title: Multi-site saturation by OmniChange yields a pH- and thermally improved phytase.
abstract: 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%).
authors: Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany; School of Engineering and Science, Jacobs University Bremen gGmbH, Campus Ring 1, 28759 Bremen, Germany.
authors: Amol V
authors: AV
authors: Shivange
authors: Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany.
authors: Alexander
authors: A
authors: Dennig
authors: Lehrstuhl für Biotechnologie, RWTH Aachen University, Worringerweg 1, D-52074 Aachen, Germany; School of Engineering and Science, Jacobs University Bremen gGmbH, Campus Ring 1, 28759 Bremen, Germany. Electronic address: u.schwaneberg@biotec.rwth-aachen.de.
authors: Ulrich
authors: U
authors: Schwaneberg
mesh_terms: D010832:6-Phytase / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D004795:Enzyme Stability; D014644:Genetic Variation; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D013379:Substrate Specificity; D013696:Temperature; D015007:Yersinia / Q000201:enzymology*
chemical_list: D001426:Bacterial Proteins; D010832:6-Phytase
keywords: Directed evolution; Multiple site saturation; Phytase; Protein engineering; pH stability