Hh. Jiang et al., Covalent immobilization of trypsin on glycidyl methacrylate-modified cellulose membrane as enzyme reactor, CHEM J CH U, 21(5), 2000, pp. 702-706
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.