Kinetic Studies on β-Galactosidase Isolated from Apricots ( Prunus armeniaca kaisa )

β-galactosidase was extracted from apricots (Prunus armeniaca kaisa) and characterized biochemically. Three isoenzymes (β-gal I, β-gal II and β-gal III) were obtained by salt fractionation and ion-exchange and Sephadex G-100 column chromatography. β-galactosidase II showed a high ability to hydrolyze the substrate p-nitrophenyl β-D-galactopyranoside than that of β-galactosidase I and III. The individual peaks showed charge homogeneity as revealed by single band on polyacrylamide gel. The molecular weight of β-gal I, β-gal II and β-gal III as determined by gel filtration was found to be 44.15, 34.70 and 23.71 KDa respectively. The optimum pH for the activity different isozymes was found between 4 and 6. The isoenzymes were determined to be thermally stable up to 40 ̊C. The Km value for β-gal I was 1.85 mM which was higher than that of β-gal II (Km = 1.7), and β-gal III (Km = 1.19). The Vmax value for β-gal I, β-gal II and β-gal III was found to be 0.52, 0.70 and 0.38 μmole/min respectively.


Introduction
Fruits constitute a commercially important and nutritionally indispensable food commodity.Excessive ripening of fruits is a serious problem in the market and/or during storage.This is a major cause of quality loss and a significant problem for the food industry.
Ripening of fruit is usually accompanied by loss of firmness or softening, a process that changes the color and aroma of the fruit.The process may be mediated by the action of hydrolytic enzymes on the polysaccharides of the cell wall.Pectin modifying enzymes, polygalacturonase (PG) and pectin methylesterase (PME) are known to be involved in the solubilization of pectin during fruit softening processes.However, molecular evidence has suggested that these enzymes alone are not responsible for causing softening in tomato [1].Further investigations demonstrated that pectin solubilization by β-galactosidase is actively involved in fruit softening [2][3][4].Although some loss of galactosyl residues could result indirectly from the action of PG, β-galactosidase is the only enzyme identified in higher plants capable of directly cleaving β- (1, 3 and 4) galactan bonds, and probably plays a role in galactan side chain loss [5][6][7].
In this study, β-galactosidase from apricots (Prunus armeniaca kaisa) was isolated and purified to homogeneity.Three isoforms of the enzyme were identified and their physiochemical, functional and kinetic properties were studied.

Materials
p-nitrophenyl β-D-galactopyranoside (PNGP), Sephadex G-100 cytochrome c, ovalbumin and pepsin were obtained from Sigma Chemical Co.(USA).BSA and CMcellulose were obtained from SRL Chemicals, Mumbai, India.All other reagents and chemicals were of analytical grade.Ripe mature apricots (Prunus Armeniaca kaisa) were obtained from Sher-e-Kashmir University of Agriculture, Science and Technology, Srinagar, J&K, India.rated apart.The cortical tissue was weighed (332 g) and homogenized for 10 min in ice cold extraction buffer (0.1 M sodium acetate pH 5.2) that contained 0.1% (v/v) βmercaptoethanol, 2% (w/w) polyvinyl poly pyrroli done, 12% (w/v) polyethylene glycol and 2% (w/v) sodium bisulfate.The homogenate was filtered through 4 layers of cheese cloth & the suspension obtained was stirred on magnetic stirrer for about 4 hours at 4˚C and 1 M NaCl was added.The residue left after filtration was washed twice by homogenization in 300 ml ice cold extraction buffer, followed by filtration.The final residue was weighed and brought up to 300 g with 0.5 M NaCl.This was stirred for 1 h at 4˚C, and then filtered through Whatman No.1 filter paper.The filtrate was combined with the suspension and was centrifuged on Remi C 24 cooling at 13,000 rpm for 10 min at 4˚C.From the supernatant, the proteins were precipitated by ammonium sulphate at 85% saturation.The crude extract was further purified involving ion exchange chromatography on CMcellulose column using 0.1 M sodium acetate buffer, pH 5.2, using linear sodium chloride gradient from 0.1 to 0.5 M. The fractions eluted from ion-exchange column were chromatographed separately on Sephadex G 100 column in 0.1 M sodium acetate buffer, pH 5.2.The charge homogeneity of the fractions eluted from gel filtration column was checked by polyacrylamide gel electrophoresis.

Polyacrylamide Gel Electrophoresis
Polyacrylamide gel electrophoresis was carried out with a discontinuous buffer system at pH 8.3 on 10% gel with a discontinuous buffer system.The gels were stained with Coomassie Brilliant Blue R-250.

Protein Estimation
Protein concentration was determined by the method of Lowry et al. [25].Bovine serum albumin (BSA) was used as standard.

Assay of β-Galactosidase
The activity of β-galactosidase was determined by using the method of Ali et al [16].The β-galactosidase was assayed by following the release of p-nitrophenol from p-nitrophenyl β-D-galactopyranoside substrate.The reaction mixture consisted of 0.5 ml of 6 mM substrate, 1 ml of 0.1 M sodium acetate buffer (pH 5.2), 0.45 ml of 0.01 mg% BSA and 50 µl of diluted enzyme solution.The reaction was allowed to proceed for 1hour at 37˚C and was terminated by adding 1.0 ml of 1.0 M sodium carbonate.For substrate and the enzyme preparation separate blanks were used.The absorbance of liberated pnitrophenol was measured at 405 nm.
One unit of enzyme activity was defined as that amount of enzyme that catalyzes the liberation of 1 µmol of pnitrophenol/min at 37˚C.The enzyme activity was calcu-

Molecular Weight Determination
Molecular weight was determined by Sephadex gel chromatography.The Sephadex G 100 column was pre equilibrated with standard proteins.

Effect of Substrate Concentrations on Enzyme Activity
In order to determine the V max and K m of β-galactosidases for PNGP as substrate, the effect of substrate concentration on enzyme activity was studied at pH 5.2 at 37˚C.Keeping the amount of enzyme constant in assay mixture, the concentration of PNGP was increased from 0.5 mM to 9 mM.For each substrate concentration and the enzyme preparation separate blanks were used.The enzyme activity was assayed by measuring the change in absorbance at 405 nm.Reciprocal plots were used to determine V max and K m .

Effect of pH on Enzyme Activity
The pH optima of apricot β-galactosidases was determined by incubating the enzyme at 30˚C in various pH adjusted buffers e.g.0.1M glycine HCl buffer, pH 2.0 and 3.0, 0.1 M NaoAc buffer pH 4.2, 4.6, 5.0 and 5.2, 0.1 M phosphate buffer pH 6.0 and 7.0, 0.1 M Tris HCl buffer pH 8 and 9 0.1 M sodium carbonate buffer, pH 10.The enzyme assay was performed separately in each buffer system.

Effect of Temperature on Enzyme Activity
To determine the temperature optima the enzyme assay was performed by incubating reaction mixtures at various temperatures from 10˚C to 70˚C.

Isolation and Purification of β-Galactosidase
The soluble form of β-galactosidase was isolated from apricot by homogenization.After the extraction of soluble enzyme the cell wall bound β-galactosidase was extracted successively with 2 M NaCl.The isolated enzyme which was subjected to 85% ammonium sulfate saturation showed 17.2 fold increase in purity of β-galactosidase.The crude preparation showed an increase in enzyme activity and specific activity over the homogenate.When applied on CM-cellulose column in 0.1 M sodium acetate buffer, pH 5.2 the protein eluted in two major peaks, I and II (Figure 1).The fractions under both peaks showed β-galactosidase activity.The fractions were pooled and chromatographed separately onto gel filtration column.Peak I was resolved into two enzymatically active protein peaks labeled as β-gal II & β-gal III, where as peak II was eluted as a single enzymatically active protein peak (β-gal I); the chromatographic profile is shown in Figure 2. The activity of β-gal I, II and III was found to be 0.39, 0.52 and 0.30 units respectively.At each purification step the specific activity of enzymes increased over the homogenate (Table 1).

Electrophoresis and Molecular Weight Determination
Purity of β-gal I, β-gal II and β-gal III was checked by Series 1 represent absorbance at 280 nm and series 2 represent β-galactosidase activities 65 milligram of protein was applied on the column (22 × 2 cm) equilibrated with 0.1 M sodium acetate buffer pH 5.2.The protein was eluted using a linear sodium chloride gradient from 0 to 0.5 M in 5 ml fractions at a flow rate of 30 ml/hr.β-galactosidase was assayed by following the release of p-nitrophenol from p-nitrophenyl β-D-galactopyranoside substrate.One unit of enzyme activity was defined as that amount of enzyme that catalyzes the liberation of 1 µmol of p-nitrophenol/min at 37˚C.One unit of β-galactosidase was defined as the amount of enzyme that liberated one µmole of nitrophenol/min/ml at 37˚C.polyacrylamide gel electrophoresis.All the three isoenzymes gave single band indicating that the preparation was homogeneous with respect to charge (Figure 3).The molecular weight of isoforms of β-galactosidase was determined by gel filtration chromatography on Sephadex G 100 column.The column was pre equilibrated with standard proteins.The purified isoenzymes displayed a molecular weight of 44.15, 34.70 and 23.71 KDa for β-gal I, β-gal II and β-gal III respectively.

Effect of Substrate Concentrations
All the three isoforms of β-galactosidase showed the Michaelis-Menten kinetics (Figures 4, 6 and 8).The data Series 1 represent absorbance at 280 nm and series 2 represent β-galactosidase activities.Twenty milligram of protein was applied on the column (40 × 2 cm) equilibrated with 0.1 M sodium acetate buffer pH 5.2.The protein was eluted at a rate of 30 ml/hr in 3 ml fractions.The void volume of column was 39 ml.β-galactosidase was assayed by following the release of p-nitrophenol from p-nitrophenyl β-D-galactopyranoside substrate.One unit of enzyme activity was defined as that amount of enzyme that catalyzes the liberation of 1 µmol of p-nitrophenol/min at 37˚C.One unit of β-galactosidase was defined as the amount of enzyme that liberated one µmole of nitrophenol/min/ml at 37˚C.The effect of substrate concentration on enzyme activity was studied at pH 5.2 and 37˚C.Keeping the amount of enzyme constant in assay mixture, the concentration of PNPG was increased from 0.5 mM to 9 mM.For each substrate concentration and the enzyme preparation separate blanks were used.The enzyme activity was assayed by measuring the change in absorbance at 405 nm.four values obtained from reciprocal plots.The K m value for β-gal I was found to be 1.85 mM which was higher than that of β-gal II and β-gal III.The V max value for β-gal II (0.70 µmole•min -1 ) was found to be higher than that of β-gal I and β-gal III.The results are given in Table 5.

Effect of pH
The isozymes responded differently when exposed to different pH buffers.The optimum pH for the enzymes were  The effect of substrate concentration on enzyme activity was studied at pH 5.2 and 37˚C.Keeping the amount of enzyme constant in assay mixture, the concentration of PNPG was increased from 0.5 mM to 9 mM.The enzyme activity was assayed by measuring the change in absorbance at 405 nm.

Effect of Temperature
The thermal stability of β-galactosidase isozymes was determined by incubating reaction mixtures at temperatures ranging from 10˚C to 70˚C.The optimum temperature was found to be 40˚C for β-gal I, β-gal II and β-gal III (Figure 11).The enzymes showed a sharp decrease in activity afterward.At 60˚C there was complete loss of enzyme activity.

Discussion
β-galactosidase in fruits have been to exist in two forms: soluble and wall bound [2].In the present study the soluble form of the enzyme was isolated by homogenization.
After the extraction of soluble enzyme the wall bound enzyme was extracted successively with 2 M NaCl.The enzyme e separated into three peaks when subjected to ion exchange chromatography on CM-cellulose column suggesting that the β-galactosidase in apricot exists in three isoforms viz β-gal I, II and III.The results are comparable to those obtained from other fruit [17,18,20,21].
The molecular weight of β-galactosidase I, II and III as determined by Sephadex gel chromatography was found to be 44.157,34.700 and 23.713 kDa respectively.In cortical tissue of ripe apples and persimmon fruit two polypeptides of 44 kDa and 32 kDa of β-galactosidase has been found [3,14].In avocado 2 polypeptides of 21.5 kDa and 66.5 kDa were found [5].
β-galactosidase I, II and III showed different abilities to hydrolyze synthetic substrate p-nitrophenyl-β-galactoside.β-galactosidase II showed a high ability to hydrolyze the substrate PNGP than that of β-galactosidase I  that in turn showed a higher ability than that of β-galactosidase III suggesting that during apricot ripening the levels of the individual forms of β-galactosidase change markedly.The K m value for β-gal I was higher than that of β-gal II and β-gal III.However, the K m value of β-gal II was higher than reported earlier 0.17 mM for carrot [26] 1.25 mM for sweet cherry [15], 1.77 mM for tomato fruit [7] and 1.66 mM for musk melon [2] but was lower than that of other fruits such as 5.16 mM for peach βgalactosidase [17].The V max value for β-gal I, β-gal II and β-gal III was found to be 0.52, 0.70 and 0.38 µmol/min respectively.The optimum pH of different isozymes was found between 4 and 6 which were consistent with earlier reports [3,6,7,15].Moreover, β-gal I and β-gal II showed maximum activity at pH 5.2 where as β-gal III showed maximum activity at pH 4.2.These results indicate that the apricot β-galactosidases possesses a wide range of pH stability.The optimum temperature was found to be 40˚C.However the enzymes were active up to 50˚C but were completely inactivated at 60˚C.These results are in close agreement with that obtained from other fruits [16,17].
The above properties of β-galactosidase isoforms suggest that enzymes may be variably adaptable to temperature and pH fluctuations which are associated with physiological changes that are caused by the fruit softening.Earlier reports [16][17][18][21][22][23] and the results presented here indicate that the β-galactosidases may be involved in the modification of the cell wall structural polysaccharides of apricot fruits, suggesting its important role in softening.A detailed analysis of the cell wall structure of apricot will provide a better picture of the exact functions of different β-galactosidase isoforms in cell wall degradation of apricot.

Figure 1 .
Figure 1.Elution profile of β-galactosidase extracted from apricot on CM cellulose column.

Figure 2 .
Figure 2. Elution profile of peak I and II obtained from cellulose column.

Figure 6 .
Figure 6.Effect of substrate concentration on the activity of apricot β-gal II.
between 4.2 to 6 for β-gal I and 4 to 6 for β-gal II and 4 to 5.2 for β-gal III.However β-gal I and β-gal II showed maximum activity at pH 5.2 and β-gal III at pH 4.2.A sharp decrease in activity of β-gal I, β-gal II and β-gal III was observed afterwards (Figure 10).

Figure 8 .
Figure 8.Effect of substrate concentration on the activity of apricot β-gal III.

Figure 11 .
Figure 11.Temperature-activity profiles of different isoforms of β-galactosidase.The enzyme assays were performed by incubating reaction mixtures at various temperatures from 10˚C to 70˚C.