Directed Evolution Of An Alkaline Phytase And Its Expression In Different Hosts

As a widely used feed additive, phytase can hydrolyze phytate into myo-inositol and inorganic phosphate. Based on the features of production technology and application environment, alkaline phytases from Bacillus subtilis are promising candidates because of their high inherent thermostability, substrate specificity and protease resistance. But compared with acid phytases, the application of alkaline phytases is restricted due to their poor specific activity in acidic and neutral conditions. So the experimental considerations in this paper consist of two aspects. The first one is improving the specific activity of alkaline phytases in acidic and neutral environments by directed evolution. And the other one is comparison of the enzyme properties and laws of directed evolution among phytases expressed by three different hosts (Escherichia coli BL21, Pichia pastoris GS115, B. subtilis WB800N).An alkaline phytase (phy168) was cloned from B. subtilis 168 and expressed in E. coli BL21. The expression system and induction condition used for directed evolution were determined by comparison among different expression methods, namely pET30a as the expression plasmid, BglⅡ and XhoI as the restriction enzyme cutting sites, the phytase gene without signal peptide and 25 ℃ as the inducing temperature.Through a combined strategy of directed evolution, combinational mutation and site saturation mutagenesis, mutants with improved specific activity in neutral and acidic conditions were obtained, such as D24G, D24G/K70R/K111E/N121S, D24G/K265N. Compared with the wild-type phytase, the specific activity of the above-mentioned mutants showed 40.35%,52.06% and 55.22% improvement at pH 7.0 and 37℃, and 76.61%,121.05% and 84.21% improvement at pH 4.5 and 37℃, respectively. Then the enzyme properties of the three mutants, including pH profile, temperature profile, thermostability, pH stability and kinetic parameters, were characterized. The results showed that in comparison with the wild type, the three mutants showed the same optimal reaction temperature, optimal reaction pH, pH stability but higher thermostability. The overall catalytic efficiency (kcat/Km) at pH 7.0 was 132%,131%,110% higher than that of the wild type, and 98%,163.3%,114.5% higher at pH 4.5, respectively.At last, five mutants (D24G、S51A、K265E、S51A/K265E D24G/K70R/K111E/N121S) with relatively large specific activity differences were secretorily expressed in P. pastoris GS115 and B. subtilis WB800N. We found that the law of directed evolution was generally same in the three hosts. The mutants with improved activity when expressed in E. coli BL21, also showed increased activity in P. pastoris GS115 and B. subtilis WB800N. But maybe due to glycosylation, the specific activity and the optimal reaction temperature of the wild type and mutants expressed in P. pastoris GS115 decreased compared to those expressed in E. coli BL21. For example, the optimal reaction temperature of D24G and S51A decreased from 60 ℃ to 55℃ and from 60℃ to 50℃, respectively. The alkaline phytase gene came from B. subtilis 168, so the specific activities of the wild type and mutants expressed in B. subtilis WB800N were generally higher than those expressed in E. coli. When expressed in B. subtilis WB800N, D24G showed 68.32% higher specific activity than that expressed in E. coli BL21 at pH 7.0 and 37 ℃. And the optimal reaction temperature of D24G increased from 60 ℃ to 65 ℃.In summary, alkaline phytase, as an ideal and green feed additive, is limited in industrial application due to low acticity. To solve this problem, we obtained several mutants with improved specific activity in acidic and neutral conditions by directed evolution and made a comparison among three different expression hosts. The results provided reference for further exploration of alkaline phytases.