Production, purification and characterization of neutral phytase from thermotolerant Aspergillus flavus ITCC 6720

Abstract

A novel thermotolerant strain of Aspergillus flavus (isolated from rhizosphere of Pisum sativum) was exploited for phytase production by solid state fermentation using mustard cake as growth substrate. Optimization of production parameters revealed that supplementation of de-oiled mustard cake (Brassica juncea) with malt extract and glucose at 0.5% each, moisture – 58.0%, inoculum level – 10%, inoculum age-72 h old, cultivation period – 4 d, incubation temperature – 37 °C and pH – 6.0 resulted in 2.3 fold enhancement in phytase activity titre (34 U g−1 to 112.25 U g−1 fermented substrate). The cell free extract showed the simultaneous production of acid and alkaline phosphatase (12.00 and 12.24 mg pNP g−1 respectively), carboxy methyl cellulase (1.494 IU g−1) and amylase activity (2.33 IU g−1). Partial purification and characterization of phytase showed its molecular weight as 30 kDa, substrate specificity for sodium phytate and p-nitrophenyl phosphate and its optimal thermal and pH activity at 45 °C and 7.0 respectively with thermal stability at pH ranging from 6.5 to 7.5. Enzyme with neutral pH optima was competent to reduce the phytic acid content of mustard cake by 80%.Fungal phytase can find application in hydrolyzing the phytate phosphorus content of animal feed.