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Páginas1583 –1600

GLUTARALDEHYDE IN BIO-CATALYSTS DESIGN: A useful crosslinker and a versatile tool in enzyme immobilization

Autores:Roberto Fernandez-Lafuente, Oveimar Barbosa
Grupos de investigación:Optimización de biocatalizadores y bioprocesos enzimáticos
Oveimar Barbosaa, Claudia Ortizb, Ángel Berenguer-Murciac, Rodrigo Torresa,
Rafael C. Rodrigues*,d Roberto Fernandez-Lafuentee,*.
a Escuela de Química, Grupo de investigación en Bioquímica y Microbiología (GIBIM), Edificio Camilo Torres 210, Universidad Industrial de Santander, Bucaramanga, Colombia.
b Escuela de Bacteriología y Laboratorio Clínico, Universidad Industrial de Santander, Bucaramanga, Colombia.
c Instituto Universitario de Materiales, Departamento de Química Inorgánica, Universidad de Alicante, Campus de San Vicente del Raspeig, Ap. 99 - 03080 Alicante, Spain.
d Biocatalysis and Enzyme Technology Lab, Institute of Food Science and Technology, Federal University of Rio Grande do Sul, Av. Bento Gonçalves, 9500, P.O. Box 15090, ZC 91501-970, Porto Alegre, RS, Brazil.

Glutaraldehyde is one of most widely used reagents in the design of biocatalysts. It is a powerful crosslinker, able to react with itself, with the advantages that this may bring forth. In this review, we intend to give a general vision of its potential and the precautions that must be taken when using this effective reagent. First, the chemistry of the glutaraldehyde/amino reaction will be commented. This reaction is still not fully clarified, but it seems to be based on the formation of 6-membered heterocycles formed by 5 C and one O. Then, we will discuss the production of intra and inter-molecular enzyme crosslinkings (increasing enzyme rigidity or preventing subunit dissociation in multimeric enzymes). Special emphasis will be placed on the preparation of cross-linked enzyme aggregates (CLEAs), mainly in enzymes that have low density of surface reactive groups and, therefore, may be problematic to obtain a final solid support. Next, we will comment on the uses of glutaraldehyde in enzymes previously immobilized on supports. First, the treatment of enzymes immobilized on supports that cannot react with glutaraldehyde (only inter and intramolecular cross-linkings will be possible) to prevent enzyme leakage and obtain some enzyme stabilization via cross-linking. Second, the cross-linking of enzymes adsorbed on aminated supports, where together with other reactions enzyme/support crosslinking is also possible; the enzyme is incorporated to the support. Finally, we will present the use of aminated supports preactivated with glutaraldehyde. Optimal glutaraldehyde modifications will be discussed in each specific case (one or two glutaraldehyde molecules for amino group in the support and/or the protein). Using preactivated supports, the heterofunctional nature of the supports will be highlighted, with the drawbacks and advantages that the heterofunctionality may have. Particular attention will be paid to the control of the first event that causes the immobilization depending on the experimental conditions to alter the enzyme orientation regarding the support surface. Thus, glutaraldehyde, an apparently old fashioned reactive, remains as the most widely used and with broadest application possibilities among the compounds used for the design of biocatalyst.
Palabras clave:Enzyme stabilization, Enzyme immobilization, inter and intramolecular crosslinking, CLEAs, heterofunctional supports, glutaraldehyde
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