Microbial phytases are of immense importance due to their application in food/feed industry by enhancing the availability of essential minerals such as phosphorous, iron, calcium etc. required for normal human physiology and also have commercial and environment significance. Therefore, in present study an attempt was made to enhance the production of phytase from isolated probiotic Pediococcus acidilactici BNS5B by employing both one variable at a time approach and statistically based design of experiments such as Plackett-Burman and Response Surface Methodology. Interestingly, phytase production was enhanced 94 fold at an optimised condition of galactose, yeast extract, beef extract and ammonium sulphate. Further, the phytase enzyme was purified and had apparent molecular weight of 43 KDa, pH optima of with pH stability in the range of , temperature optima of 40°C and retaining an activity of 76% at a temperature range of 20-80°C and followed normal Michael-Menten curve with the kinetic parameter Km and Vmax of and respectively. | Optimization, purification and characterization of phytase from isolated probiotic Pediococcus acidilactici BNS5B