Immobilization of amyloglucosidase onto macroporous cryogels for continuous glucose production from starch

Poly(methyl methacrylate-glycidyl methacrylate) [Poly(MMA-GMA)] cryogels were synthesized using monomers of methylmethacrylic acid and epoxy group bearing GMA via radical cryopolymerization technique. Synthesized cryogels were used for the immobilization of amyloglucosidase to the cryogel surface using epoxy chemistry. Characterizations of the free and immobilized amyloglucosidase were carried out by comparing the optimum and kinetic parameters of enzymes. For this, pH and temperature profiles of free and immobilized preparation were studied and, it was found that, optimum pH of enzyme was not change upon immobilization (pH 5.0), while optimum temperature of the enzyme shifted 10 °C to warmer region after immobilization (optimum temperatures for free and immobilized enzyme were 55 and 65 °C, respectively). Kinetic parameters of free and immobilized enzyme were also investigated and Km values of free and immobilized amyloglucosidase were found to be 2.743 and 0.865 mg/mL, respectively. Vmax of immobilized amyloglucosidase was found to be (0.496 µmol/min) about four times less than that of free enzyme (2.020 µmol/min). Storage and operational stabilities of immobilized amyloglucosidase were also studied and it was showed that immobilized preparation had much more stability than free preparation. In the present work, amyloglucosidase immobilized poly(MMA-GMA) cryogels were used for continuous glucose syrup production from starch for the first time. Efficiency of immobilized enzyme was investigated and released amount of glucose was found to be 2.54 mg/mL at the end of the 5 min of hydrolysis. The results indicate that the epoxy functionalized cryogels offer a good alternative for amyloglucosidase immobilization applications with increased operational and thermal stability, and reusability. Also, these cryogels can be used for immobilization of other industrially valuable enzymes beyond amyloglucosidase.