NDLI: Directed evolution of a highly active Yersinia mollaretii phytase

Content Provider	Springer Nature Link
Author	Shivange, Amol V. Serwe, Annegret Dennig, Alexander Roccata, Danilo Haefner, Stefan Schwaneberg, Ulrich
Copyright Year	2011
Abstract	Phytase improves as a feed supplement the nutritional quality of phytate-rich diets (e.g., cereal grains, legumes, and oilseeds) by hydrolyzing indigestible phytate (myo-inositol 1,2,3,4,5,6-hexakis dihydrogen phosphate) and increasing abdominal absorption of inorganic phosphates, minerals, and trace elements. Directed phytase evolution was reported for improving industrial relevant properties such as thermostability (pelleting process) or activity. In this study, we report the cloning, characterization, and directed evolution of the Yersinia mollaretii phytase (Ymphytase). Ymphytase has a tetrameric structure with positive cooperativity (Hill coefficient was 2.3) and a specific activity of 1,073 U/mg which is ∼10 times higher than widely used fungal phytases. High-throughput prescreening methods using filter papers or 384-well microtiter plates were developed. Precise subsequent screening for thermostable and active phytase variants was performed by combining absorbance and fluorescence-based detection system in 96-well microtiter plates. Directed evolution yielded after mutant library generation (SeSaM method) and two-step screening (in total ∼8,400 clones) a phytase variant with ∼20% improved thermostability (58°C for 20 min; residual activity wild type ∼34%; variant ∼53%) and increased melting temperature (1.5°C) with a slight loss of specific activity (993 U/mg).
Starting Page	405
Ending Page	418
Page Count	14
File Format	PDF
ISSN	01757598
Journal	Applied Microbiology and Biotechnology
Volume Number	95
Issue Number	2
e-ISSN	14320614
Language	English
Publisher	Springer-Verlag
Publisher Date	2011-12-11
Publisher Place	Berlin, Heidelberg
Access Restriction	Subscribed
Subject Keyword	Directed evolution High-throughput screening SeSaM Phytase Thermostability Microbiology Biotechnology Microbial Genetics and Genomics
Content Type	Text
Resource Type	Article
Subject	Medicine Applied Microbiology and Biotechnology Biotechnology

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