Laboratoire de Microorganismes et de Biomolécules, Centre de Biotechnologie de Sfax, Université de Sfax, Route de Sidi Mansour Km 6, BP 1177 3018 Sfax, Tunisia.
Int J Biol Macromol. 2013 Mar;54:9-15. doi: 10.1016/j.ijbiomac.2012.11.020. Epub 2012 Nov 23.
We have previously cloned and characterized the thermostable phytase (PHY US417) from Bacillus subtilis US417. It differs with PhyC from B. subtilis VTTE-68013 by the R257P substitution. PHY US417 was shown to be more thermostable than PhyC. To elucidate the mechanism of how the Pro 257 changes the thermostability of Bacillus phytases, this residue was mutated to Arg and Ala. The experimental results revealed that the thermostability of the P257A mutants and especially P257R was significantly decreased. The P257R and P257A mutants recovered, respectively, 64.4 and 81.5% of the wild-type activity after incubation at 75 °C for 30 min in the presence of 5mM CaCl(2). The P257R mutation also led to a severe reduction in the specific activity and catalytic efficiency of the enzyme. Structural investigation, by molecular modeling of PHY US417 and PhyC focused on the region of the 257 residue, revealed that this residue was present in a surface loop connecting two of the six characteristic β sheets. The P257 residue is presumed to reduce the local thermal flexibility of the loop, thus generating a higher thermostability.
我们之前已经从枯草芽孢杆菌 US417 中克隆并鉴定了耐热植酸酶（PHY US417）。它与来自枯草芽孢杆菌 VTTE-68013 的 PhyC 不同之处在于 R257P 取代。结果表明 PHY US417 比 PhyC 更耐热。为了阐明 Pro 257 如何改变芽孢杆菌植酸酶的热稳定性的机制，将该残基突变为 Arg 和 Ala。实验结果表明，P257A 突变体和特别是 P257R 的热稳定性显著降低。在 5mM CaCl2 存在下，P257R 和 P257A 突变体在 75°C 孵育 30 分钟后，分别恢复了野生型活性的 64.4%和 81.5%。P257R 突变还导致酶的比活性和催化效率严重降低。通过对 PHY US417 和 PhyC 的分子建模进行结构研究，集中在 257 残基区域，表明该残基存在于连接六个特征β片的两个表面环中。假定 P257 残基降低了环的局部热灵活性，从而产生更高的热稳定性。
