Institute of Biotechnology, National Taiwan University, Taipei 106, Taiwan.
Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.
J Biotechnol. 2014 Apr 10;175:1-6. doi: 10.1016/j.jbiotec.2014.01.034. Epub 2014 Feb 8.
Escherichia coli phytase (EcAppA) which hydrolyzes phytate has been widely applied in the feed industry, but the need to improve the enzyme activity and thermostability remains. Here, we conduct rational design with two strategies to enhance the EcAppA performance. First, residues near the substrate binding pocket of EcAppA were modified according to the consensus sequence of two highly active Citrobacter phytases. One out of the eleven mutants, V89T, exhibited 17.5% increase in catalytic activity, which might be a result of stabilized protein folding. Second, the EcAppA glycosylation pattern was modified in accordance with the Citrobacter phytases. An N-glycosylation motif near the substrate binding site was disrupted to remove spatial hindrance for phytate entry and product departure. The de-glycosylated mutants showed 9.6% increase in specific activity. On the other hand, the EcAppA mutants that adopt N-glycosylation motifs from CbAppA showed improved thermostability that three mutants carrying single N-glycosylation motif exhibited 5.6-9.5% residual activity after treatment at 80°C (1.8% for wild type). Furthermore, the mutant carrying all three glycosylation motifs exhibited 27% residual activity. In conclusion, a successful rational design was performed to obtain several useful EcAppA mutants with better properties for further applications.
大肠杆菌植酸酶（EcAppA）能够水解植酸盐，已被广泛应用于饲料工业，但仍需要提高其酶活性和热稳定性。在这里，我们采用两种策略进行了合理设计，以增强 EcAppA 的性能。首先，根据两种高效 Citrobacter 植酸酶的共识序列，对 EcAppA 底物结合口袋附近的残基进行了修饰。十一个突变体中有一个 V89T，其催化活性提高了 17.5%，这可能是由于蛋白质折叠更稳定。其次，根据 Citrobacter 植酸酶对 EcAppA 的糖基化模式进行了修饰。在靠近底物结合位点的位置破坏 N-糖基化模体，以消除植酸盐进入和产物离开的空间障碍。去糖基化突变体的比活性提高了 9.6%。另一方面，采用 CbAppA 的 N-糖基化模体的 EcAppA 突变体显示出了更好的热稳定性，携带单个 N-糖基化模体的三个突变体在 80°C（野生型为 1.8%）处理后仍保留了 5.6-9.5%的残余活性。此外，携带所有三个糖基化模体的突变体保留了 27%的残余活性。总之，成功地进行了合理设计，获得了一些具有更好性能的有用 EcAppA 突变体，可进一步应用。
