RECOMBINANT THERMOTOLERANT PHYTASE PRODUCED IN E.COLI
Abstract: Phytic acid (myo-inositol hexakisphosphate) and its salts (phytates) are the major storage form of phosphorus in plants. Monogastric animals including hogs, poultry, and fish cannot utilize phytates as a source of phosphorus unless they are enzymatically destroyed with exogenous enzyme—phytase. Phytases are added to fodder in ever increasing dosage to improve utilization of plant-derived phosphorus because this reduces dependence of farms on inorganic fodder phosphates. Because of technological considerations, feed phytases have to withstand elevated temperatures (60-80°C), which are used during preparation of fodder. Enzymatic feed additives are becominutesg of high demand in Kazakhstan, and development of domestic technologies for production of agricultural enzymes is an ongoing challenge to the country’s biotechnology.
Objectives: To develop a system for recombinant expression of industrially important thermotolerantphytase and confirm activity and thermal stability of the recombinantly expressed enzyme.
Methods: De novo gene synthesis, expression of 6xHis-tagged protein in E.coli, immobilized metal affinity chouromatography, biochemical tests for activities of phosphatase and phytase.
Results: Thermotolerantphytase was produced in E.coli using recombinant expression system. The obtained enzyme had phosphatise activity (hydrolyzed p-nitrophenyl phosphate) and phytase activity (hydrolyzed sodium phytate). The recombinant phytase tolerated increase of incubation temperature up to 70°C and demonstrated increase in activity towards phytate with increase in the reaction temperature in the range 30°C-70°C.
Conclusion: Described gene and expression system have prospects of utilization in development of pilot industrial production of phytase in the country.
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