Reddy Chinreddy Subramanyam, Achary V Mohan Murali, Manna Mrinalini, Singh Jitender, Kaul Tanushri, Reddy Malireddy K
Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110 067, India,
Appl Biochem Biotechnol. 2015 Mar;175(6):3058-67. doi: 10.1007/s12010-015-1487-4. Epub 2015 Jan 15.
The thermostable phytase gene was isolated from Bacillus subtilis ARRMK33 (BsPhyARRMK33). The gene has an ORF of 1152 bp and that encodes a protein of 383 amino acids. Sequence analysis showed high homology with Bacillus sp. phytase proteins, but no similarity was found with other phytases. SDS-PAGE analysis exhibited a predicted molecular mass of 42 kDa. Homology modeling of BsPhyARRMK33 protein based on Bacillus amyloliquefaciens crystal structure disclosed its β-propeller structure. BsPhyARRMK33 recombinant plasmid in pET-28a(+) was expressed in Rosetta gami B DE3 cells and the maximum phytase activity 15.3 U mg(-1) obtained. The enzyme exhibits high thermostability at various temperatures and broad pH ranges. The recombinant protein retained 74% of its original activity after incubation at 95 °C for 10 min. In the presence of Ca(2+), the recombinant phytase activity was maximal where as it was inhibited by EDTA. The optimal pH and temperature for the recombinant phytase activity is achieved at 7.0 and 55 °C, respectively. Thermostable nature and wide range of pH are promising features of recombinant BsPhyARRMK33 protein that may be employed as an efficient alternative to commercially known phytases and thereby alleviate environmental eutrophication.
从枯草芽孢杆菌ARRMK33（BsPhyARRMK33）中分离出热稳定植酸酶基因。该基因具有1152 bp的开放阅读框，编码一个由383个氨基酸组成的蛋白质。序列分析表明，它与芽孢杆菌属的植酸酶蛋白具有高度同源性，但与其他植酸酶没有相似性。SDS-PAGE分析显示预测分子量为42 kDa。基于解淀粉芽孢杆菌晶体结构对BsPhyARRMK33蛋白进行同源建模，揭示了其β-螺旋桨结构。pET-28a(+)中的BsPhyARRMK33重组质粒在Rosetta gami B DE3细胞中表达，获得的最大植酸酶活性为15.3 U mg(-1)。该酶在不同温度和较宽的pH范围内均表现出高热稳定性。重组蛋白在95℃孵育10分钟后仍保留其原始活性的74%。在Ca(2+)存在下，重组植酸酶活性最高，而EDTA会抑制其活性。重组植酸酶活性的最佳pH和温度分别为7.0和55℃。热稳定特性和较宽的pH范围是重组BsPhyARRMK33蛋白的有前景的特性，可作为商业已知植酸酶的有效替代品，从而减轻环境富营养化。
