No abstract

Immobilization and purification of enzymes are usual requirements for their industrial use. Both purification and immobilization have a common factor: they use a solid activated support. Using a support for enzyme purification means having mild conditions for enzyme release and a selective enzyme–support interaction is interesting. When using a support for immobilization, however, enzyme desorption is a problem. The improvement of enzyme features through immobilization is a usual objective (e.g., stability, selectivity). Thus, a support designed for enzyme purification and a support designed for enzyme immobilization may differ significantly. In this review, we will focus our attention on the requirements of a support surface to produce the desired objectives. The ideal physical properties of the matrix, the properties of the introduced reactive groups, the best surface activation degree to reach the desired objective, and the properties of the reactive groups will be discussed.

Novozym 435 (N435) is a commercially available immobilized lipase produced by Novozymes with its advantages and drawbacks.

27A heterofunctional support for enzyme immobilization may be defined as that which 28 possesses several distinct functionalities on its surface able to interact with a protein. We will 29 focus on those supports in which a final covalent attachment between the enzyme and the 30 support is achieved. Heterofunctionality sometimes has been featured in very old 31 immobilization techniques, even though in many instances it has been overlooked, giving rise to 32 some misunderstandings. In this respect, glutaraldehyde activated supports are the oldest 33 multifunctional supports. Their matrix has primary amino groups, the hydrophobic 34 glutaraldehyde chain, and can covalently react with the primary amino groups of the enzyme. 35Thus, immobilization may start (first event of the immobilization) via different causes and may 36 involve different positions of the enzyme surface depending on the activation degree and 37 immobilization conditions. Other "classical" heterofunctional supports are epoxy commercial 38 supports consisting of reactive covalent epoxy groups on a hydrophobic matrix. Immobilization 39 is performed at high ionic strength to permit protein adsorption, so that covalent attachment may 40 take place at a later stage. Starting from these old immobilization techniques, tailor-made 41 heterofunctional supports have been designed to permit a stricter control of the enzyme 42 immobilization process. The requirement is to find conditions where the main covalent reactive 43 moieties may have very low reactivity towards the enzyme. In this review we will discuss the 44 suitable properties of the groups able to give the covalent attachment (intending a multipoint 45 covalent attachment), and the groups able to produce the first enzyme adsorption on the support. 46Prospects, limitations and likely pathways for the evolution (e.g., coupling of site-directed 47 mutagenesis and thiol heterofunctional supports of enzyme immobilization on heterofunctional 48 supports) will be discussed in this review. 49 50