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Purification and Kinetic Properties of Purified Peroxidase from Proso Millet Seeds
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| Author:
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ABDULHAKEEM HUSSIEN, KHALID F. AL-RAWI, GAZI M. AZIZ, ZAZALI BIN ALIAS
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| Abstract:
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This study was carried out to purify and characterize peroxidase from proso millet seeds. Peroxidase was
purified by some chromatographic methods with precipitation: saturation by ammonium sulphate 80%, then
using the DEAE-Cellulose ion exchanger by fast protein liquid chromatography (FBLC). The specific activity
was 39.49 U/mg Protein, with purification times 5.94 and yield 43.38%. This was followed by the use of gel
filtration by SephadexG100 column in the same FBLC technique. The specific activity of the enzymatic extract
increased to 61.22 U/mg protein, and the purification times were 9.2 with an enzyme yield of 25.75%. The
molecular weight of the enzyme was estimated by electrophoreses with SDS and it was 34 kD. The pH
optimum of enzyme was 5 and for stability was 7. The optimum temperature was 40 ° C and for stability was
30-45 ° C. Michaelis constant (Km) and Vmax value were estimated using five different calculation methods
(Michaelis-Menten, Linweaver-Burk, Hanes-Woolf, Woolf - Augustinson - Hofstee and Eadie - Schatchard
equations). When fixing the concentration of hydrogen peroxide and changing the concentration of guaiacol,
Km and Vmax of the enzymatic reaction were 18.8 mM and 16.4 mM/min, respectively. In the case of fixing the
concentration of guaiacol at different concentrations of hydrogen peroxide, the arithmetic mean of Km and
Vmax values were 7.07 mM and 16.46 mM/min, respectively. The effect of the metal ions on the activity of
enzyme was studied at 10 and 50 mM. The enzymatic activity of the peroxidase slightly decreased with sodium,
potassium, zinc, manganese and magnesium ions. The effect of inhibition was moderate in the presence of
copper and iron ions, while the ferric, mercury and lead ions were more inhibitive. Calcium has already shown
an activation of enzymatic activity in both concentrations. 2-Mercaptaethanol (2ME), ethylene diamine tetra
acetate (EDTA), urea and ascorbic acid significantly inhibited enzyme activity. The degree of inhibition at a
concentration of 50 mM was higher than that of the 10 mM concentration in all cases.
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| Keyword:
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Peroxidase, Purification, Characterization, Proso Millet.
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| EOI:
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| DOI:
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https://doi.org/10.31838/ijpr/2020.12.02.0143
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| Download:
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