Heterofunctional supports in enzyme immobilization: from traditional immobilization protocols to opportunities in tuning enzyme properties.

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 toward 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.