Reversible enzyme immobilization via a very strong and nondistorting ionic adsorption on support-polyethylenimine composites.
New tailor-made anionic exchange resins have been prepared, based on films of large polyethylenimine polymers (e.g., MW 25,000) completely coating, via covalent immobilization, the surface of different porous supports (agarose, silica, polymeric resins). Most proteins contained in crude extracts from different sources have been very strongly adsorbed on them. Ionic exchange properties of such composites strongly depend on the size of polyethylenimine polymers as well as on the exact conditions of the covalent coating of the solids with the polymer. On the contrary, similar coating protocols yield similar matrices by using different porous supports as starting material. For example, 77% of all proteins contained in crude extracts from Escherichia coli were adsorbed, at low ionic strength, on the best matrices, and less than 15% of the adsorbed proteins were eluted from the support in the presence of 0.3 M NaCl. Under these conditions, 100% of the adsorbed proteins were eluted from conventional DEAE supports. Such polyethylenimine-support composites were also very suitable to perform very strong and nondistorting reversible immobilization of industrial enzymes. For example, lipase from Candida rugosa (CRL), beta-galactosidase from Aspergillus oryzae and D-amino acid oxidase (DAAO) from Rhodotorula gracilis, were adsorbed on such matrices in a few minutes at pH 7.0 and 4 degrees C. Immobilized enzymes preserved 100% of catalytic activity and remained fully immobilized in 0.2 M NaCl. In addition to that, CRL and DAAO were highly stabilized upon immobilization. Stabilization of DAAO, a dimeric enzyme, seems to be due to the involvement of both enzyme subunits in the ionic adsorption.
Mateo
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C
Departamento de Biocatalisis, Instituto de Catalisis, CSIC, Campus Universidad Autonoma, Cantoblanco, 28049 Madrid, Spain.
Abian
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O
Fernandez-Lafuente
R
R
Guisan
J M
JM
D000327:Adsorption; D000837:Anion Exchange Resins; D001709:Biotechnology / Q000379:methods*; D004800:Enzymes, Immobilized; D011094:Polyethyleneimine
D000837:Anion Exchange Resins; D004800:Enzymes, Immobilized; D011094:Polyethyleneimine