Background and Aim: Phytases (myo - inositol hexakisphosphate phosphohydrolase) are a class of phosphatase catalyzing the stepwise hydrolysis of phytate to less phosphorylated myo - inositol derivatives and inorganic phosphate, phytate is the primary form of phosphate storage in plants. Phytases are used as an animal feed additive to improve phosphate bioavailability. Among microbial phytase, E. coli phytase has gain commercial application in broiler feed formulation due to high specific activity, acidic pH op timum, and pepsin resistance. This study focused on cloning and expression of mutant E. coli phytase using site - directed mutagenesis.
Methods: For this purpose, selection of desired mutation was first conducted through bioinformatic studies, Argenine 92 was replaced to Lysine (R92K) in AppA2 encoding sequence using overlap extension PCR (SOE - PCR). The native and mutant AppA2 genes were cloned and expressed in E. coli Bl21(DE3) host. Subsequently, both phytases were purified.
Results: Here, we mutated the N - te rminal of conserved active site sequence motif RHGXRXP in AppA2. Both Arginine 92 substitution to Lysine and the native phytase genes were cloned in pET - 26b(+) as fusion with His6 - tag and transferred to E. coli Bl21(DE3). Considerable amount of proteins we re expressed after IPTG induction and purified using affinity chromatography.
Conclusion: It is desirable to develop phytases with higher stability and specific activity to apply in animal feed industry, to decrease the use of inorganic phosphorous in animal feeds, and lower phosphorous levels in the excreted manure. Here, production of the purified recombinant phytase, enabled us to investigate the effects of point mutation on enzyme kinetics.