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

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 (993U/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; 20min 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 (2-3h 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 3.35×10(7) variants, yielded a Ymphytase variant with 32% improved residual activity (58°C for 20min), 2°C increased apparent melting temperature (Tm), and 2-fold higher pH stability (pH 2.8; 3h incubation time) when compared to the wild-type Ymphytase. Compared to the M1 variant, the pH stability (pH 2.8; 3h 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%).