Screening, expression, and characterization of recombinant phytase from Bacillus subtilis KM-BS for biodegradation of phytic acid in animal feeds

Abstract

Two extracellular phytases derived from Bacillus subtilis KM-BS were cultured at 37 and 55°C. These phytases were successfully expressed in Escherichia coli BL21 (DE3) cells and biochemically characterized for potential industrial applications. Recombinant PhyC-37 and PhyC-55 were shown to be catalytically active, with activities of 3.73 and 2.51 U/ml and specific activities of 4.12 and 1.43 U/mg, respectively. SDS-PAGE analysis indicated that the purified enzymes had a theoretical molecular weight of 58 kDa. Similar to other Bacillus phytases, the optimal temperature of the recombinant enzymes was 55°C. Purified PhyC-37 and PhyC-55 were optimally active at pH 7.0 and 5.0, respectively. They exhibited good stability at their optimal pH values and showed possible thermostability, retaining 46.22 and 50.05% of their activity after incubation for 360 min at 55°C. Addition of 1 mM of metal ions such as Na+, K+, Mg2+, Ca2+, Mn2+, Co2+, and Zn2+ stimulated PhyC-37 and PhyC-55, while enzyme activities were moderately to completely inhibited in the presence of Cu2+, SDS, EDTA, Triton X-100, and Tween-80. The Km and Vmax values of PhyC-37 were determined as 0.18 mM and 448.60 µmol/min, while for PhyC-55 the values were 0.19 mM and 355.60 µmol/min, respectively. Crude PhyC-37 and PhyC-55 also demonstrated remarkable rice bran digestion efficiency. Overall, findings highlighted the appealing features of these two purified phytase enzymes, which showed significant potential as candidates for application in the animal feed industry to mitigate environmental eutrophication.