Directed evolution of an acid Yersinia mollaretii phytase for broadened activity at neutral pH

Phytases are phosphohydrolases that initiate the sequential hydrolysis of phosphate from phytate, which is the main storage form of phosphorous in numerous plant seeds, especially in cereals and grains. Phytate is indigestible for most monogastric animals, such as poultry, swine, fish, and humans; therefore, microbial phytases have been widely used in plant (specially soy)-based animal feeding to improve nutrition by enhanced phosphorus, mineral, and trace element absorption, and reducing phosphorus pollution by animal waste. Most phytases used as animal feed additives have an acid pH optimum (pH 2.5 and 5.5 for Aspergillus and pH 4.5 for E. coli phytases) and show a sharp decrease in performance at neutral pH, correlating with intestinal digestion. Directed evolution of phytases has been previously reported to improve enzyme thermostability, pH, or specific activity. In this manuscript, we report a directed evolution campaign of the highly active bacterial phytase from Yersinia mollaretii (YmPh) towards a broadened pH activity spectrum. Directed evolution identified the key positions T44 and K45 for increased YmPh activity at neutral pH. Both positions are located in the active site loop of the phytase and have a synergistic effect on activity with a broadened pH spectrum. Kinetic characterization of the improved variants, YmPh-M10 and -M16, showed up to sevenfold increased specific activity and up to 2.2-fold reduced K_half at pH 6.6 under screening conditions compared to Yersinia mollaretii phytase wild type (YmPhWT).