Extraction, Purification and Characterization of Β-Galactosidase from Tomato (Lycopersicom Esculentum) and
It’s Application in Milk
1Sari Ali Hussien1,Kifahsaed doosh *2
College of Agriculture- University of Anbar, 2College of Agriculture- University of Baghdad
*Corresponding author: [email protected]
Abstract: β-galactosidase (EC 3.2.1.23) is important in the formation of a medicinal plant tomato (Lycopersicom esculentum). The plant extracts of tomato (Lycopersicom esculentum) were used to characterize the enzyme in the term of pH, temperature, enzyme kinetic and effects of some heavy metals on its activity. The enzyme activity was measured by its ability to hydrolyze the substrate 2-nitrophenyl β-D-galactopyranoside (ONPG). the
isoelectric point for enzyme was 4.4. Carbohydrate concentration of enzyme was found to be 19.5 % by employing phenol-sulfuric acid method. The Km and Vmax values of the enzyme were 3.65 mM and 0.18μmol/min, respectively. The activities of β-galactosidase from tomato (Lycopersicom esculentum)is completely inhibited by HgCl2 and KCN. After 4h incubation, lactose in milk was reduced by 38.5 and 70% by β-galactosidase from tomato (Lycopersicom esculentum), respectively. The study showed that Hg+2 was the most potent inhibitor while Cu+2 exhibited the least inhibition degree on β-galactosidase activity in the tomato (Lycopersicom esculentum). These finding indicated that the enzyme β-galactosidase in the leaves extract of tomato (Lycopersicom esculentum) can be used in industrial and medical applications.
Keywords: tomato (Lycopersicom esculentum) β-galactosidase, Enzymatic Kinetics, Heavy Metals
Introduction
β-galactosidase (also β-D-galactohydrolase) called lactase and transglycosylases [1] are group of enzymes able to cleave β linked galactose residues from various compounds and is commonly used to cleave lactose into galactose and glucose [2], it was widely distributed in nature and found in many microorganisms, plant and animal tissues [3, 4].β-galactosidases have many biological roles include degradation of structural polysaccharides in plant cell walls; thereby can promote their loosening and th consequent elongating of the cell [5]. They have many medical and industrial applications include treatment of lactose malabsorption and production of lactose hydrolyzed milk [6, 7, 8]. These enzymes have two important applications: the removal of lactose from milk products for lactose intolerant people and production of galactosylated products [12, 13].β-galactosidases have been detected in a wide range ofplant organs and tissues and are described by their ability to hydrolyze terminal non-reducing β-D-galactosyl residues from β-D-galactosides [9]. It has been purified from various plant sources, like chick pea [10], almond [11], apricots [12], Vigna unguiculata [13], apricot seed [14]. β-galactosidase play key roles in fruit ripening. β-galactosidase activity was reported during fruit development and ripening for rice [15], pepper [16] and Arabidopsis [17]. Many studies have indicated remarkable increase in expression level of mRNA β-galactosidase during fruit ripening in many fruits [18]. It was reported that β-galactosidases are widely distributed in many plant tissues, like seeds [11,
19], stems [19], and meristem zones of roots, trichomes, cotyledons, vascular tissues, and pollens [20, 21]. On the other hand, it also participates in the cell wall modification during elongation and differentiation of plant cells [22, 23]. Plant β- galactosidase would be best suited for industrial applications because of its easy availability, cost effectiveness and easy adaptability [24]. β-galactosidase from almond seeds was used for the preparation of delactosed milk for those lactose intolerants individuals [11].Heavy metals are essential and important for plants growth, and play a great role in many vital compounds [25]. Some of these metals are micronutrients necessary for plant growth, such as Zn2+, Cu2+, Mn2+, Ni2+, and Co2+, while others have unknown biological function, such as Cd2+, Pb2+ and Hg2+ [32]. At high concentrations, all heavy metals have strong toxic effects and are regarded as environmental pollutants [26]. They may alter the reaction rates and influence the kinetics properties of enzymes which cause changes in metabolism of plant, or any excessive amount of heavy metals may drive the oxidative stress [27]. (Lycopersicom esculentum) tomato in Arabic, is a perennial fragrant shrub, bushy herbs, grows in the valley bottoms in the desert areas particularly at the Southern part nearest Saudi-Jordan borders (Al- Mudawarah) and in Sinai Peninsula in Egypt [36, 37, 38]. It is used traditionally as a medicinal herb in Jordan and Egypt. Artemisia has multiple beneficial bioactivities such as antiviral, antipyretic, antihemorhagic, anticoagulant, antitumor, anti-anginal, anti-ulcerogenic and anti-hepatitis [39]. In this original study, the plant extracts of tomato (Lycopersicom esculentum). were used as a source for the enzyme β- galactosidase. The enzyme activities, kinetics and the effects of heavy metals were investigated as a primary step for the use of β-galactosidase in industrial, biotechnological and medical applications in future.
Materials and Methods Materials
Fresh plant sample of tomato (Lycopersicom esculentum). was collected from Iraqi local markets. Characterization of β-galactosidase was conducted at the Biochemistry lab in Baghadad University.
Plant Extract Preparation
plant extract was prepared from tomato (Lycopersicom esculentum) and used as source for β- galactosidase. Plant were homogenized in 0.2 M sodium-phosphate buffer (pH 6.0) in a blender for 4 min. The homogenate was filtered using cloth sheet and then was centrifuged for 20 min at 10,000 rpm. The supernatants were used for β-galactosidase assay as crude enzyme solution [28].
Protein Estimation
The protein content has been specified in a spectro-photometric way at 595nm using the Bradford method [29] with the use of the Bovine Serum Albumin (BSA) as standard.
Enzyme Assay
Crude plant extract was prepared tomato (Lycopersicom esculentum) and used as source for β- galactosidase β-galactosidase activity was assayed by measuring the rate at which it hydrolyzes ONPG by the method described by Sekimata et al. [33]. In the presence of β- D-galactosidase, ONPG is hydrolyzed to D-galactose (colorless) and o-nitrophenol (ONP) (yellow). The reaction mixture of β-galactosidase contained 0.4 ml of 0.1 M acetate buffer (pH 4.0), 0.5 ml of 2 mM of
substrate and 0.1 ml of enzyme solution. After incubation for 15 min at 37°C, the reaction was terminated by addition of 1 ml of 0.1 mM Na2CO3 and monitored at 420 nm. One unit of enzyme activity is defined as the amount of enzyme that liberates 1.0 μmol of ONP per minute under the assay conditions.
Figure1: standard curve of ONP using to quantified o-nitrophenol released in the presence of β-D-Galactosidase
Determination of Kinetic Parameters
To determine the maximum velocity (Vmax) and Michaelis-Menten constant (Km) of β- galactosidases, ONGP was used as substrate, and the effect of substrate concentration on enzyme activity were studied at pH 6.0 and at temperature 50°C. The concentration of ONGP was increased from 1 mM to 9 mM. The enzyme activity was assayed by monitoring the absorbance at 420 nm. Line weaver-Burk Plot (Reciprocal plots) used to determine Vmax, and Km values [33].
Activation energy determination:
Activation energy of enzyme for converting ONPG to product was determined using Arrhenius equation.
Determination of Isoelectric point (pI): Method that described by [16] in preparation of solutions and pI determination by isoelectric focusing, was used.
Estimation of Carbohydrate content Phenol-sulfuric acid method which described by Dubois et al wasused. [17]
Effect of metals ions, inhibitors and other substance on the enzyme activity
Stock solutions of CaCl2, HgCl2, CuSO4, FeCl3, KCN, NiCl2, MnCl2 and FeSO4 were prepared in acetate buffer (at optimum pH for each β-galactosidase extract) were added separately to the reaction mixture to a final concentration of 0.001 mol/Liter. Inhibitors such as beta-mercapt-oethanol (0.01 mol/Liter) and ethylene-diamine tetracetic acid (EDTA) (0.0001 mol/Liter) were also assayed. The residual enzyme activity was assayed and expressed as a percentage of the activity determined in acetate buffer alone (control).
Statistical Analyses
Every experiment has been applied in triplicates and results have been represented as values of average ± standard deviations (SD) with the use of the Microsoft excel 2007.
Results and Discussion . Protein Content
The protein content in the extract of tomato (Lycopersicom esculentum) was measured by Bradford method using BSA as standard protein (Figure 2). The result showed that the extract of tomato (Lycopersicom esculentum) has (0.56 mg/ml) amount of protein.
Figure 2. Determination of protein content (mg/ml) in crude extract of using BSA as standard.
Isoelectric Point of β-Galactosidase:
One protein band was appeared after coloring the gel with Commassie Brilliant Blue R-250 as shown in figure 4. Isoelectric point of β-galactosidase was found to be 4.4 when estimated by Isoelectric focusing which depends on progressive hydrogen number (pH) of the gel that is sustainable and stable due to the charged small particles (Ampholytes) and that means the existence of higher percentage of acidic amino acids in the protein as compared to basic amino acids. Our result is in agreement with [26].
y = 1.601x + 0.002 R² = 0.997 0
0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
0 0.1 0.2 0.3 0.4 0.5 0.6
Absorbance (595 nm)
Concentration (mg/ml
Figure (3): Isoelectric point pI of β-galactosidase purified from tomato Carbohydrate content:
Figure (5) shows that the carbohydrate peak at 490nm is approximately identical to the protein peak at
280nm and activity peak at 420nm, this is an evidence of presence carbohydrates in the protein structure of lactase and this means that apricot lactase is a glycoprotein. Carbohydrate concentration of apricot β- galactosidase was found to be 19.6 % by employing phenol-sulfuric acid method and this result approximately agree with [27]. Carbohydrate percentage in lactase enzyme differs from one source to another.
Table (1): Carbohydrates are% of protein
Enzyme Absorbance at
490 nm
Carbohydrate content Carbohydrates
are% of protein
B galactosidase 0.25
20.011 19.6
Activation energy of β-galactosidase:
Activation energy (Ea) of lactase purified from apricot for conversion of ONPG to product was measured to be 16.56 kcal/ mol; and this value is within the range that stated by [33] which is located between 6-18Kcal/mol. Low value of Ea is considered as an indicator of the catalytic efficiency of the enzyme to convert substrate to products. Whereas, it was found that inactivation energy of purified enzyme from apricot equals 48.46kcal/mol, this value gives an idea about un-
0 10 20 30 40 50 60 70 80
0 1 2 3 4 5 6 7 8 9 10
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24
Enzyme activity U/m)
ph
tube namber
IP
pH
stability of enzyme at high temperatures. Inactivation energy of enzymes ranged 40-175 Kcal/mol. [34]
Figure (4): Arrhenius plot for determining activation energy of apricot β-galactosidase.
Kinetics Analysis
To determine the enzyme kinetic parameters (Km and Vmax of β-galactosidase), initial reaction rates at different ONPG concentration ranging from 1 mM to 9 mM were measured. The data were analyzed according to Line weaver Burk plot by plotting 1/V value against 1/[S] value and kinetic parameters were calculated from the graph. The results of Km and Vmax values of the enzyme were 3.65 mM and 0.18 μmol/min, respectively (Figure 4).
Figure 5. Determination of Vmax and Km values of the enzyme β-galactosidase in the extracts of tomato (Lycopersicom esculentum) using ONPG as a substrate.
0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4
2.9 3 3.1 3.2 3.3 3.4
Log K
0bs1/Tx( 1000))
-0.4 0.1 0.6 1.1 1.6
-5 -4 -3 -2 -1 0 1 2 3 4 5 6 7 8 9 10 11
1/ S 1/ Vo
Km is the concentration of substrate at which the reaction rate is half- maximum. Km is important in enzyme kinetic because its value includes not only the affinity of substrate for the enzyme but also the rate at which the enzyme-bound substrate is converted to the product in the catalytic reaction. Thus, Km value can be interpreted as a crude measurement of the affinity of the substrate for the enzyme [55]. The Km value of the enzyme was higher than reported earlier, 1.67 mM for carrot [56], 1.77 mM for tomato fruit [48], 1.85 mM for apricot β-galactosidase I [11], 1.73 mM for chick pea [13] and 1.19 mM for radish seed [33], but it was lower than that of other plants such as 5.16 mM for peach [46] and 10.53 mM for almond [6]. The rate or velocity of a reaction (V) is the number of substrate molecules converted to product per unit time. The rate of an enzyme-catalyzed reaction increases with substrate concentration until a maximal velocity (Vmax) is reached. The leveling off of the reaction rate at high substrate concentrations reflects the saturation with substrate of all available binding sites on the enzyme molecules present [49]. However, the Vmax value of the enzyme β-galactosidase in the crude extract was higher than reported earlier. The Vmax value for β-galactosidase I, β-galactosidase II and β- galactosidase III isolated from apricots was found to be 0.52, 0.70 and 0.38 μmol/min, respectively [18], but it was lower than that of other plants such as 5.2 μmol/min for rice [46].
Effect of metal ions and inhibitors on β-galactosidase Activity in tomato (Lycopersicom esculentum) Extract
figures (5) show effects of some metal ions and inhibitors on the β-galactosidase activity extracted from tomato (Lycopersicom esculentum). The data indicated that the activities of β- galactosidase isolated from tomato (Lycopersicom esculentum) are completely inhibited by HgCl2 and KCN. CaCl2, NiCl2 KCN and CuSO4 reduced the activities of β-galactosidase by 63.12, 34.18 , 0 and 33.5% respectively. FeCl3 and FeSO4 increased the activities of β- galactosidase by 149 and 152.4%, respectively. Ag , EDTA and Beta-M1 increased the activities of β-galactosidase by 0 , 118.5 and 104.3 %, respectively.
Fig. 6: Effect of metal ions on the β-galactosidase activity extracted from tomato (Lycopersicom esculentum)
0 50 100 150 200
10097.3
0 149
112.8
44.168.5 152.4
0
104.3118.5
Relative activity
MnCl2 increased the activities of β-galactosidase from tomato (Lycopersicom esculentum) by 12.8%, [40] . noted that the presence of divalent actions such as Fe2+ and Mn2+ in the reaction mixture increased the activity of β -galactosidase from Lactobacillus crispatus [41].
extracted β-galactosidase from Kluyveromyces marxianus IFoo541 and K. marxianus var. lactase 1-2. They found that the crude enzyme from K. marxianus IFoo541 was activated largely by Mn2+ and inhibited by Ca2+. However, the crude enzyme from K. marxianus var. lactis 1-2 was strongly activated by Ca2+ and minimally activated by Mn2+. β-galactosidase from mung bean reduced by Fe2+ (15.1%), Ca2+ (15.1%), Cu2+ (23.4%) and Mn2+ (21.1%), but strongly inhibited by Hg2+ [42]. The activity of β-galactosidase from Bacillus coagulans RC53 was inhibited by Fe2+ (17.1%), Cu2+(91.1%), Ni2+ (76.3%) and Hg2+ (65.7%), while the activity of the enzyme was activated by Ca2+ (7.5%) and Mn2+(19.6%) [43]. [44] noted that the activity of β-galactosidase from Bifidobacterium infants HL96 was activated by Fe2+ and Mn2+ while, the enzyme was partially inhibited by Ca2+ and it was completely inhibited by Hg2+ and Cu2+. [42]
reported that the activity of β-galactosidase from Bacterium pseudoalteromonas increased by Mn2+ (40%), but strongly inhibited by Cu2+.
Figure (6) shows the effect of inhibitors on the β-galactosidase activity extracted from tomato (Lycopersicom esculentum). The data indicated that the beta-mercaptoethanol had very little effect on the activity of β-galactosidase tomato (Lycopersicom esculentum). However, EDTA inhibited the activity of the enzyme from tomato (Lycopersicom esculentum) by 18.5 and 4.3%
respectively [44] . found that the activity of β-galactosidase from mung bean was inhibited by EDTA. The activity of β-galactosidase from chickpea was not affected by EDTA [45]. The activity of β-galactosidase from persimmon fruit was inhibited greatly by EDTA [46].
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