A multi-factors rational design strategy for enhancing the thermostability ofEscherichia coliAppA 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.

Iterative key-residues interrogation of a phytase with thermostability increasing substitutions identified in directed evolution

“…Reports on phytase reengineering comprise improved thermostability (directed evolution and semi-rational design approaches) to match demands in the feed pelleting process (60-80°C). We recently reported two experiments improving Ymphytase thermostability (Shivange et al 2014;Shivange et al 2012), and only four other reports on bacterial phytase (α/ß-fold) improvement by directed evolution are available (Fei et al 2013;Zhao et al 2010;Zhu et al 2010). The residual activity of E. coli phytases (appA) was improved by 23.3 % (80°C for 5 min) (Zhu et al 2010), and appA2 phytase was improved by 20 % (80°C for 10 min) in directed evolution experiments using error-prone PCR for diversity generation.…”

Evolution of E. coli Phytase for Increased Thermostability Guided by Rational Parameters

“…However, to cut the labor-associated cost of the high false-positive rate of non-rational methods, a number of rational protein engineering methods were established. Rational strategies such as molecular dynamics (MD) simulation [11,19] and structural dynamics [20,21] have been applied to redesign thermophilic proteins. Based on the crystal structure and rational cascade methods, Fei et al constructed a series of engineered phytases, and the mutants showed a great increase of heat resistance compared to the wild-type [19].…”

“…Rational strategies such as molecular dynamics (MD) simulation [11,19] and structural dynamics [20,21] have been applied to redesign thermophilic proteins. Based on the crystal structure and rational cascade methods, Fei et al constructed a series of engineered phytases, and the mutants showed a great increase of heat resistance compared to the wild-type [19]. Furthermore, under the guidance of 3D structural information, analysis and calculation of some types of molecular bonds, such as hydrogen bonds [22], salt bridges [23] and disulfide bonds [24], have also been utilized to improve the heat and pH resistance of phytase.…”

Current Progresses in Phytase Research: Three‐Dimensional Structure and Protein Engineering

“…In another report, the authors identified uncharged residues in the highly flexible areas on the protein surface with molecular dynamic simulation and 3D structures. Then, these unstable residues were mutated to form salt bridges to neighboring residues [57]. The selected mutants with higher residual activity at 80°C were further mutated to carry the previous identified I405L.…”