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A novel phytase of Acidobacteria was identified from a soil metagenome, cloned, overexpressed, and purified. It has low sequence similarity (<44%) to all the known phytases. At the optimum pH (2.5), the phytase shows an activity level of 1,792 μmol/min/mg at physiological temperature (37°C) and could retain 92% residual activity after 30 min, indicating the phytase is acidophilic and acidostable. However the phytase shows poor stability at high temperatures. To improve its thermal resistance, the enzyme was redesigned using Disulfide by Design 2.0, introducing four additional disulfide bridges. The half-life time of the engineered phytase at 60°C and 80°C, respectively, is 3.0× and 2.8× longer than the wild-type, and its activity and acidostability are not significantly affected.

Four novel phytases of the histidine acid phosphatase family were identified in two publicly available metagenomic datasets of an acidic peat-soil microbiome in northeastern Bavaria, Germany. These enzymes have low similarity to all the reported phytases. They were overexpressed in Escherichia coli and purified. Catalytic efficacy in simulated gastric fluid was measured and compared among the four candidates. The phytase named rPhyPt4 was selected for its high activity. It is the first phytase identified from unculturable Acidobacteria. The phytase showed a longer half-life than all the gastric-stable phytases that have been reported to date, suggesting a strong resistance to low pH and pepsin. A wide pH profile was observed between pH 1.5 and 5.0. At the optimum pH (2.5) the activity was 2,790 μmol/min/mg at the physiological temperature of 37°C and 3,989 μmol/min/mg at the optimum temperature of 60°C. Due to the competent activity level as well as the high gastric stability, the phytase could be a potential candidate for practical use in livestock and poultry feeding., (© 2016 S. Karger AG, Basel.)