Enhanced activity of an alkaline phytase from Bacillus subtilis 168 in acidic and neutral environments by directed evolution

• An alkaline phytase from Bacillus subtilis 168 was engineered using directed evolution.
• Catalytic activity in acidic and neutral environments was successfully improved.
• Underlying mechanism for the enhanced activity was revealed by molecular docking.

As a feed additive, the desirable features of phytase include thermostability, strict substrate specificity, and high stability in a wide pH range. Alkaline phytases from Bacillus subtilis are promising candidates because of their high inherent thermostability and substrate specificity, but the application is restricted due to their poor specific activity in acidic and neutral conditions. Directed evolution was utilized in this paper to improve the activity of phytase from B. subtilis 168 in neutral and acidic environments. After two rounds of evolution, a number of positive mutants were picked out from the mutant library. Compared with the wild type, variants D24G, S51A, D24G/K265N, D24G/K70R/K111E/N121S showed 29.7%, 13.5%, 42.7%, 42.8% improvement in specific activity under the optimal reaction conditions (pH 7.0, 60 °C) and 76.6%, 79.5%, 84.2%, 121.1% improvement at pH 4.5 and 37 °C, respectively. The catalytic efficiency (kcat/Km) of D24G, D24G/K265N, D24G/K70R/K111E/N121S at pH 7.0 was increased by 132%, 110%, 131% as compared with that of the wild type, and the improvement at pH 4.5 was 98.0%, 114.5%, 163.3%, respectively. Molecular docking results revealed that D24, S51 and K70 had a relatively large influence on the catalytic activity probably because they are adjacent to the active site cleft.