The development of an immobilized enzyme reactor containing glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma cruzi: the effect of species' specific differences on the immobilization.
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) plays an important role in the life cycle of the Trypanosoma cruzi, and an immobilized enzyme reactor (IMER) has been developed for use in the on-line screening for GAPDH inhibitors. An IMER containing human GAPDH has been previously reported; however, these conditions produced a T. cruzi GAPDH-IMER with poor activity and stability. The factors affecting the stability of the human and T. cruzi GAPDHs in the immobilization process and the influence of pH and buffer type on the stability and activity of the IMERs have been investigated. The resulting T. cruzi GAPDH-IMER was coupled to an analytical octyl column, which was used to achieve chromatographic separation of NAD(+) from NADH. The production of NADH stimulated by d-glyceraldehyde-3-phosphate was used to investigate the activity and kinetic parameters of the immobilized T. cruzi GAPDH. The Michaelis-Menten constant (K(m)) values determined for d-glyceraldehyde-3-phosphate and NAD(+) were K(m) = 0.5 +/- 0.05 mM and 0.648 +/- 0.08 mM, respectively, which were consistent with the values obtained using the non-immobilized enzyme.
Cardoso
Carmen Lúcia
CL
Departamento de Química, Faculdade de Filosofia, Ciências e Letras de Ribeirão Preto, Universidade de São Paulo, 14040-901, São Paulo, Brazil.
de Moraes
Marcela Cristina
MC
Guido
Rafael Victorio Carvalho
RV
Oliva
Glaucius
G
Andricopulo
Adriano Defini
AD
Wainer
Irving William
IW
Cass
Quezia Bezerra
QB
D000818:Animals; D019149:Bioreactors / Q000382:microbiology*; D014355:Chagas Disease / Q000382:microbiology; D004791:Enzyme Inhibitors / Q000032:analysis; D004800:Enzymes, Immobilized / Q000378:metabolism*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D006801:Humans; D013045:Species Specificity; D014349:Trypanosoma cruzi / Q000201:enzymology*
D004791:Enzyme Inhibitors; D004800:Enzymes, Immobilized; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases