Multi-site saturation by OmniChange yields a pH- and thermally improved phytase

• We improved thermostability and pH resistance (pH 2.8) of Yersinia mollaretii phytase without affecting catalytic efficiency.
• Improved phytase variant was obtained by multi-site saturation mutagenesis of five sites identified in directed evolution.
• Only <0.004% of the theoretical sequence space (3.35 × 107 variants) was sampled to achieve the improvement.
• Current approach allows exploring key positions discovered in directed evolution campaign or by rational protein design.

Directed evolution of Yersinia mollaretii phytase (Ymphytase) yielded an improved variant SM2P3E4 (also named M1; D52N, T77K, K139E, G187S, V298M) in our previous study. Variant M1 retained high specific activity (993 U/mg; equivalent to 93% of wild-type activity) and improved thermal resistance (T50 improved by 1.5 °C compared to wild-type at 58 °C; 20 min incubation time), making variant M1 an attractive enzyme for industrial applications. Recently, the OmniChange method was developed for multi-site saturation mutagenesis. The five sites identified in variant M1 were subjected to OmniChange saturation in order to explore whether a variant with higher activity, higher thermal resistance, and higher resistance at low pH (2–3 h incubation was performed to mimic the gastric residence time of phytase) could be identified. Screening of a small library of 1100 clones, covering <0.004% of the theoretical sequence space of 3.35 × 107 variants, yielded a Ymphytase variant with 32% improved residual activity (58 °C for 20 min), 2 °C increased apparent melting temperature (Tm), and 2-fold higher pH stability (pH 2.8; 3 h incubation time) when compared to the wild-type Ymphytase. Compared to the M1 variant, the pH stability (pH 2.8; 3 h incubation time) was improved by 3-fold, and thermal resistance as well as activity was improved slightly (residual activity: 32% compared to 20%; apparent Tm: 2 °C compared to 1.5 °C; activity difference <4%).

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