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PublicationBIOMACROMOLECULES
Year2013
Volume14
Pages2433-2462
International

HETEROFUNCTIONAL SUPPORTS IN ENZYME IMMOBILIZATION: FROM TRADITIONAL IMMOBILIZATION PROTOCOLS TO OPPORTUNITIES IN TUNING ENZYME PROPERTIES

Authors:Roberto Fernandez-Lafuente
Groups of research:Optimización de biocatalizadores y bioprocesos enzimáticos
Oveimar Barbosaa, Rodrigo Torresa, Claudia Ortizb, Ángel Berenguer-Murciac,
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.
e Departamento de Biocatalisis. Instituto de Catálisis-CSIC. Campus UAM-CSIC. Cantoblanco. 28049 Madrid. Spain.
A heterofunctional support for enzyme immobilization may be defined as that which possesses several distinct functionalities on its surface able to interact with a protein. We will focus on those supports in which a final covalent attachment between the enzyme and the support is achieved. Heterofunctionality sometimes has been featured in very old immobilization techniques, even though in many instances it has been overlooked, giving rise to some misunderstandings. In this respect, glutaraldehyde activated supports are the oldest multifunctional supports. Their matrix has primary amino groups, the hydrophobic glutaraldehyde chain, and can covalently react with the primary amino groups of the enzyme. Thus, immobilization may start (first event of the immobilization) via different causes and may involve different positions of the enzyme surface depending on the activation degree and immobilization conditions. Other “classical” heterofunctional supports are epoxy commercial supports consisting of reactive covalent epoxy groups on a hydrophobic matrix. Immobilization is performed at high ionic strength to permit protein adsorption, so that covalent attachment may take place at a later stage. Starting from these old immobilization techniques, tailor-made heterofunctional supports have been designed to permit a stricter control of the enzyme immobilization process. The requirement is to find conditions where the main covalent reactive moieties may have very low reactivity towards the enzyme. In this review we will discuss the suitable properties of the groups able to give the covalent attachment (intending a multipoint covalent attachment), and the groups able to produce the first enzyme adsorption on the support. Prospects, limitations and likely pathways for the evolution (e.g., coupling of site-directed mutagenesis and thiol heterofunctional supports of enzyme immobilization on heterofunctional supports) will be discussed in this review.
Keywords:heterofunctional supports; enzyme immobilization; enzyme stabilization; multipoint covalent attachment;
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