Fungal tyrosinase immobilized on chitosan, calcium alginate, and silica gel for phenol elimination and dye decolorization

Aspergillus nidulans (AUMC No. 7147) was utilized for tyrosinase (EC 1.14.18.1) production. In this study, we purified the enzyme, immobilized it on various beads, analyzed its kinetics, and applied it for phenol removal and dye decolorization, including Crystal Violet (CV), Congo Red (CR), Methyl Red (MR), and Malachite Green (MG). Tyrosinase was isolated from Aspergillus nidulans (AUMC No. 7147) with a specific activity of 230.76 units mg⁻¹ protein. The purified enzyme was then immobilized on chitosan, Ca-alginate, and silica gel, achieving immobilization efficiencies of 89.6 %, 75.0 %, and 69.4 %, respectively. After 10 reuse cycles, the immobilized tyrosinase on chitosan and Ca-alginate retained 41 % and 15 % of its initial activity, respectively. The K_m values were determined to be 0.23, 0.37, and 0.38 mM, while the V_max values were 32.5, 33.3-, and 27.5-units mg⁻¹ protein for the free enzyme and the two immobilized forms, respectively. After 30 days of storage at 25°C, the residual activities of tyrosinase immobilized on silica gel, alginate, and chitosan were 14 %, 27 %, and 52 %, respectively. The optimal temperatures for the free and immobilized tyrosinase were 40°C and 50°C, respectively. The activation energies were calculated as 59.66, 54.43, and 48.86 KJ mol⁻¹ . The free tyrosinase exhibited an optimal pH of 7.0, which shifted to pH 8.0 upon immobilization. The enzyme could be reused for at least eight cycles. Phenol was effectively removed by chitosan-immobilized tyrosinase; however, the removal efficiency declined in subsequent cycles. The highest removal percentage was achieved using the chitosan-immobilized enzyme at pH 8.0, and Temp., 50°C, after 2 hours. Additionally, the immobilized enzyme was effective in decolorizing the dyes Crystal Violet (CV), Congo Red (CR), Methyl Red (MR), and Malachite Green (MG). These findings support bioremediation strategies for the safe environmental removal of toxic dyes, and phenol from wastewater.