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Glutathione transferases immobilized on nanoporous alumina: Flow system kinetics, screening, and stability

Author:
Kjellander, Marcus, Mazari, Aslam M.A., Boman, Mats, Mannervik, Bengt, Johansson, Gunnar
Source:
Analytical biochemistry 2014 v.446 pp. 59-63
ISSN:
0003-2697
Subject:
Drosophila melanogaster, aluminum oxide, glutathione transferase, humans, immobilized enzymes, nanopores, prostaglandins, screening
Abstract:
The previously uncharacterized Drosophila melanogaster Epsilon-class glutathione transferases E6 and E7 were immobilized on nanoporous alumina. The nanoporous anodized alumina membranes were derivatized with 3-aminopropyl-triethoxysilane, and the amino groups were activated with carbonyldiimidazole to allow coupling of the enzymes via ε-amino groups. Kinetic analyses of the immobilized enzymes were carried out in a circulating flow system using CDNB (1-chloro-2,4-dinitrobenzene) as substrate, followed by specificity screening with alternative substrates. A good correlation was observed between the substrate screening data for immobilized enzyme and corresponding data for the enzyme in solution. A limited kinetic study was also carried out on immobilized human GST S1-1 (also known as hematopoietic prostaglandin D synthase). The stability of the immobilized enzymes was virtually identical to that of enzymes in solution, and no leakage of enzyme from the matrix could be observed.
Agid:
5983798