Covalent immobilization of trypsin on glycidyl methacrylate-modified cellulose membrane as enzyme reactor

The membrane of the activated glycidyl methacrylate (GMA)-modified cellulos e was prepared and packed into a column piece by piece to make a microreact or by immobilization of trypsin. The microreactor based on the membrane med ium showed the advantages of being cheaper, mechanically strong and chemica lly stable. The activity of immobilized trypsin towards N-benzoyl-L-arginin e ethyl ester(BAEE) was 17 800 U/g dry membrane, and was 52% of that for fr ee enzyme. Besides, the effects of pH value of buffer, temperature, ionic s trength, organic modifier, and protein denaturants on the activity of immob ilized trypsin were investigated in comparison to the free trypsin, and the rmal stability also was found especially to be improved after immobilizatio n. The activity of the immobilized trypsin showed no decay after continuous ly pumping BAEE through immobilized trypsin microreactor for 24 h at 40 deg rees C and 0. 5 mL/min. Finally, the cytochrome C was digested by microreac tor and the products were analysed by MALDI-TOF-MS. The peptide mapping for a protein by combination of the immobilized enzyme membrane microreactor a nd MALDI-TOF-MS has been developed.