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.

Poly(2-hydroxyethyl methacrylate-co-glycidyl methacrylate) [poly(HEMA-GMA)] cryogel was synthesized by cryopolymerization technique at frozen temperature. Iminodiacetic acid (IDA) was then attached covalently to the cryogel as a chelating agent. Then, poly(HEMA-GMA)-IDA cryogel was chelated with Ni(II) ions and this novel metal affinity support was used for adsorption of urease from its aqueous solution. Urease adsorption experiments were carried out in a continuous system by using a peristaltic pump. Maximum urease adsorption onto poly(HEMA-GMA)-IDA-Ni(II) cryogel was found to be 11.30 mg/g cryogel at pH 5.0 acetate buffer and in 25 °C medium temperature. Urease adsorption capacity decreased with increasing ionic strength and increasing chromatographic flow rate. Adsorption kinetics of urease onto poly(HEMA-GMA)-IDA-Ni(II) cryogel was also investigated and it was found that Langmuir adsorption model is applicable for this adsorption study. This novel immobilized metal affinity chromatography support was used 10 times without any decrease at their adsorption capacity. It was also observed that urease enzyme was repeatedly adsorbed and desorbed without significant lost in enzymatic activity.

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In this study, poly (methyl methacrylate-glycidyl methacrylate) [poly(MMA-GMA)] cryogels were prepared by radical cryocopolymerization of MMA with GMA as a functional comonomer. Reactive Green 19 dye was then attached to the cryogel by nucleophilic substitution reaction, and this dye-attached cryogel column was used for lysozyme adsorption. Characterization of the cryogel was performed by Fourier transform infrared spectroscopy, environmental scanning electron microscopy, Brunauer-Emmett-Teller, and energy dispersive X-ray analysis. Pore size of the cryogels was 15-30 μm and pores were interconnected structure. Attached amount of Reactive Green 19 to cryogel support was calculated as 106.25 μmol/g cryogel. Lysozyme adsorption studies were carried out by using a continuous system. It was found that the maximum amount of lysozyme adsorption (32 mg/g cryogel) obtained from experimental results was found to be approximately same with the calculated Langmuir adsorption capacity (33 mg/g cryogel). Desorption of adsorbed lysozyme was carried out by using 1.5 M NaCl in pH 4.5 acetate buffer, and desorption yield was found to be 97.4%. Cryogels were very stable, and it was found that there was no remarkable reduction in the adsorption capacity at the end of ten adsorption-desorption cycles. As a result, Reactive Green 19-attached cryogels have great advantages such as easy preparation, rapid adsorption, and desorption, being economic and allowing the direct separation of proteins.

Supermacroporous poly(2-hydroxyethyl methacrylate) [poly(HEMA)] monolithic cryogel was prepared by radical cryocopolymerization of HEMA with N,N'-methylene bisacrylamide as crosslinker. Reactive Green 5 dye was immobilized to the cryogel with nucleophilic substitution reaction, and this dye attached cryogel column was used for affinity purification of papain from Carica papaya latex. Reactive Green 5-immobilized poly(HEMA) cryogel was characterized by swelling studies, Fourier transform infrared spectroscopy, scanning electron microscopy, and energy dispersive X-ray analysis. Maximum papain adsorption capacity was found to be 68.5 mg/g polymer while nonspecific papain adsorption onto plain cryogel was negligible (3.07 mg/g polymer). Papain from C. papaya was purified 42-fold in single step with dye attached cryogel, and purity of papain was shown by silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis.