Thermostable alkaline phytase from Bacillus sp. MD2: effect of divalent metals on activity and stability |
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Authors: | Tran Thuy Thi Hashim Suhaila Omar Gaber Yasser Mamo Gashaw Mattiasson Bo Hatti-Kaul Rajni |
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Institution: | a Department of Biotechnology, Lund University, P. O. Box 124, SE-221 00 Lund, Swedenb Biotechnology and Microbiology Department, Hanoi National University of Education, 136 Xuan Thuy, Hanoi, Viet Namc Department of Microbiology, Faculty of Pharmacy, Beni-Suef University, Beni-Suef, 62111, Egypt |
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Abstract: | Phytate, the major source of phosphorus in seeds, exists as a complex with different metal ions. Alkaline phytases are known to dephosphorylate phytate complexed with calcium ions in contrast to acid phytases that act only on phytic acid. A recombinant alkaline phytase from Bacillus sp. MD2 has been purified and characterized with respect to the effect of divalent metal ions on the enzyme activity and stability. The presence of Ca2+ on both the enzyme and the substrate is required for optimal activity and stability. Replacing Ca2+ with Ba2+, Mn2+, Mg2+ and Sr2+ in the phytase resulted in the expression of > 90% of the maximal activity with calcium-phytate as the substrate, while Fe2+ and Zn2+ rendered the enzyme inactive. On the other hand, the calcium loaded phytase showed significant activity (60%) with sodium phytate and lower activity (17-20%) with phytate complexed with only Mg2+, Sn2+ and Sr2+, respectively. On replacing Ca2+ on both the enzyme and the substrate with other metal ions, about 20% of the maximal phytase activity was obtained only with Mg2+ and Sr2+, respectively. Only Ca2+ resulted in a marked increase in the melting temperature (Tm) of the enzyme by 12-21 °C, while Ba2+, Mn2+, Sr2+ or Cu2+ resulted in a modest (2-3.5 °C) increase in Tm. In the presence of 1-5 mM Ca2+, the optimum temperature of the phytase activity was increased from 40 °C to 70 °C, while optimum pH of the enzyme shifted by 0.4-1 pH unit towards the acidic region. |
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Keywords: | Alkaline phytase Phytate Divalent metal ions Differential scanning calorimetry |
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