title: A multi-factors rational design strategy for enhancing the thermostability of Escherichia coli AppA phytase.
abstract: Despite recent advances in our understanding of the importance of protein surface properties for protein thermostability,there are seldom studies on multi-factors rational design strategy, so a more scientific, simple and effective rational strategy is urgent for protein engineering. Here, we first attempted to use a three-factors 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 (61.7 %, and was compared to 30.97 % after being heated at 80 °C for 10 min) and catalytic efficiency (4.46 was compared to 2.37) improved more than the wild-type AppA phytase almost without hampering catalytic activity. These multi-factors of rational design strategy can be applied practically as a thermostabilization strategy instead of the conventional single-factor approach.
authors.affiliation: Microbiology and Metabolic Engineering Key Laboratory of Sichuan Province, College of Life Science, Sichuan University, Chengdu 610064, Sichuan, People's Republic of China.
authors.forename: Baojin
authors.initials: B
authors.lastname: Fei
authors.forename: Hui
authors.initials: H
authors.lastname: Xu
authors.forename: Yu
authors.initials: Y
authors.lastname: Cao
authors.forename: Shuhan
authors.initials: S
authors.lastname: Ma
authors.forename: Hongxiu
authors.initials: H
authors.lastname: Guo
authors.forename: Tao
authors.initials: T
authors.lastname: Song
authors.forename: Dairong
authors.initials: D
authors.lastname: Qiao
authors.forename: Yi
authors.initials: Y
authors.lastname: Cao
mesh_terms: D010832:6-Phytase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D055162:Biocatalysis; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006358:Hot Temperature; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D011487:Protein Conformation; D015202:Protein Engineering
chemical_list: D029968:Escherichia coli Proteins; D010832:6-Phytase