Current Organic Chemistry - Volume 19, Issue 17, 2015

The development of silica-based sol-gel techniques compatible with the retention of protein structure and function started more than 20 years ago, mainly for the design of biotechnological devices or biomedical applications. Silica gels are optically transparent, exhibit good mechanical stability, are manufactured with different geometries, and are easily separated from the reaction media. Biomolecules encapsulated in silica gel normally retain their structural and functional properties, are stabilized with respect to chemical and physical insults, and can sometimes exhibit enhanced activity in comparison to the soluble form. This review briefly describes the chemistry of protein encapsulation within the pores of a silica gel three-dimensional network, the mechanism of interaction between the protein and the gel matrix, and its effects on protein structure, function, stability and dynamics. The main applications in the field of biosensor design are described. Special emphasis is devoted to silica gel encapsulation as a tool to selectively stabilize subsets of protein conformations for biochemical and biophysical studies, an application where silica-based encapsulation demonstrated superior performance with respect to other immobilization techniques.

Biohybrid materials combine the natural diversity of biomolecules with the flexibility of the inorganic sol-gel process. Association of biopolymers with silica is particularly useful for the preparation of threedimensional hosts for protein immobilization with tailored physical and chemical stability. They are easily tunable in terms of composition, structure and shape, including silicified hydrogels, core-shell particles, and thin films. Such a versatility also allows for the fine tuning of the bio-mineral interface so as to insure the integrity of the encapsulated species, and even improve their biological activity. Biohybrid materials are currently widely explored as drug delivery systems, in biocatalysis as well as to develop new biorecognition devices. They also raise more fundamental questions related to organic-inorganic interactions, both from a chemical and biological perspective.

Organically modified silica (Ormosil) gels are three-dimensional matrices obtained by co-polymerizing tetraalkyl orthosilicates and organoalkoxysilanes through the sol-gel technology. Ormosil matrices are highly flexible with respect to macroscopic size and shape, as they can be prepared in the form of monoliths, layers, powders and nanoparticles, and to their physical-chemical properties, with special regard to hydrophobicity or specific functionalization of their alkyl and aryl groups. This makes Ormosils a very promising matrix, though not widely exploited so far, for the immobilization of enzymes and other biomolecules either by encapsulation within the gel matrix or by surface decoration of nanoparticles. Potential applications range from the development of biocatalysts and bioremediation devices to intracellular drug delivery and photodynamic therapy.

Immobilization of proteins and other biomolecules in saccharide matrices leads to a series of peculiar properties that are relevant from the point of view of both biochemistry and biophysics, and have important implications on related fields such as food industry, pharmaceutics, and medicine. In the last years, the properties of biomolecules embedded into glassy matrices and/or highly concentrated solutions of saccharides have been thoroughly investigated, at the molecular level, through in vivo, in vitro, and in silico studies. These systems show an outstanding ability to protect biostructures against stress conditions; various mechanisms appear to be at the basis of such bioprotection, that in the case of some sugars (in particular trehalose) is peculiarly effective. Here we review recent results obtained in our and other laboratories on ternary protein- sugar-water systems that have been typically studied in wide ranges of water content and temperature. Data from a large set of complementary experimental techniques provide a consistent description of structural, dynamical and functional properties of these systems, from atomistic to thermodynamic level. In the emerging picture, the stabilizing effect induced on the encapsulated systems might be attributed to a strong biomolecule-matrix coupling, mediated by extended hydrogen-bond networks, whose specific properties are determined by the saccharide composition and structure, and depend on water content.

Supports based on poly-styrene-divinylbenzene (PSD) are commercially available since a long time ago. However, they are not commonly used as enzyme immobilization matrices. The main reason for this lies in the negative effect of the very hydrophobic surface on enzyme stability that produces the instantaneous enzyme inactivation in many instances. However, they have recently regained some impact in enzyme immobilization. They are easy to modify, and have been prepared with different modifiers. We will pay special attention to the coating of these supports with ionic liquids, which permits to have the ionic liquid phase anchored to the solid and modulate the enzyme properties without risk of losing these expensive and potentially toxic compounds. Thus, this review will present the covalent or physical immobilization of enzymes on PSD supports, submitted to different modifications. Moreover, lipases immobilized via interfacial activation on some naked PSD supports have shown some unexpected improvement in their catalytic properties, with uses in reactions like hydrolysis, esterification or transesterification.

The binding of enzymes on carriers with a high degree of activation with glyoxyl groups is an excellent method for improving enzyme stability by multipoint covalent attachment on a pre-existing carrier. Glyoxyl groups are short aliphatic aldehyde groups (Support – O-CH2 – CHO) that can be obtained by periodate oxidation of glyceryl groups (Support –O-CH2-CHOH-CH2OH). The unique features of glyoxyl groups are as follows: a.- The immobilization of enzymes through their amino groups has to occur via multipoint attachment. b.- The glyoxyl groups are stable at pH 10, which allows for the participation of Lys in the immobilization process. c.- The glyoxyl groups are very stable at pH 10, which allows for a long-term incubation between the immobilized enzyme and the activated support to promote a very intense enzyme-support multipoint covalent attachment. Using this protocol, more than 100 industrial enzymes were highly stabilized. In many cases, stabilizations of greater than 1000-fold compared with immobilized derivatives generated by conventional methods were obtained. Although dramatic stabilization was achieved, the immobilized enzymes maintained only 50 to 90 % of the catalytic activity of the corresponding soluble enzyme. Stabilization of industrial enzymes is a key step in immobilization protocols. Enzymes are immobilized for use at industrial scales for a number of reaction cycles.

Alginate has revolutionized the way in which proteins and enzymes are used in daily life, right from food, textiles, medicines and surgical advancement to environment. Alginate is a biopolymer with unique physical and chemical properties that makes it functionally an ideal material for attachment with proteins. Immobilization of enzymes on alginate is well known to show altered catalytic functions and improved operational stability with no or minimal drawbacks. The GRAS (Generally Recognized as Safe) status of alginate enables it to be readily used as an encapsulation material for food proteins for their better gastrointestinal absorbance, and also in delivery of several proteins and peptides for their therapeutic effects. The exclusive interactions of alginate with proteins as a function of ionic strength, pH and metallic ions make it a support of choice for purification of enzymes and proteins by simple chromatographic separations and affinity precipitation. However biomedical in vivo use of alginate, specifically in tissue engineering, necessitates the use of extra pure form of alginate. This review highlights the impact of interactions between proteins and alginate on the functional properties of the proteins. The review also emphasizes certain core applications areas where alginate is used to a large extent.