Preparation of novel cryogel for lysozyme adsorption and determination of some optimization parameters for enzyme

In this study, 2-hydroxyethyl methacrylic acid-methacryloyl antipyrin-Ce3+[P(HEMA MAAP-Ce3+)] cryogel was prepared by free radical polymerization method based on 2-hydroxyethyl methacrylic acid (HEMA) and methacryloyl antipyrin-Ce3+(MAAP Ce3+) complex. The MAAP-Ce3+ complex was characterized by UV-near infrared (UV-NIR) and energy dispersive X-ray (EDX) techniques, and the p(HEMA-MAAP Ce3+) cryogel was characterized by scanning electron microscopy (SEM), FT-IR and swelling tests. Five different peaks were seen at 916, 1037, 1081, 1109 and 1141 nm for the MAAP-Ce3+ complex in the UV-NIR spectrum. In the EDX spectrum, the presence of 15.12% N and 2.89% Ce3+ in the MAAP-Ce3+ complex showed nitrogen and Ce3+ in the structure. In FT-IR spectrum studies, cryogel showed –OH vibrations at 3412 cm-1 and characteristic –NH vibrations at 3104 cm 1 , and also at 1768-1712 cm-1 , ester, amide and cyclic carbonyl groups appears to have C=O bonds. The cryogel was used for lysozyme adsorption from the aqueous solution. Equilibrium swelling ratio of P(HEMA-MAAP-Ce3+) cryogel was calculated as 7.39 g H2O/g cryogel, and the macropore amount was calculated as 80.8%. The cryogel pore size was found to be 30-50 µm. The effects of the flow rate, pH, temperature, initial enzyme concentration and ionic strength on lysozyme adsorption were studied. Maximum adsorption capacity reached with 57.84 mg/g of prepared cryogel under 0,5 flow rate (mL/min), at pH 6.0 and at 25 oC. The result of the effect of ionic strength, it was determined that the lysozyme adsorption capacity decreased from 41.31 mg/g to 2.01 mg/g as the salt concentration increased from 0.05 M to 1.0 M. The correlation coefficient (R2 : 0.9939) obtained as a result of the values calculated for the Langmuir and Freundlich isotherms showed that the Langmuir adsorption model, could be suitable for this system. The adsorbed enzyme was desorbed 95.7% with 1.0 M NaCl solution containing 20 mM, pH 6.0 phosphate buffer in 120 minutes. In the prepared cryogel reusability studies, no significant decrease was observed in the adsorption capacity of the cryogel used 7 times in succession. Keywords: Cryogel, IMAC, Cerium3+ , Adsorption, Lysozyme