Phytase from Citrobacter koseri PM-7: Cost-Effective Production Using Agro-Industrial Residues, Biochemical Characterization and Application in de-Phytinization

Abstract

The study explored the potential of low-cost agro-industrial residues in the production of phytase from bacterial sources which was (1) active at high temperature and broad range of pH and, (2) can act as a supplement for animal feed in order to improve its nutritional efficiency. Optimization of various factors (incubation period, initial pH, inoculum age/size, and carbon/nitrogen source) under submerged fermentation resulted in 21.55-fold (0.431 U/ml) improvement in phytase (Phy-Ck) production from Citrobacter koseri PM-7 [isolated from the rhizosphere of black sesame (Sesamum indicum)]. Under solid state fermentation using wheat bran, 9.372 U/g dry substrate of Phy-Ck production was obtained after 120 h of incubation. Partially purified Phy-Ck exhibited maximum activity at pH 5 and was stable in a pH (3–6) range by retaining more than 40 % of its activity after 3 h of incubation. Phy-Ck was optimally active at 70 °C. Ca2+ was found to marginally stimulate Phy-Ck while, moderate to complete inhibition of enzyme activity was observed in presence of Mn2+, Mg2+, Ni2+, Li2+, Hg2+, Cu2+, and EDTA. Thermal inactivation kinetics of Phy-Ck at 50–63 °C in the presence of Ca2+ led up to 12.33-fold improvement in half-life (t1/2) and better values for thermodynamic parameters [deactivation energy (Ed), enthalpy (H) and entropy (S)]. The K m and V max values of Phy-Ck were 0.408 mM and 3.586 μmol/ml/min, respectively. Phy-Ck displayed good tolerance (up to 98.5 % activity) towards proteolytic enzymes (pepsin, trypsin, and chymotrypsin). Phy-Ck was able to dephytinize agro-industrial residues with concomitant liberation of inorganic phosphate and soluble protein. The study demonstrated valorization of agro-residues for production of phytase useful for feed industry.