Identification and characterization of a mesophilic phytase highly resilient to high-temperatures from a fungus-garden associated metagenome.

Phytases are enzymes degrading phytic acid and thereby releasing inorganic phosphate. While the phytases reported to date are majorly from culturable microorganisms, the fast-growing quantity of publicly available metagenomic data generated in the last decade has enabled bioinformatic mining of phytases in numerous data mines derived from a variety of ecosystems throughout the world. In this study, we are interested in the histidine acid phosphatase (HAP) family phytases present in insect-cultivated fungus gardens. Using bioinformatic approaches, 11 putative HAP phytase genes were initially screened from 18 publicly available metagenomes of fungus gardens and were further overexpressed in Escherichia coli. One phytase from a south pine beetle fungus garden showed the highest activity and was then chosen for further study. Biochemical characterization showed that the phytase is mesophilic but possesses strong ability to withstand high temperatures. To our knowledge, it has the longest half-life time at 100 °C (27 min) and at 80 °C (2.1 h) as compared to all the thermostable phytases publicly reported to date. After 100 °C incubation for 15 min, more than 93 % of the activity was retained. The activity was 3102 μmol P/min/mg at 37 °C and 4135 μmol P/min/mg at 52.5 °C, which is higher than all the known thermostable phytases. For the high activity level demonstrated at mesophilic temperatures as well as the high resilience to high temperatures, the phytase might be promising for potential application as an additive enzyme in animal feed.