Enzyme immobilization on epoxy- and 1,1'-carbonyldiimidazole-activated methacrylate-based monoliths.
Monolithic Convective Interaction Media (CIM) have been activated with epoxide and imidazole carbamate functionalities and used as supports for covalent immobilization of protein A, deoxyribonuclease I, and trypsin. The efficiency of immobilization for these proteins was determined from the amount of bound IgG, degradation of DNA, and hydrolysis of Nalpha-benzoyl-L-arginine ethyl ester, respectively. The respective biological activities of trypsin and the binding capacity of protein A immobilized via imidazole carbamate groups were 11.45 and 2.25 times higher than those obtained for epoxide matrix while they were practically equal for deoxyribonuclease I. The kinetics of immobilization was studied in detail for trypsin under dynamic conditions and revealed that the enzyme immobilized via imidazole carbamate groups already reached its highest activity in 5 min. In contrast, a much longer time was required for immobilization via epoxy groups.
Bencina
Katja
K
BIA Separations doo, Teslova 30, Ljubljana, Slovenia.
Podgornik
Ales
A
Strancar
Ales
A
Bencina
Mojca
M
D000818:Animals; D001120:Arginine / Q000031:analogs & derivatives / Q000737:chemistry; D002138:Calibration; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000378:metabolism; D003850:Deoxyribonuclease I / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004800:Enzymes, Immobilized / Q000737:chemistry*; D004852:Epoxy Compounds / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007093:Imidazoles / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry; D007700:Kinetics; D008024:Ligands; D008689:Methacrylates / Q000737:chemistry*; D008956:Models, Chemical; D010179:Pancreas / Q000378:metabolism; D013205:Staphylococcal Protein A / Q000737:chemistry; D013997:Time Factors; D014357:Trypsin / Q000737:chemistry
D004800:Enzymes, Immobilized; D004852:Epoxy Compounds; D007093:Imidazoles; D007074:Immunoglobulin G; D008024:Ligands; D008689:Methacrylates; D013205:Staphylococcal Protein A; C006900:N,N-carbonyldiimidazole; D004247:DNA; D001120:Arginine; C489258:arginine ethyl ester; D003850:Deoxyribonuclease I; D014357:Trypsin