Covalent immobilization of α-amylase on magnetic particles as catalyst for hydrolysis of high-amylose starch

Enzyme immobilized on magnetic particles can be used as efficient recoverable biocatalysts under strong magnetic response. To enable re-use of enzyme, modified Fe 3 O 4 particles were used as carrier to immobilize α-amylase in this paper. Firstly, the surface of Fe 3 O 4 particles were coated with amino groups by direct using TEOS (tetraethoxysilane) followed by treatment with APTES (3-aminopropyltriethoxysilane) and then carboxylated by reacting it with succinic anhydride. In addition, the effect of the immobilization condition on enzyme activity recovery and immobilization efficiency were investigated. The results showed that the optimal immobilization occurred under following conditions: pH 5.5, 40   °C, enzyme concentration of 20   mg   mL −1 , reaction time for 36   h. Using immobilized α-amylase as biocatalyst, the optimum pH and temperature for hydrolysis were observed to be 6.5 and 60   °C. The kinetics of hydrolysis reaction were studied using Michaelis–Menten equation. The affinity constant ( K m ) and maximum reaction rate ( v max ) of magnetic particles immobilization α-amylase (MPIA) was 0.543   mg   mL −1 and 1.321   mg   min −1 compared to those of 0.377   mg   mL −1 and 6.859   mg   min −1 of free enzyme. After immobilization, enzymatic activity, storage stability, thermo-stability, and reusability of MPIA were found superior to those of the free one. MPIA maintained 86% enzyme activity after 30 days and maintained 78% enzyme activity after recycling six times.