Department of Biotechnology, Lund University, Lund, Sweden.
J Inorg Biochem. 2011 Jul;105(7):1000-7. doi: 10.1016/j.jinorgbio.2011.04.005. Epub 2011 Apr 21.
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 Ca(2+) on both the enzyme and the substrate is required for optimal activity and stability. Replacing Ca(2+) with Ba(2+), Mn(2+), Mg(2+) and Sr(2+) in the phytase resulted in the expression of >90% of the maximal activity with calcium-phytate as the substrate, while Fe(2+) and Zn(2+) 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 Mg(2+), Sn(2+) and Sr(2+), respectively. On replacing Ca(2+) on both the enzyme and the substrate with other metal ions, about 20% of the maximal phytase activity was obtained only with Mg(2+) and Sr(2+), respectively. Only Ca(2+) resulted in a marked increase in the melting temperature (T(m)) of the enzyme by 12-21°C, while Ba(2+), Mn(2+), Sr(2+) or Cu(2+) resulted in a modest (2-3.5°C) increase in T(m). In the presence of 1-5mM Ca(2+), 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.
植酸是种子中磷的主要来源，它以与不同金属离子形成的复合物形式存在。已知碱性植酸酶可以使与钙离子结合的植酸复合物去磷酸化，而酸性植酸酶则只能作用于植酸。从芽孢杆菌 MD2 中纯化并表征了一种重组碱性植酸酶，研究了二价金属离子对酶活性和稳定性的影响。酶和底物上都需要存在 Ca(2+)才能达到最佳活性和稳定性。用 Ba(2+)、Mn(2+)、Mg(2+)和 Sr(2+)取代植酸酶中的 Ca(2+)，会导致以钙植酸盐为底物时，酶活性表达超过 90%，而 Fe(2+)和 Zn(2+)则使酶失活。另一方面，负载钙的植酸酶对植酸钠显示出显著的活性（60%），而对仅与 Mg(2+)、Sn(2+)和 Sr(2+)结合的植酸盐的活性较低（分别为 17-20%）。在分别用其他金属离子取代酶和底物上的 Ca(2+)时，仅用 Mg(2+)和 Sr(2+)可获得约 20%的最大植酸酶活性。只有 Ca(2+)会使酶的熔点（Tm）显著升高（12-21°C），而 Ba(2+)、Mn(2+)、Sr(2+)或 Cu(2+)则会使 Tm 适度升高（2-3.5°C）。在 1-5mM Ca(2+)存在下，植酸酶活性的最适温度从 40°C升高到 70°C，而酶的最适 pH 值向酸性区域移动了 0.4-1 pH 单位。
