Abstract <p>In this study, phytase genes obtained from <i>Bacillus subtilis</i> KM-BS cultured at 37°C (<i>PhyC-37</i>) and 55°C (<i>PhyC-55</i>) were successfully expressed in <i>Escherichia coli</i> BL21 (DE3). The over-expression of the recombinant phytase was optimal within just 2 h after using 0.1 mM IPTG. SDS-PAGE analysis showed that the purified enzymes had a predicted molecular mass of 58 kDa and exhibited high activities at 3.73 and 2.51&#xa0;U/mL for the PhyC-37 and PhyC-55, respectively. Optimal temperature for both enzymes was 55°C, and these enzymes displayed the highest activity at pH 7.0 and 5.0 for purified PhyC-37 and PhyC-55, respectively. Metal ions such as Na⁺, K⁺, Mg²⁺, Ca²⁺, Mn²⁺, Co²⁺, and Zn²⁺ stimulated enzyme activities, while their activities were moderately to completely inhibited in the presence of Cu²⁺, SDS, EDTA, Triton X-100, and Tween-80. <i>K</i>_M and <i>V</i>_max values for PhyC-37 using sodium phytate as substrate were 0.18 mM and 0.45 µmol/min, respectively, while for PhyC-55, the corresponding values were 0.17 mM and 0.35 µmol/min. The hydrolysis of rice bran using the in-house crude phytases increased phytic acid digestion efficiency by 3.9−5.9-fold compared to without enzyme addition, with high hydrolysis rates within the first 2 h. The data emphasizes their potential application in the animal feed industry, offering a promising solution to mitigate environmental eutrophication.</p>