Authors: Baojin Fei Hui Xu Yu Cao Shuhan Ma Hongxiu Guo Tao Song Dairong Qiao Yi Cao
Publish Date: 2013/03/14
Volume: 40, Issue: 5, Pages: 457-464
Abstract
Despite recent advances in our understanding of the importance of protein surface properties for protein thermostabilitythere are seldom studies on multifactors rational design strategy so a more scientific simple and effective rational strategy is urgent for protein engineering Here we first attempted to use a threefactors rational design strategy combining three common structural features protein flexibility protein surface and salt bridges Escherichia coli AppA phytase was used as a model enzyme to improve its thermostability Moreover the structure and enzyme features of the thermostable mutants designed by our strategy were analyzed roundly For the single mutants two Q206E and Y311K in five exhibited thermostable property with a higher success rate of prediction 40 For the multiple mutants the themostable sites were combined with another site I427L we obtained by directed evolution Q206E/I427L Y311K/I427L and Q206E/Y311K/I427L all exhibited thermostable property The Y311K/I427L doubled thermostability 617 and was compared to 3097 after being heated at 80 °C for 10 min and catalytic efficiency 446 was compared to 237 improved more than the wildtype AppA phytase almost without hampering catalytic activity These multifactors of rational design strategy can be applied practically as a thermostabilization strategy instead of the conventional singlefactor approachThis work was supported by National Science Funds Committee 30740055 31171447 C130404 J1103518 National twelfth fiveyear science and technology support program 2011BAD14B05 2013BAD10B01 and Sichuan Science and Technology Bureau 2012GZ0008
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