of graph between log D versus T (C) using equation-
1Slope
z
= /5/
The activation energy for denaturation (Ed) of -galactosidase was determined by an
Arrhenius plot of log denaturation rate constants (ln kd) versus reciprocal of the absolute
temperature (K) using the equation-
dESlope
R
= /6/
The change in enthalpy (H, kJ mol-1
), free energy (G, kJ mol-1
) and entropy (S, J mol-
1K
-1) for thermal denaturation of -galactosidase were determined using the following equation-
dH E RT = /7/
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.ln
.
d
B
k hG RT
k T = /8/
H GS
T
= /9/
Where Edis the activation energy for denaturation, T is the corresponding absolute temperature (K),
R is the gas constant (8.314 J mol-1
K-1
), h is the Planck constant (11.04 X 10-36
J min), kBis the
Boltzman constant (1.38 X 10-23
J K-1
) and kdis the deactivation rate constant (min-1
).
Effect of metal ions and chelator
To determine the requirement of metal ions for enzymatic activity, effect of various metal
ions and chelator (EDTA) on -galactosidase activity was determined by incorporating them in the
standard assay mixture with the final concentrations ranging from 5-30 mM. The activity was
expressed as relative activity compared to control.
Effect of reducing agents
The effect of various reducing agents on -galactosidase activity was determined by
incorporating them in the standard assay mixture with the final concentrations ranging from 5-30
mM. The activity was expressed as relative activity compared to control.
Individual and combined effect of end products
To study the individual effect of end products, glucose and galactose were added into the
reaction mixture in the range 20-140 mM while their equimolar mixture (1:1) was added in the
range of 40-280 mM to evaluate their combined effect on enzyme activity.
Hydrolysis of milk lactose using purified -galactosidase and almond seed powder
Skimmed milk was prepared by centrifuging cold milk at 10, 000 g for 15 min. The fat layer
was removed and treated with purified -galactosidase (100 IU / 100 mL). In a separate set of
experiment, different concentrations of almond seed powder were tried for milk lactose hydrolysis
at different temperatures. Aliquots of the reaction mixture were removed at different time intervals
and hydrolysis was terminated by placing in a boiling water bath for 5 min. The hydrolysis of
lactose was calculated by estimating the amount of glucose released by method as described by
Joshi et al. (13).
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Results and Discussion
Optimization of extraction conditions for -galactosidase
Initially, the extraction was performed in phosphate buffer (0.05 M, pH=7.0). Once the
enzyme activity was detected and the enzyme assay was developed, the extraction conditions were
optimized to yield maximum -galactosidase activity by manipulating buffer pH, ionic strength and
PVP concentration.
Effect of buffer pH
Results pertaining to the extraction of enzyme in buffers of varied pHs are shown in Fig. 1a.
As maximum activity was extracted in acetate buffer (0.05 M, pH=5.0), it was considered as the
best extractant. Enzyme extraction in alkaline pH showed diminished activity indicating the labile
nature of enzyme in alkaline conditions.
Effect of buffer ionic strength
Acetate buffer (100 mM, pH=5.0) was found best extractant for the almond -galactosidase
(Fig. 1b). However, the ionic strength required in present study was high as compared to that
required for the extraction of other plant enzymes (14,15).
Effect of PVP
Most of the plant products are good source of polyphenols which are known to form
insoluble complexes with enzyme. The complexes in turn reduce the extraction and activity of
enzymes. PVP helps in the absorption of phenolic compounds after tissue disintegration and act as
enzyme stabilizer. Therefore, its effect on extraction of -galactosidase was studied and results are
shown in Fig. 1c. The enzymatic activity was observed maximum in the presence of 0.75 % PVP in
extraction mixture. The protective effect of PVP during enzyme extraction has also been observed
in earlier studies (16).
Purification and characterization of -galactosidase
The -galactosidase from almond seeds was purified up to electrophoretic homogeneity
using a three-step chromatographic procedure. A summary of purification procedures is presented
in Table 1. The crude enzyme extract having total activity of 146.8 IU and specific activity of 0.09
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was subjected to (NH4)2SO4precipitation (15-60 %) at 4 C. During this step, enzyme was purified
4.2 fold with 81.54 % recovery. The precipitates recovered were then dissolved in 0.1 M acetate
buffer (pH=5.0), dialyzed against same buffer (diluted 1:10) and concentrated against solid sucrose.
The concentrate was loaded on to the ion-exchange column and eluted with a linear gradient of 0.5
M KCl after the removal of unbound proteins. The enzyme got eluted at 0.18 M KCl and the level
of recovery and fold purification in this step were 52.04 % and 26.3, respectively. The active
fractions were finally loaded on a pre-equilibrated sephadex G-100 column for gel filtration
chromatography. Elution profiles of the proteins and enzymes on DEAE-cellulose and sephadex G-
100 columns are presented in Fig. 2a, b. This purification scheme resulted in the final enzyme
preparation which was purified 50.9 fold with 33.72 % recovery of the total activity as compared to
crude extract.
Although numerous -galactosidases have been purified from microorganisms (1,3) but only
few reports are available from plant sources. Moreover, the plant -galactosidases have been studied
for deciphering their role in plant growth and development and not for the production of de-lactosed
milk. The -galactosidase I and II have been purified 7.4 and 53.5 folds from cowpea cotyledons
(17) and more recently, another -galactosidase has been purified from barley (Hordeum vulgare)
by Hemavathi and Raghavarao (18).
Purity and molecular mass determination
The molecular mass of purified -galactosidase as determined by gel filtration was ~62 kDa
(Fig. 2c). The purified preparation yielded a single protein band on SDS-PAGE (Fig. 2d) and
migrated with an apparent molecular mass ~62 kDa, as determined by relative mobility. The
comparable molecular mass of purified -galactosidase as determined by gel filtration and SDS-
PAGE confirms the monomeric nature of -galactosidase from almond seeds.
-Galactosidases of different molecular masses have been reported from plant sources. Five
isozymes of molecular masses 87, 87, 87, 73 and 45 kDa have been reported from mung bean
seedlings (19) while a 45 kDa -galactosidases has been purified from radish seeds (20).
Determination of kinetic constants
With increasing concentration of substrate in an otherwise standard assay mixture, the
enzyme showed a typical hyperbolic velocity saturation curve (Fig. 3a) revealing that it followed
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Michaelis-Menten kinetics. From the double reciprocal Lineweaver-Burk plot (Inset Fig. 3a), Kmof
the enzyme for ONPG was calculated as 10.53 mM with a Vmaxof 2.67 IU mL-1
.
Effect of pH on activity and stability of -galactosidase
The enzyme was highly active in a wide range of pH, showing more than 50 % of the
maximum activity in the pH range 3.5 to 6.5. The optimum pH of the purified -galactosidase was
5.5 (Fig. 3b). The results indicate that enzyme was quite stable over the broad pH range and is
suitable for hydrolysis of lactose present in whey or milk where pH varies from 4.5 to 6.8. It has
earlier been reported that the pH optima of plant -galactosidases are in the acidic pH range while
those derived from bacteria are in the neutral pH range (21).
A pH stability study is an essential part of any enzyme characterization before it can be
exploited commercially. The result shows that pre-incubation of the -galactosidase in the pH range
5.0 to 6.0 has no effect on the activity measured at pH=5.5. However, pre-incubation at 5.0>pH>6.0
resulted in decreased activity of -galactosidase (Fig. 3b). Thus, the decline in activity between
pH=5.0 and 5.5 and between pH=5.5 and 6.0 must result from the formation of an improper ionic
form of the -galactosidase and/or substrate. When the enzyme is pre-incubated at pH>6.0 or
pH
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3c). It is also obvious from the Arrhenius plot that the enzyme had a single conformation up to the
transition state.
The thermostability of -galactosidases incubated at various temperatures is depicted in Fig
3d. The enzyme showed no loss in activity after incubation at 35 C during the test period of 6 h. At
40 C and above, the activity decreased with increasing temperature. The plots of residual activity
versus incubation time for the enzyme were linear, with R2>92 %, indicating that the inactivation
could be expressed as first order kinetics in the temperature range 40-60 C (Table 2a). The half-life
(t1/2) determinations are more accurate and reliable especially when computing stability properties
of an enzyme at different temperatures. With increasing temperature, the t1/2and D-value decreased
and the first order thermal deactivation rate constants (kd) increased (Table 2a). From the results, it
is clear that enzyme is less thermostable at higher temperatures as a higher rate constant means the
enzyme is less thermostable (23). Thez-value of -galactosidase, calculated from the slope of graph
between log D versus temperature, was 15.8 C (Fig. 3e). In general, high z-value indicates more
sensitivity to the duration of heat treatment while lower z-value indicates more sensitivity to the
increase in temperature (24).
The activation energy (Ed) of the thermo inactivation mechanism is equal to 29.72 kcal / mol
(124.53 kJ / mol). The higher value found for the Ed in comparison to Ea means that a higher
amount of energy is needed to initiate denaturation as compared to catalysis (24). The enzyme had a
range of 121.93 to 121.76 kJ / molenthalpy of denaturation (H) at 40-60 C showing a decreasing
trend with increase in temperature (Table 2b). The high values of H obtained for the thermo
inactivation of -galactosidase indicate that enzyme undergoes a considerable change in
conformation during denaturation (25). The fact that H value decreases with increase in
temperature reveals that less energy is required to denature enzyme at high temperature (26). The
value of free energy of thermal denaturation (G) for -galactosidase was 104.96 kJ / mol at 40 C
which initially increased and then decreased with increase in temperature. When entropy of
inactivation (S) was calculated at each temperature, it showed positive values which indicate that
there are no significant processes of aggregation, since had this happened, the values would have
been negative (25). In contrast to present study, we have earlier reported that xylanase purified from
Aspergillus nigerDFR-5 undergoes significant processes of compaction/aggregation with increase
in temperatures (27,28). A decreasing H and negative Swith increase in temperature has also
been observed by Rajoka and Riaz (29) in parent and mutant xylosidase of Kluyveromyces
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marxianus PPY 125. It is also worth mentioning that G values, which are measures of the
spontaneity of the inactivation processes, are lower than the H values. This is due to the positive
entropic contribution during the inactivation process (30).
Based on the thermodynamic observation, it can be postulated that thermal denaturation of
enzyme may occur in two steps as shown below-
Where, N is native enzyme, U is unfolded enzyme that could be reversibly refolded upon
cooling and D is the denatured enzyme formed after prolonged exposure to heat and therefore
cannot be recovered on cooling. The thermal denaturation of enzymes is accompanied by the
disruption of non-covalent linkages, including hydrophobic interactions. The opening up of the
enzyme structure is accompanied by an increase in the disorder, randomness or entropy of
inactivation.
Effect of metal ions and chelator
The divalent metal ions such as Ca+2
and Mn+2
stimulated the enzyme activity by 62 and 49
%, respectively while monovalent cations like Na+1
and K+1
had small stimulating effect on the
activity at variable concentrations. Li+1
was found to inhibit the enzyme activity substantially (Table
3a). The addition of EDTA inhibited the enzyme activity, suggesting that metal ions are needed forthe enzymatic reaction.
Ca+2
is one of the important intrinsic components of milk and stimulation of enzyme activity
by this divalent cation is good at practical point of view since it will facilitate the hydrolysis of
lactose. In contrast to almond -galactosidase, microbial -galactosidases have been reported to be
inhibited by Ca+2
, thereby restricting their application (1).
Effect of reducing agent
The effect of a number of reducing agents, most widely used for the reduction of disulfide
bonds, was tested on -galactosidaseactivity (Table 3b). At a concentration of 15 and 25 mM in the
reaction mixture, ascorbic acid and vanillin activated the enzyme activity maximally to an extant of
10 and 16 %, respectively. The other reductive agents such as DTT, glutathione, cysteine, and -ME
also displayed identical behaviors but effect was not substantial. The stimulation of -galactosidase
N U D
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activity by reducing agents indicates that there is a relationship between the reduced form of the
cysteine residues and the activity of the -galactosidase.
Individual and combined effect of end products
Glucose and galactose are the end products of -galactosidase catalyzed hydrolysis of
lactose and therefore their effect on -galactosidase activity is worth studying. -Galactosidase
activity from almond seeds was considerably decreased in the presence of high concentration of
glucose and galactose indicating that lactose hydrolysis gets slowed down as the reaction products
build-up (Fig. 3f). It has earlier been observed by us that the end product of hydrolysis particularly
galactose competitively inhibits the enzyme activity (1,7). When glucose and galactose were
incorporated at a ratio of 1:1 in the reaction mixture, neither additive nor synergistic effect on
enzyme activity was observed (Fig. 3g). This may be due to well established second activity i.e.
transgalactosylation activity of this bifunctional enzyme by which it synthesizes
galactooligosaccharides (GOS) using glucose as a primer. So, in the presence of both glucose and
galactosidase, GOS are synthesized thereby decreasing the effective concentration of these
metabolites and subsequent enzyme inhibition.
Hydrolysis of milk lactose using purified -galactosidase and almond seeds
Lastly, lactose hydrolysis in skimmed milk was studied using purified -galactosidase and
hydrolysis up to the extant of 95 % was observed after 4 h of incubation at room temperature (data
not shown). The 100 % hydrolysis could not be estimated probably because once the end products
build-up, the transgalactosylation activity dominates which blocks glucose molecules in GOS
thereby making it unavailable for estimation.
To prepare low/de-lactosed milk at home, powered almond seeds were added into the milk
instead of purified -galactosidase and the results obtained are shown in fig. 3h. Our study shows
that ~50 % lactose hydrolysis can be achieved by incubating 100 mL milk with 10 g powder for 5 h
at 42 C. Apart from low lactose content, the antioxidant activity of almond will additionally make
the milk more nutritious. Moreover, the proteolytic enzymes of almond might partially digest the
milk protein to further aid in its easy digestion.
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Conclusions
A 50.9 fold purification and 33.72 % recovery of -galactosidase was achieved from almond
seeds using neutral salt fractionation (15-60 %), gel permeation (sephadex G-100) and ion-
exchange chromatography (DEAE-cellulose). The enzyme was monomeric in nature with molecular
mass ~62 kDa as estimated by gel filtration and SDS-PAGE. H, S and G of thermal
denaturation revealed that enzyme undergoes through the significant processes of unfolding during
denaturation. Milk lactose up to the extant of 95 % could be hydrolyzed using purified preparation
while ~50 % hydrolysis was observed using almond seed powder. The findings indicate the almond
seeds can successfully be employed for the production of low/de-lactosed milk for lactose intolerant
people.
Acknowledgement
The authors are grateful to Dr. A.S. Bawa, Ex-Director and Dr. H.V. Batra, Director Defence
Food Research Laboratory, Mysore, for providing all the necessary facilities, constant guidance and
encouragement during this investigation.
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23. A.G. Marangoni, Enzyme kinetics: A modern approach. John Wiley and sons, NJ (2003) 140-147.
24. H. Tayefi-Nasrabadi, R. Asadpour, Effect of heat treatment on buffalo (Bubalus bubalis)lactoperoxidase activity in raw milk,J. Biol. Sci. 8(2008) 1310-1315.
25. E. Marin, L. Sanchez, M.D. Perez, P. Puyol, M. Calvo, Effect of heat treatment on bovinelactoperoxidase activity in skim milk: kinetic and thermodynamic analysis, J. Food Sci. 68
(2003) 89-93.
26. H.N. Bhatti, A. Zia, R. Nawaz, M.A. Sheikh, M.H. Rashid, A.M. Khalid, Effect of copper ionson thermal stability of glucoamylase from Fusariumsp,Int. J. Agric. Biol. 7(2005) 585-587.
27. A. Pal, F. Khanum, Purification of xylanase from Aspergillus niger DFR-5: Individual andinteractive effect of temperature and pH on its stability, Process Biochem. 46 (2011a) 879-
887.
28. A. Pal, F. Khanum, Covalent immobilization of xylanase on glutaraldehyde activated alginatebeads using response surface methodology: Characterization of immobilized enzyme, Process
Biochem.46(2011b) 1315-1322.
29. M.I. Rajoka, S. Riaz, Hyper-production of a thermotolerant -xylosidase by a deoxy-D-glucose and cycloheximide resistant mutant derivative of Kluyveromyces marxianusPPY 125,
Electronic J. Biotechnol. 8(2005) 177-184.
30. A. Tanaka, E. Hoshino, Calcium-binding parameter of Bacillus amyloliquefaciens-amylasedetermined by inactivation kinetics,Biochem. J. 364(2002) 635-639.
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0
20
40
60
80
100
120
4 4,5 5 5,5 6 6,5 7 7,5 8
Relativeactivity(%)
pH
Fig. 1 (a). Effect of buffer pH on extraction and activity of almond -galactosidase.
0
20
40
60
80
100
120
50 100 150 200
Ionic strength (mM)
Relativeactivity(%)
Fig. 1b. Effect of buffer ionic strength on extraction and activity of almond -galactosidase.
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18
60
80
100
120
140
0 0.25 0.5 0.75 1
PVP (%)
Relativeactivity(%)
Fig. 1c. Effect of PVP on extraction and activity of almond -galactosidase.
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10
30
50
70
90
110
1 7 13 19 25 31 37 43 49 55 61 67 73 79 85 91 97
Fraction no.
Relativeactivity(%)
0
0.22
0.44
0.66
0.88
1.1
Absorbance(280nm)andKClgradient(M)
Activity Protein Gradient
Fig. 2a. Elution profile of -galactosidase on DEAE-cellulose column.
0
0.2
0.4
0.6
0.8
1
1.2
1 5 9 13 17 2 1 25 29 33 3 7 41 45 49 53 5 7 61 65 6 9 73 77 81 85 8 9
Fraction no.
Absorbance(280nm)
10
30
50
70
90
110
Relativeactivity(%)
Protein Activity
Fig. 2b. Elution profile of -galactosidase on sephadex G-100 column.
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Fig. 2c. Estimation of molecular mass of -galactosidase through gel filtration on sephadex G-100
column.
Fig. 2d. SDS-PAGE analysis of purified -galactosidase. Lane 1: purified -galactosidase; lane 2:
molecular mass marker.
y = -0.0103x + 5.6696
R2= 0.9925
3.8
4.2
4.6
5
5.4
40 60 80 100 120 140 160
Elution volume (ml)
LogMW
Ovalbumin
Alcohol dehydrogenase
Bovine serum albumin
Carbonic anh drase
-galactosidase
1 2 kDa
116.0
14.4
66.2
45.0
25.0
35.0
18.4
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Fig. 3a. Michaelis-Menten kinetics for -galactosidase. Inset is the Lineweaver-Burk plot for
estimation of kinetic parameters.
0
20
40
60
80
100
120
3.5 4.5 5.5 6.5 7.5
pH
Relativeactivity(%
pH optima pH stability
Fig. 3b. Effect of pH on activity and stability of -galactosidase.
0
0.3
0.6
0.9
1.2
1.5
1.8
0 3 6 9 12 15 18 21
Substrate (mM)
Enzymeactivity(IU/ml)
y = 3.8435x + 0.3762
R2= 0.9769
0
0.4
0.8
1.2
1.6
-0.15 0.05 0.25 0.45
1/[S]
1/v
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Fig. 3c. Effect of temperature on -galactosidase activity. Inset is the Arrhenius plot to calculate
activation energy (Ea) of catalysis.
y = -2.2456x + 7.1332
R2= 0.9494
-0.4
-0.2
0
0.2
0.4
2.97 3.06 3.15 3.24 3.33
1000/T (K)
logV
0
20
40
60
80
100
120
25 35 45 55 65 75 85
Temperature (C)
Re
lativeactivity(%)
log V = -2.2456 (1000/T) + 7.1332
R2= 0.9494
-0.4
-0.2
0
0.2
0.4
2.97 3.06 3.15 3.24 3.33
1000/T (K)
logV
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Fig. 3d. First order thermal deactivation of purified -galactosidase. Inset is the Arrhenius plot to
calculate activation energy (Ed) for thermal denaturation.
-4.5
-3.5
-2.5
-1.5
-0.5
0.5 Time (min)
ln(A/Ao)
40C 45C 50C 55C 60C
40 80 160120
ln Kd = -15.013 (1/T) + 40.981
R2= 0.9583-7
-6.25
-5.5
-4.75
-42.99 3.04 3.09 3.14
1/T (K)
lnKd
-4.5
-3.5
-2.5
-1.5
-0.5
0.5 Time (min)
ln(A/Ao)
40C 45C 50C 55C 60C
40 80 160120
ln Kd = -15.013 (1/T) + 40.981
R2= 0.9583-7
-6.25
-5.5
-4.75
-42.99 3.04 3.09 3.14
1/T (K)
lnKd
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Fig. 3e. Temperature dependence of the decimal reduction of -galactosidase to calculate z-value.
0
20
40
60
80
100
20 40 60 80 100 120 140
Concentration (mM)
Relativeactivity(%ofcontrol)
Glucose Galactose
Fig. 3f. Individual effect of glucose and galactose on -galactosidase activity.
1.9
2.1
2.3
2.5
2.7
2.9
3.1
3.3
38 40 42 44 46 48 50 52 54 56 58 60 62
Temperature (C)
LogD
Z-value
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0
20
40
60
80
100
40 80 120 160 200 240 280
Concentration (mM, glc:gal::1:1 )
Relativeactivity(%ofcontrol)
Fig. 3g. Combined effect of glucose and galactose on -galactosidase activity.
0
15
30
45
60
0 1 2 3 4 5
Time (h)
Lactosehydrolysis(%)
37C 42C 45C
Fig. 3h. Almond seed powder catalyzed lactose hydrolysis at different temperatures.
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Table 1. Purification and activity of -galactosidase.
Fraction Total activity
(IU)
Total protein
(mg)
Specific activity
(IU / mg)
Purification
fold
Yield
(%)
Crude extract
146.8 1642.2 0.09 1 100
Ammonium sulphate
(15-60 %)
119.7 314.5 0.38 4.2 81.54
Ion-exchange
chromatography
76.4 32.2 2.37 26.3 52.04
Gel permeation
chromatography
49.5 10.8 4.58 50.9 33.72
Table 2.
(a) Inactivation kinetic parameters of -galactosidase towards thermal processes.
Temperature (C) kd(min-1
) R2
(%) t1/2 (min) D-value (min)
40 0.0012 99.7 577 1919
45 0.0015 99.1 462 1535
50 0.004 92.8 173 576
55 0.0071 95.7 98 324
60 0.0208 93.3 33 111
z-value = 15.8 C; Ea= 10.23 kcal / mol (42.86 kJ / mol)
kd= Thermal inactivation rate constant; R2= Coefficient of correlation; t1/2 = Half-life; D-value =
Decimal reduction time
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(b)Thermodynamic parameters for thermal inactivation of -galactosidase.
Temperature (C) H (kJ mol-1
) G (kJ mol-1
) S (J mol-1
K-1
)
40 121.93 104.96 54.21
45 121.89 106.09 49.68
50 121.85 105.16 51.67
55 121.8 105.26 50.43
60 121.76 103.94 53.51
Ed(Activation energy for denaturation) = 29.72 kcal / mol (124.53 kJ / mol)
H = Variations in enthalpy; G = Variations in free energy; S = Variations in entropy
Table 3.
(a) Effect of metal ions and chelator on -galactosidase activity
Additives
Enzyme activity (% of control)a
Concentration (mM)
5 10 15 20 25 30
Ca+2
122.87.8 124.66.2 126.54.7 162.212.2 ppt ppt
Mn+2
121.35.4 129.35.8 138.86.2 149.18.9 148.56.2 147.08.5
Mg+2
100.55.2 101.34.3 104.74.9 110.14.6 119.95.4 123.43.9
K+1
102.54.9 106.85.8 109.83.8 112.54.8 115.84.9 119.45.2
Na+1 100.95.2 104.34.2 106.24.1 108.16.2 111.56.7 112.84.2
Li+1
92.64.6 86.36.2 83.93.7 78.84.7 72.94.3 64.23.7
EDTA 79.53.4 67.24.8 60.44.2 54.82.9 49.83.4 46.72.8
aValues are meanSD of 3 experiments.
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(b) Effect of reducing agents on -galactosidase activity
Reducing
agents
Enzyme activity (% of control)a
Concentration (mM)
5 10 15 20 25 30
Ascorbic acid 102.56.7 106.87.1 110.44.8 109.67.5 108.77.2 109.24.9
Vanillin 104.85.9 109.13.8 112.64.2 115.504.9 116.85.8 114.88.2
Cysteine 102.64.8 104.84.8 103.87.5 104.23.8 103.84.6 104.36.4
Glutathione 101.56.1 103.87.5 104.86.4 105.24.8 105.86.1 105.25.7
-ME 100.95.6 101.56.4 100.84.8 101.53.7 101.34.8 100.76.7
DTT 106.93.8 105.84.8 104.66.4 105.65.8 104.92.9 105.86.7
aValues are meanSD of 3 experiments.
