PURIFICATION AND CHARACTERIZATION OF PHYTASE PRODUCED BY BACILLUS AMYLOLIQUEFACIENS AUPPB02 AND ITS PROBIOTICS POTENCY

The anti nutritional property of phytic acid is one of major challenge with food but phytase producing probiotic strain could be the best alternative for this issue. In the present study, phytase from Bacillus amyloliquefaciens AUPPB02 was isolated, purified by ammonium salt precipitation, ion exchange chromatography and gel filtration chromatography, which showed molecular mass of 47 kDa by SDS-PAGE. The purification fold of purified phytase was 5.13, with a specific activity of 10.15 IU mg-1 protein. The purified enzyme showed its optimum activity at 7.0 pH and 40 oC temperature for sodium phytate,as specific natural substrate. To enhance the enzyme’s, activity, the additives, which were used (EDTA, BME, sodium azide, oxalate and citrate), didn’t respond positively, hence theywere not required. The Km and Vmax values of phytase produced by this strain were calculated from the Lineweaver - Burk plot, as 1.057 mM and 0.20 µmoles/sec respectively. Further, B. amyloliquefaciens AUPPB02 was evaluated for the probiotic activity of. It showed much resistance toward high acidic(pH 2 and 3) and bile concentration (0.3 – 2.0 %), with MIC of 2.0 % bile. It also exhibited good hydrophobicity, when treated with xylene (70.2 %), chloroform (65.69 %) and ethyl acetate (68.32 %). The performance of this strain was exceptionally good for autoaggregation (68.3 %) along with digestibility for casein and lipid. It has non haemolytic and non lecithinase producing ability. Subsequently, it revealed exceptional antibacterial activity against S. aureus(32 ± 0.72 mm) and P. aeruginosa (27 ± 0.21 mm). The strain B. amyloliquefaciens AUPPB02 also displayed susceptibility against commonly used antibiotics likes, azithromycin, tetracycline, erythromycin, amoxicillin, norfloxacin and levofloxacin. Considering the outstanding biochemical qualities of isolated phytase, which B. amyloliquefaciens AUPPB02 possess, along with its significant probiotic traits, it could be a suitable candidate to be used in food and feed applications. Yet, in order to reap the promised benefits from the strain, secreting phytase, its in vivo research in animals is required.