Improved thermostability and catalytic performance of bacterial phytase via chitosan hydrogel immobilization

Phytase enzymes are essential for enhancing phosphorus bioavailability and mitigating the impacts of phytic acid in agriculture. In this study, a phytase-producing bacterium was isolated from rhizospheric soil and poultry waste and identified via 16S rRNA sequencing as Bacillus pumilus (PersiPhytase1). The purified enzyme, classified as an alkaline phytase, exhibited a molecular weight of ∼39 kDa and was characterized by high thermal and proteolytic stability. The enzyme was biochemically characterized, followed by immobilization within a chitosan-based hydrogel to enhance its stability. Post-immobilization analyses revealed improvements in operational and thermal stability. The immobilized phytase demonstrated broad pH activity across pH 5.0-9.0, with maximum activity at pH 7.0 and retained over 90% activity at pH 9.0. Temperature-profile analysis showed optimal activity at 50 °C, maintaining over 85% activity even at 80 °C. Kinetic parameters indicated enhanced catalytic efficiency: K_m decreased from 11.41 to 5.14 mM, and V_max increased from 0.054 to 0.063 mM·min^-1 upon immobilization. Furthermore, catalytic turnover (k_cat) and efficiency (k_cat/K_m) increased from 0.108 to 0.127 min^-1 and from 0.00954 to 0.024 mM^-1·min^-1, respectively. The immobilized enzyme retained 39.52% activity after 10 reuse cycles, with minimal leaching; even at 80 °C, enzyme release was 24.67%. Thermodynamic studies confirmed improved half-life and conformational stability, with a ΔG of 91.9 kJ/mol and ΔS of -0.157 kJ/mol·K at 70 °C. These findings indicate that chitosan-based hydrogel immobilization improves phytase stability and catalytic performance under laboratory conditions, highlighting its potential for feed-related applications pending further validation under processing and matrix conditions.