International Journal of Agricultural Policy and Research Vol.1 (4), pp. 093-102, June 2013 Available online at http://www.journalissues.org/journals-home.php?id=1 © 2013 Journal Issues

Original Research Paper

Legume phytases: Characteristics and changes in activity during germination

Accepted 16 May,2013

A. S. *Abdel-Gawad1 B. R. Ramadan1

and R. E. A. Oraby2

1Department of Food Science and Technology Faculty of

Agriculture Assiut University. Assiut ,Egypt.

2Department of Food Science and Technology Faculty of

Agriculture Sohag University. Sohag, Egypt.

Corresponding author

E-mail: abdallasaleh25@yahoo.com Tel: +21222438266

The activity of phytases from raw, soaked, and germinated Faba bean, lentil, kidney bean and pea were assayed followed by characterization of partial purified phytases from these legumes. The optimal pH value was 5.0 for lentil and pea and 5.2 for Faba bean and kidney bean. The optimal temperature of Faba bean, lentil and kidney bean was 50°C, while of pea it was 45°C. Both optimal pH and temperature of phytases of different legume seeds did not affected by soaking or germination processes. The maximal activity of phytase was found to be at 2.0 mM sodium phytate concentration from Faba bean, lentil and pea, and at 1.5 mM from kidney bean. The calculated Michael's constant (Km) were 0.14, 0.18, 0.07 and 0.71 mM, and their corresponding Vmax values were 1.7, 1.7, 1.6 and 1.04 µM Pi of Faba bean, lentil, kidney bean and pea; respectively. The enzyme activity was decreased with extending the incubation time more than 1 hr for Faba bean, lentil and kidney bean, and more than 2 hrs for pea phytase. Calcium ions addition at concentration of 10-2 M caused increases in the phytase activity of kidney bean by 12 and pea 13%. The phytase activity of all studied legumes was diminished by addition of Fe-3, Fe-2 and Zn++ ions; however, these metal ions had variable effects on decreasing activity which was found to be depending on the added metal ion concentration. Faba bean and pea showed high increases in their phytase activity by 10.3 and 8.5- folds after 144-hrs germination, whereas lentil and kidney bean by 7.5 and 7.7-folds after 120-hrs germination, after that the enzyme activity decreased with prolonging time of germination. Key words: phytase, activity, legume, seeds, soaking, germination,characterization

INTRODUCTION Phytate-degrading enzymes (phytases) catalyse the hydrolysis of phytate (myo-inositol hexa-phosphate, IP6), the major storage form of phosphorus in the plant kingdom. Phytases belong to a special group of phosphatases, that are chemically known as myo-inositol (1,2,3,4,5,6) hexakisphosphate phosphohydrolase, and catalyze the sequential release of phosphate from phytate (Frias et al., 2003; Tran et al., 2011). Phytase sequesters orthophosphate groups from the inositol ring of phytic acid to produce free inorganic phosphorus, along with a chain of intermediate myo-inositol phosphates (inositol

pentaphosphate to inositol monophosphate) (Debnath et al., 2005; Ma et al., 2009). Phytase not only releases the phosphorus from plant-based diets, but also makes available calcium, magnesium, protein and lipid. Thus, by releasing bound phosphorus in feed ingredients of vegetable origin, phytase makes more phosphorus available for bone growth and protects the environment against phosphorus pollution (Baruah et al., 2007; da Luz et al., 3013). Phytase has been categorized on two bases, depending on the site where the hydrolysis of the phytate molecule is initiated and on the pH of activity (Selle et al.,

Int.J.Agric.Policy Res. 094 2007). These are 3-phytase (EC 3.1.3.8 or myo-inositol hexakisphosphate 3-phosphohydrolase) and 6-phytase (EC 3.1.3.26 or myo-inositol hexakisphosphate 6-phosphohydrolase). The former liberates the P moiety at position C3, whereas the latter releases it from position C6 of the myo-inositol hexaphosphate ring (Selle and Ravindran 2007, Rui-juan et al., 2012). Phytases can also be broadly categorized into two major classes based on their optimum pH: the histidine acid phosphatases and alkaline phytases. The former showed the optimum activity at pH around 5.0, whilst the latter are more pronounced at pH near to 8.0 (Baruah et al., 2007).

Phytates are widely distributed in nature in plants ( Konietzny et al., 1995) and microorganisms (Mullaney and Ullah, 2003; Osman et al., 2012 ). They have been studied intensively in the last few years because there is a great interest in using the enzymes for reducing phytate content in animal feedstuff (Zyle 1980). Phytic acid is an inhibitor of mineral absorption because the negative charges of phosphate groups form insoluble salts upon interaction with di- and tri-valent cation such as Ca, Fe, Mg and Zn. It is also the principal storage form of phosphate in plant seeds (Enujiugha, 2005; Luo and Xie 2012). According to Sandberg (2002), mineral content of legumes is generally high, but the bioavailability is poor due to the presence of phytate, which is a main inhibitor for Fe and Zn absorption. Phytase is responsible for phytate degradation thereby leading to increased bioavailability of the affected elements in food.

Sprouting is the practice of soaking and leaving seeds until they germinate and begin to sprout. This practice is reported to be associated with improvements in the nutritive value of seeds (Greiner et al. 2001; Zanabria et al. 2006; Kumar et al., 2010). At the same time, there are indications that germination is effective in reducing phytic acid (Kalpanadevi and Mohan 2013). In case of white kidney beans, Faba beans and chickpeas; sprouting improved the protein/ amino acid digestibility by decreasing anti-nutritional factors and increasing the true/ apparent protein/ amino acid digestibility (Rubio et al., 2002).

Legumes provide a large amount of protein, carbohydrates, dietary fiber, minerals and water-soluble vitamins in human diets. In some areas of the world, where the predominant diet pattern is vegetarian or animal meat is available in only small amounts, legumes provide the major source of proteins. Therefore, legumes can be considered as foods with health benefits, but their phytate contents can limit the availability of minerals.

Although some studies have reported on the phytase, a specific acid phosphatases for phytic acid in germinated Faba bean (Eskin and Weibe 1983; Rizk 1991; Greiner et al., 2001) and pea seeds (Beal and Mehta 1985), but there are a

lack or no information about phytases of some legume seeds such as lentil and kidney bean in literatures. The present research work was conducted, therefore, to investigate the changes of phytase activities during germination of Faba bean, lentil, kidney bean and pea that are cultivated and commonly consumed in Egypt, in addition to study the characteristics of phytases extracted from these sprouted legumes. MATERIALS AND METHODS Materials Faba bean seeds (Vicia faba cv. Giza 3) were obtained from Agronomy Research Institute (Shandaweel Agricultural Research Center, Sohag – Egypt). Seeds of lentil (Lens eulimaris cv. Giza 9), kidney bean (Phaseolus vulgaris var. Swiss Blanc), and peas (Pisum sativum var. Alaska ) were obtained from Vegetable Research Institute (Agricultural Research Center, Giza – Egypt). Chemicals Dodecasodium phytate, acetic acid, sodium acetate, calcium chloride, Ferric chloride, Ferrous sulfate and zinc sulfate were obtained from Sigma (Germany). Imidazol (C3H4N2) was purchased from Alderich (Germany). Methods Soaking and germination Legume seeds were cleaned, washed and soaked for 12 hrs in tap water at room temperature (25±2°C) as reported by Abdel-Gawad (1993). After soaking, a part of soaked samples was used for phytase activity assay. The another part was used for germination in the dark till 168 hrs (for lentil and kidney bean, and 192 hrs for Faba bean and pea). Seeds were sprayed every 12 hrs by sterilized water as needed. Produced seedlings were dried at 60°C for 48 hrs and ground in an electric grinder to pass through a 100-mesh (0.15 mm) sieve and stored in closed bottles in a refrigerator at 5°C until analysis. Extraction of phytase The crude enzyme was extracted as described by Abdel-Gawad and Hamada (2002) by stirring the fine sample in 0.1 M acetate buffer, pH 5.2, (using 1 flour : 10 buffer, W/V) at 5-10°C for 30 min, then centrifuged for 20 min at 4200 xg and finally filtering the supernatant through four layers of filter cloth. The obtained filtrate was mixed with cold

acetone to precipitate the enzyme. The produced precipitate was re-dissolved in acetate buffer (pH 5.2), dialyzed over night against the same buffer and centrifuged as mentioned above. The obtained supernatant was the partial purified phytase. Moisture content Moisture content of soaked and germinated legume seed samples that were previously dried (at 60°C, for 48 hrs) were finally performed at 105°C for 3 hrs according to AOAC (1990) methods. Phytase activity The activity of partial purified phytase was measured as described by Lolas and Markakis (1977). The reaction mixture contained 4.0 ml 0.1M acetate and 0.1M imidazol-HCl buffers (pH from 4.0 to 6.5), 0.2 ml 2 mM dodecasodium phytate and 0.5 ml enzyme extract. After incubation at temperatures (35 to 65°C) for 60 min, the reaction was stopped by addition of 0.5 ml 10% TCA. Inorganic phosphate liberated by phytase was determined by measuring the absorbance at 680 nm after 10 min as stated by Chen et al., (1956). Potassium dihydrogen phosphate was used as a standard. The activity of phytase is expressed as micromole (µM) inorganic phosphorus (Pi) liberated in one minute per 1g sample. Characteristics of phytase Phytase properties were carried out using dodecasodium phytate as a substrate as described by Abdel-Gawad and Hamada(2002). The effect of pH on enzyme activity was determined at pH from 4.0 to 6.5 in 0.1 M acetate buffer (pH 4.0 to 5.5) and 0.1 M imidazol-HCl buffer (pH 6.0 to 6.5) using 2 mM dodecasodium phytate. Incubation was carried out at 40°C for 60 min. To study the effect of temperature, enzyme assay mixtures were incubated at its optimal pH with the substrate at temperature ranged from 35-65°C for 60 min. The effect of substrate concentration on phytase activity, the reaction mixtures were incubated at its optimal temperature and pH for 60 min using different sodium phytate levels (0 to 4 mM). Km and Vmax were calculated from the relation between 1/ substrate concentrations and 1/ velocity of reaction using Lineweaver-Burck Plot method (Lineweaver and Burck, 1934). The enzyme assay was performed at optimal pH, temperature and substrate concentration for various times (1 to 8 hrs) to identify an optimal incubation time.

The thermal inactivation of phytase in 0.1 M acetate buffer (at optimal pH) was estimated after heating of enzyme extract at a temperature from 30 to 75°C for 10

Abdel-Gawad et al 095 min in a water bath, cooling, adding substrate at an optimal concentration and the activity was assayed after incubation for 60 min at optimal temperature. The influence of the following salts: CaCl2, FeCl3, FeSO4 and ZnSO4 at concentrations of 10-5 and 10-2 M on enzyme activity were carried out at optimal pH, temperature and substrate concentration, and at 60 min incubation. The relative activity of the enzyme was calculated from the following equation: Enzyme activity in presence of salt X 100 Relative activity = ـــــــــــــــــــــــــــــــــــــــــــــــــــــــــــــ ـ Enzyme activity in absence of salt Effect of germination period on phytase activity Phytase was extracted from legume seeds after 24, 48, 72, 96, 120, 144, 168 and 192-hrs germination and its activity was determined at the optimal pH, temperature, and substrate concentration after 60 min incubation. RESULTS AND DISCUSSION Characteristics of legume phytases Effect of pH The effect of pH on phytase activity of Faba bean, lentil, kidney bean and pea seeds (Table 1) showed an increase in its activity with increasing the pH up to the optimal values and then decreased. The optimal pH value was 5.0 for lentil and pea, and 5.2 for Faba bean and kidney bean. Soaking and germination processes have no effect on optimal pH of enzyme activity. The highest activity of phytase was noticed after 120-hrs germination of lentil and kidney bean, whereas was after 144-hrs germination of Faba bean and pea seeds. Similar results for optimal pH values were reported for Faba bean (pH 5.3) by Eskin and Wiebe (1983) and (pH 5.2) by Greiner et al., (2001), and pea (pH 5.2) by Beal and Mehta (1985). The optimal pH of phytases from other legumes such navy beans (Lolas and Markakis 1977) and germinated soybean (Gibson and Ullah 1988) ranged from 4.8 to 5.3. Effect of temperature Phytase activity of raw, soaked and germinated Faba bean, lentil, kidney bean and pea at different temperatures (35 - 65°C) were presented in Table 2. An optimal temperatures of phytase activity was 50°C for Faba bean, lentil and kidney bean, and 45°C for pea. Such temperatures of phytase did not altered by soaking or germination treatments and confirm the fact that the character of enzyme is stable and can not changed with any treatments.

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Table1: Phytase activity of raw, soaking and germinating legume seeds at different pH values

Legume samples pH value

4.0 4.5 5.0 5.2 5.5 6.0 6.5 Phytase activity (U/g sample)

Faba bean: Raw

Soaked for 12 hrs Germinated for (h):

24 48 72 96

120 144 168 192

0.72 1.35

2.20 2.70 3.28 4.20 4.78 5.29 4.15 2.80

0.90 1.67

3.70 5.00 6.55 8.80

10.45 11.92 10.60 9.35

1.08 1.95

4.35 5.73 8.00

10.50 11.75 13.68 12.44 11.20

1.27 2.20

4.65 6.00 8.38

11.34 13.50 15.00 13.37 11.69

1.00 1.95

3.40 5.39 7.30 9.66

12.20 13.25 11.50 10.29

0.70 1.35

1.85 2.35 2.90 3.78 4.35 5.29 4.15 2.80

0.55 1.10

1.55 2.00 2.50 3.35 3.90 4.40 3.20 2.33

Lentil: Raw

Soaked for 12 hrs Germinated for (h):

24 48 72 96

120 144 168

0.53 1.25

3.56 4.00 5.22 6.10 7.39 5.92 4.95

0.72 1.58

4.27 4.74 6.72 8.77 9.72 8.68 7.42

1.10 2.22

4.63 5.46 7.10 9.53

10.90 9.10 7.84

0.89 1.90

4.27 4.74 6.72 8.77 9.72 8.68 7.42

0.72 1.58

3.56 4.37 5.97 7.63 8.94 7.50 6.19

0.53 1.25

1.78 2.18 2.60 3.05 3.50 2.80 2.10

0.00 0.95

1.42 1.82 2.23 2.66 3.11 2.40 1.65

Kidney bean: Raw

Soaked for 12 hrs Germinated for (h):

24 48 72 96

120 144 168

0.70 1.59

2.22 2.64 3.08 3.94 4.47 3.71 2.48

1.00 2.23

2.96 3.77 5.00 6.30 7.73 6.18 4.56

1.26 2.55

3.33 4.91 6.56 7.87 9.36 7.83 5.39

1.45 2.88

4.07 5.28 6.95 8.27

10.58 8.24 6.63

1.08 2.23

3.33 4.53 5.79 7.08 9.36 7.00 5.39

0.70 1.59

2.22 2.64 3.00 3.54 4.00 3.30 2.48

0.55 1.87

1.85 2.26 2.70 3.15 3.66 2.47 1.66

Pea: Raw

Soaked for 12 hrs Germinated for (h):

24 48 72 96

120 144 168 192

0.70 1.66

5.20 5.73 6.54 7.97 8.89 9.44 7.48 6.17

1.00 2.33

6.24 7.17 8.47

10.76 11.86 12.45 9.83 7.12

1.44 3.00

6.93 8.24

10.00 11.17 12.28 13.31 11.69 9.00

1.26 2.66

6.58 7.89 9.63

10.76 11.86 12.88 10.76 7.59

1.00 2.33

5.89 6.81 8.00 9.57

11.00 12.00 9.83 6.65

0.90 2.00

2.43 2.86 3.46 3.98 4.65 5.15 4.21 2.85

0.54 1.33

1.73 2.15 2.69 3.19 3.81 4.29 3.27 2.37

The reported optimal temperatures 50°C for Faba bean by Eskin and Wiebe (1983) and 45°C for pea by Beal and Mehta (1985) is similar to that found for Faba bean and pea in this study.

Effect of Substrate concentration Phytase activity was increased with increasing substrate concentration and the maximal activity was reached at 2

Abdel-Gawad et al 097

Table 2: Phytase activity of raw, soaking and germinating legume seeds at different temperature and optimal pH value of each.

Legume samples Temperature (°C )

35 40 45 50 55 60 65 Phytase activity (U/g sample)

Faba bean: Raw

Soaked for 12 hrs Germinated for (h):

24 48 72 96

120 144 168 192

0.73 1.67

2.20 2.69 4.37 6.30 7.84 9.27 6.92 5.61

1.27 2.20

4.65 6.00 8.38

11.34 13.50 15.00 13.37 11.69

1.45 2.50

4.97 6.74 9.11

12.60 14.37 15.89 13.83 12.16

1.63 2.78

5.28 7.42

10.20 13.44 15.67 16.78 14.29 12.63

1.27 2.23

4.00 4.72 5.83 7.98

10.00 11.00 9.68 7.95

1.00 1.95

2.48 3.00 3.65 4.62 5.66 6.20 5.00 4.20

0.55 1.11

1.55 2.00 2.55 3.36 3.90 4.40 3.68 2.33

Lentil: Raw

Soaked for 12 hrs Germinated for (h):

24 48 72 96

120 144 168

0.70 1.58

2.85 4.00 4.85 5.72 6.61 5.13 4.54

1.10 2.22

4.63 5.46 7.10 9.53

10.90 9.10 7.84

1.43 2.86

4.98 5.83 7.83 9.53

11.28 9.87 8.24

1.61 3.17

5.34 6.56 8.58

10.29 12.05 10.66 8.66

1.43 2.54

4.63 5.46 6.34 8.00 9.33 7.89 6.59

0.90 1.90

2.49 2.91 3.35 3.81 4.28 3.55 2.06

0.54 1.27

1.78 2.18 2.61 3.00 3.53 2.76 1.65

Kidney bean: Raw

Soaked for 12 hrs Germinated for (h):

24 48 72 96

120 144 168

1.08 2.23

2.96 4.15 5.40 6.70 7.32 5.77 4.97

1.45 2.88

4.07 5.28 6.95 8.27

10.58 8.24 6.63

1.63 3.19

4.45 6.42 7.72 9.00

12.21 9.90 7.88

1.80 3.52

5.18 7.55 9.25

11.00 13.43 11.14 8.29

1.26 2.55

4.45 6.42 8.10 8.66

11.80 9.50 6.63

1.00 2.23

3.70 4.53 5.79 7.00 8.54 6.18 4.96

0.70 1.60

2.22 2.64 3.00 3.54 4.00 3.30 2.50

Pea: Raw

Soaked for 12 hrs Germinated for (h):

24 48 72 96

120 144 168 192

0.90 2.00

2.77 3.94 6.16 7.79 8.47 9.87 7.95 6.17

1.44 3.00

6.93 8.24

10.00 11.17 12.28 13.31 11.69 9.00

1.80 3.66

8.32 9.32

11.17 12.36 13.97 15.45 13.56 11.87

1.60 3.33

7.28 8.60

10.39 11.56 13.13 14.17 12.63 10.44

1.44 3.00

6.24 7.89 9.63

10.37 12.28 13.31 12.16 10.44

1.00 2.33

3.12 3.58 4.62 6.38 8.00 9.87 8.88 7.12

0.54 1.33

1.73 2.15 2.69 3.19 3.81 4.30 3.74 2.85

mM sodium phytate for Faba bean, lentil and pea, and at 1.5 mM for kidney bean (Figure 1). The activity was then

gradually decreased with further increasing of substrate concentration. According to Gibbins and Norris (1963) and

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Figure 1: Effect of substrate concentration on phytase activity of germinating legume seeds

Sayed (1992), when the substrate concentration reached an optimal level, all the active sites of enzyme molecule became saturated and the activity arrives to the maximal level. Dixon et al. (1979) reported that rising substrate concentration above the optimal value reduce from the enzyme activity.

From a plot of 1/[V] vs. 1/[S] , the Michael`s constant (Km) values were calculated (Lineweaver and Burk, 1934). They were 0.14 and 0.071 mM for phytase extracted from 144-hrs germinated Faba bean and pea; 0.18 and 0.07 mM for 120-hrs germinated lentil and kidney bean; respectively. Meanwhile, the corresponding Vmax values were 1.7, 1.7, 1.6 and 1.04 mM Pi liberated/ min/ ml enzyme for Faba bean, lentil, kidney bean and pea; respectively as shown in Figure 2.

The Km values of studied legumes were ranged from 0.07 to 0.18 mM and are higher than reported for navy bean 0.018 mM (Lolas and Markakis, 1977); broad bean 1.11x 10-3 (Rizk 1991) and lower than 0.65 mM for germinating mung bean (Mandal et al., 1972).

The Michaelis constant for phytases reported in the present study was rather low; it is assumed that the investigated enzymes, in this study possesses a relatively higher affinity to the used substrate. Effect of optimal incubation time The effects of incubation time on phytase activity of germinating legumes at optimal conditions of each are shown in Figure 3. The results indicated that maximum activity of Faba bean, lentil and kidney bean was reached after one hour of incubation, and after two hours for pea phytase. The enzyme activity decreased with the extending incubation time of germinating legume phytases. The same

observation was stated by Sutardi and Buckle (1986) for soybean phytase; Rizk (1991) for broad bean and sunflower phytase and Abdel-Gawad and Hamada (2002) for rice bran phytase. Thermal inactivation Preheating of enzyme extracts of pea to 45°C and Faba bean, lentil and kidney bean to 50°C for 10 min did not depress phytase activity as shown in Figure 4. Over these temperatures, the activity of enzyme was gradually decreased. The enzyme activity of phytase after preheating of lentil extract at 65°C and of Faba bean, kidney bean and pea extracts at 70°C was practically inhibited. According to Lolas and Markakis (1977) at 65°C the activity of navy beans decreased to about 45% of the control value and at 80°C the enzyme was almost completely inactivated. Sutardi and Buckle (1986) found that the preheating of soybean phytase at 60°C decreased 95% of its activity and at 90°C the enzyme was completely inhibited. The phytase activity of broad bean was stable after preheating at 50°C and totally inhibited at 80°C (Rizk 1991). The resistance of phytase to heat treatments obviously varies depending on the source of enzyme. Effect of metal salts Most of salts tested (Table 3) showed an effect upon phytase activity. Calcium ions at concentration of 10-2 M increased the phytase activity of kidney bean and pea by 12 and 13% of the control value, without metal salts addition, respectively. Increases in navy bean phytase activity (Lolas and Markakis 1977) and in that of pollen of Typha latifolia L. (Hara et al. 1985) were reported by adding Ca2+ and Mg2+

Abdel-Gawad et al 099

Figure 2: Lineweaver-Burk plot of phytases from germinating legumes seeds

Figure 3:Optimal incubation time of phytase activity of germinating legumes seeds ions. Whereas, the Fe3+ ions concentration of 10-5 M reduced the phytase activity of Faba bean, lentil and peas by 23%, 7% and 43%; while the reduction enhanced at 10-2 M to 69%, 48%, 25% and 59% for Faba bean, lentil, kidney bean and peas, respectively. Fe2+ ions reduced the enzyme

activity by 8% for kidney bean to 19% for lentil and pea at concentration of 10-5M and by 44%, 46%, 17% and 46% at concentration of 10-2 M for Faba bean, lentil, kidney bean and peas; respectively. The values of reduction of enzyme activity by Zn2+ ions at concentrations of 10-5 M and 10-2 M

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Figure 4: Thermal inactivation of phytase activity of germinating legumes seeds

Table 3: Effect of some metal salts on the relative activity of phytase extracted from germinating legume seeds.

Relative activity*

10 -5 M 10 -2 M Metal

salts Faba bean

Lentil kidney

bean Pea Faba bean Lentil

kidney

bean Pea

Ca Cl2

Fe Cl3

Fe SO4

Zn SO4

98

77

108

85

90

93

111

86

106

103

92

72

102

57

81

43

92

31

56

62

82

52

133

57

112

75

83

52

113

41

54

33

*The relative activity without added metal salts was taken as 100%.

were 15-38, 14-43, 28-48, and 57-67% for Faba bean, lentil, kidney bean and pea, respectively. Sutardi and Buckle (1986) reported that soybean phytase decreased activity by about 25, 23 and 22% in the presence of 10-3 M zinc, cupric and mercuric, respectively. Rizk (1991) found that the percentage of broad bean phytase inhibition was about 25, 20, 20 and 28% in the presence of 10-3 M zinc, cupric, ferrous and mercuric, respectively. Effect of germination period on phytase activity Figure 5 illustrated the germination period effect of Faba bean, lentil, kidney bean and pea on their phytase activities. Faba bean and pea showed the highest increase in phytase activity up to 144-hrs germination (reached to 16.78 and 15.45 U/g sample), then decreased to 12.63 and 11.87 U/g sample; respectively after 192-hrs germination. Lentil and kidney bean showed maximal phytase activity at 120-hrs germination and thereafter their activities were lowered. Phytase activity of Faba bean, lentil, kidney bean and pea increased by 10.3, 7.5, 7.7 and 8.5-folds after 144, 120, 120,

and 144-hrs germination; respectively. In other studies on phytase legumes, Lolas and Markakis (1977) reported an increase of phytase activity of navy beans by 7.0-folds after 6-days of germination, and then it started to drop slowly. The increases in phytase activity in the range of 800-3000% have been reported for several varieties of pea during the first 5 days of germination (Chen and Pan 1977). Chang (1967) found a 40-folds increase in phytase activity of corn on the 4-days of germination. Conclusion The phytase activity of raw, soaking, and germinating Faba bean seeds, lentil, kidney bean and pea were determined followed by characterization of partial purified phytase from these legumes. The optimal pH, temperature, substrate concentration, incubation time, thermal inactivation, Km and Vmax were determined. In addition, the effect of some metal salts and germination time on phytase activity was studied.

Figure 5:Effect of germination period on phytase activity of some legumes

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