الفهرس 
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Abstract
The seeds of four legume samples, namely kidney bean variety Bronko, cowpea variety Kaha 1, Field bean variety Giza 429 and peas variety MasterB were used in the present study to investigate the levels of phytic acid and phytase in both dry and germinated seeds.
Phytic acid contents ranged from 0.9 to 1.55 % in the four legume samples used in the present work.
Only a low phytase activity was detected in the dry seeds of kidney beans and cowpeas. Neither dry seeds of field bean nor the seeds of peas revealed any phytase activity.
Effect of Germination on Phytic acid and Phytase in legume seeds.
The levels of phytate decreased slightly during the first two days of germination with rapid decline between the third and sixth day of germination. The overall reduction in phytate at the end of germination period (9 days) accounted for 87, 81.8, 77.8 and 76.3 % for the germinated seeds of kidney bean, field bean, peas and cowpea respectively.
Germination increased the phytase activities to 28-, 22-, 18-, and 17.3- fold of that corresponding dry seeds of field bean, peas, cowpea and kidney bean by the 5th, 6th, 5th, and 6th day of germination, respectively. Therefor seeds of kidney bean or peas germinated for 6 days or seeds of cowpeas and field bean germinated for 5 days were used as a source for obtaining phytate degrading enzymes.
Preparation of partially purified phytase from the germinated legume seeds.
Partially purified phytases separated from the germinated legume seeds used in the present work were prepared by extraction with 2 % CaCl2 at 40C followed by ammonium sulphate saturation.
The phytase activity of the partially purified enzyme preparations were examined after incubation in buffer with pH values ranging from 2 to 8.5., all phytases studied in this work exhibited low activities at pH 2 and tend to increase as pH raised to attend maximum level at pH 5 for kidney bean and field bean phytases and pH 5.5 for peas and cowpea.
The optimum temperatures for the activities of partially purified phytases were recorded to be 45, 50, 55, and 60 0C for peas, kidney bean, field bean and cowpea enzyme respectively. Similar to other phytate degrading enzyme, the partially purified phytases, separated from germinated legume seeds in the present work, were effective in removal of phytate from soy bean protein isolate
Purification and characterization of phytase from germinated kidney bean seeds.
Solution of 2% CaCl2 was found satisfactory in separating the phytase from phytic acid (crude phytase).
Preliminary study indicated that most of phytase activity in crude enzyme extract was separated in protein fraction obtained between 35 and 80% (NH4)2SO4 saturation.
Thus ammonium sulphate fractionation was used as a step for purification of kidney bean phytase using the protein separated between 35 and 80% saturation in next purification step (CM-Sephadex column chromatography), 1.95 fold increase in the specific activity and recovery of 97 % was achieved by this step.
CM-Sephdex peak fractions containing phytase activity were collected, desalted by passing through a Sephadex G-25 column, freeze dried and the desalted protein eluate, dissolved in 5ml of 50mM phosphate buffer, pH 7.8 and loaded on a Sephadex G-200 column.
Polyacrylamide gel electrophoresis of the phytase eluted from the Sephadex G-200 column revealed one major protein band contaminated with a minor band. Thus, an additional purification step was performed.
The phytase activity fractions eluted from the Sephdex G-200 column were pooled, freeze dried and dissolved in 3 ml of 10mM phosphate buffer, pH 6.8 and chromatographed on a hydroxyapatite column equilibrated with the same buffer. The column was eluted first with 10mM phosphate buffer and then with a linear gradient of 10-300 mM of the same buffer.
Characterization of the phytase.
The molecular weight of the native purified kidney bean phytase was determined to be 69 1 kDa on a calibrated Sephadex G-200 column with elution volume measured by determining of the enzyme activity. The molecular mass of the apparent phytase was determined to be 70 2 kDa as estimated by SDS-PAGE. Consequently, this enzyme is a monomeric protein. Measuring the activity of the purified kidney bean phytase at different pH values revealed the highest activity at pH 5 (optimum pH).
Because the pH of stomach content may fall to pH 3 or below, the stability of the enzyme was determined at pH 3 which revealed that kidney bean phytase retained most of its activity after 80 min incubation at this pH. On the other hand increases of pH to 7 resulted in inactivating the enzyme activity after one hour of treatment. More than 50 % of phytase activity remained after incubation for 80 min at pH 5.
Substrate specificity.
Substrate specificity for kidney bean phytase was examined and the results revealed that this enzyme has a broad substrate specificity and exhibited significant levels of activity with a range of phophate compounds that were not necessarily structurally similar to phytic acid(for example phenyl phosphate, p-nitrophenyl phosphate and Fructose –1,6-di phosphate).
Effect of Metal ions on kidney bean phytase activity.
Phytase activity was measured in the absence or presence of several cations at a concentration of 1mM for Mg++, Ca++ and Mn++ in the presence or absence of 1mM EDTA which revealed no significant effect. On the other hand Fe++,Fe+++ and ,Cu++ depressed cosiderably the phytase activity.It is unlikely thate the inhibitory effect of Cu++ (and to some extent the inhibitory effects of Fe++, and Fe+++ are due to direct binding of the metal ions to the phytases. Rather, metal ions were found to form poorly soluble complexes with phytic acid and may therefore decreased the active concentration of phytic acid in an activity assay.
The effect of some inhibitors on kidney bean phytase revealed that the enzymatic activity was not affected by more than 10%.
Effect of pepsin on phytase activity.
The activity of kidney bean phytase decreased with increasing pepsin concentration, and more than 50% of the starting activity remained after 60 min of incubation with 5mg pepsin.