Heterofunctional Supports in Enzyme Immobilization: From Traditional Immobilization Protocols to Opportunities in Tuning Enzyme Properties

Abstract

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

Enzyme immobilization by adsorption: a review

“…Another example are the epoxy-activated polymer supports, such as Eupergit (Katchalski-Katzir and Kraemer 2000; Erhardt and Jordening 2007) commercialized by Rhon Haas and Sepabeads commercialized by Resindion (Barbosa et al 2013). Both are available in the form of macroporous beads.…”

A Novel Oxidation of Salicyl Alcohols Catalyzed by Lipase

“…Meanwhile, Novozym 435 could be reused six times without significant loss of activity which indicated that the method has great potential for the preparation of salicylaldehydes. Generally, immobilization is a powerful technique to enhance the reusability and stability of enzyme [35][36][37][38]. Further study to improve the enzyme properties (mainly stability) of this lipase-catalyzed oxidation by using other immobilization strategies or even coupling immobilization and chemical modification is underway and will be reported in due course.…”

Strategies for the one-step immobilization–purification of enzymes as industrial biocatalysts

“…Now, we will present the development of tailor-made heterofunctional supports to get the specific immobilization of target proteins. Heterofunctional supports has have been recently reviewed (Barbosa et al, 2013); here we will focus on the prospects to use them to perform one step immobilization-purification.…”

“…Heterofunctional supports are defined as those matrices that present several functionalities on its their surface, with different physical or chemical properties, able to interact with a protein (Barbosa et al, 2013, Mateo et al, 2000.…”

“…The use of tailor-made supports to specifically immobilize proteins with certain structural particular features (large or small proteins, lipases via interfacial activation), with the final development of heterofunctional supports to achieve the specific enzyme immobilization followed by its stabilization via multipoint or multisubunit immobilization will be also an important part of this review (Barbosa et al, 2013). Finally, the coupling of site directed mutagenesis (to introduce specific domains in the desired areas of the protein) to these heterofunctional supports immobilization/stabilization of the protein will be discussed (Barbosa et al, 2013).…”