Methods of Immobilization

The selection of the proper immobilization strategy is mainly dependent on the properties of the selected support and the lipase, where the interaction between these 

An exceptional simplicity of the adsorption method of immobilization renders its application very attractive however, it is by no means acceptable in all cases because of possible desorption of the enzyme from the carrier surface and a partial denaturing of the enzyme due to the unfolding of the protein globule. 

Immobilization by Inclusion into the Space Lattice of Geis 

Immobilization through inclusion into a gel, similarly to immobilization by adsorption, is a physical method of protein fixation. Advantages of such a method are its simplicity and the absence of chemical modification of the enzyme molecules. Polyacrylamide, polyethylene glycol methacrylate, polysaccharide, polyionite, as well as various inorganic gels are used. 

Polyethylene glycol methacrylate gel is formed during copolymerization of methacrylate ethylene glycol and dimethacrylate ethylene glycol. The gels obtained are similar to the polyacrylamide ones but have better mechanical properties and transparency, and are more hydrophobic. 

Polysaccharide and polyionite gels are different in that their spatial structure is governed by hydrogen and ionic bonds rather than by covalent bonds. The formation of polyionite gels is effected with a reduction of the ionic strength of the solution containing polycations and polyanions. An amplification of electrostatic interaction between polymeric chains takes place with the formation of a gel, in the structure of which the enzyme is incorporated. 

There is a large number of methods of immobilization that can be broadly divided into those than involve the interaction of the enzyme with a matrix (usually through a chemical bond) and those in which the enzyme is contained within a restricted space, as shown in Fig. 4.1. 

Consider those methods that include the chemical binding of the enzyme molecules to an inert carrier (carrier-bound), where linkage can be covalent or non-covalent, and those in which the enzyme protein molecules are chemically linked among themselves, usually through a bifunctional reagent, without the participation of an inert carrier (carrier-free). 

A covalent bond is established between the functional groups in the activated carrier and the functional groups in the amino acid residues of the enzyme, like —OH, —SH, —NH2, and —COOH. Covalent immobilization has been thoroughly studied and detailed information on methods and procedures can be found in several publications devoted to it (Zaborsky 1973 Cao 2005b Guisan 2006). It is a rather complex method where the carrier is hardly recoverable after enzyme exhaustion, immobilization yield is rather low and the kinetic properties of the enzyme can be severely altered. However, operational stability is high and it is quite flexible, so that directed immobilization can be done to suit the particular characteristics of the process. 

It considers all kind of interactions between the enzyme and the support not involving covalent bonds, including short-range interactions like van der Waals forces, but also stronger ones like hydrophobic interactions and ionic bonds sometimes they 

Enzymes and highly hydrophilic polymers (i.e. polyethyleneimine and dextran sulfate) can be co-immobilized prior to cross-hnking, as shown in Fig. 4.4b creating 

---

Methods of immobilization have already been discussed, and various reactor configurations are possible. An enzyme immobilized on... 

All the existing methods of immobilization involve formation of a high local BAS concentration and retention of its biological activity. In this respect, the use of disperse forms of CP as carriers of BAS used for different purposes is very promising [88]. In this case, the CP-protein interaction is an important factor in controlling the structure and properties of these systems. 

They found that the methods of immobilizing flies during treatment had no effect on the relative toxicity of the pyrethrins. Strong chilling possibly increased the susceptibility of the flies to the pyrethrins, at least during the first 24 hours after treatment, but did not alter the relative toxicities. 

Another way of immobilizing catalyst complexes might be to trap them in the pores of solid particles, for instance by synthesizing the complex inside the pores of a zeolite ( ship in a bottle ). Another method could be to trap catalyst complexes in porous materials and deposit a membrane at the outer. surface. These methods of immobilizing a homogeneous catalyst do not involve chemical linkage between the catalyst and the carrier. The fixation is the result of steric hindrance. 

We will describe first the different methods of immobilization of catalysts, and highlight their advantages and disadvantages and their fields of application. We will then examine the properties of such supported complexes for the major classes of catalytic reactions. We will focus mainly on those studies where at least some characterization of the supported catalyst is given, unless the catalytic properties of the described system are outstanding the review is therefore far from being exhaustive. Finally, where possible, we will mention tests of recyclability, which are essential for the supported complex to be as a potential industrial catalyst. 

K. Nakanishi, H. Muguruma, K. Ikebukuro, and I. Karube, A novel method of immobilizing antibodies on a quartz crystal microbalance using plasma-polymerized films for immunosensors. Anal. Chem. 68, 1695-1700 (1996). 

Suaud-Chagny and Gonon [3] presented a new procedure for protein immobilization adapted to carbon microelectrode characteristics. The principle of this method of immobilization is based on the association of the protein with an inert porous film immobilized around the active tip of the electrode. For this purpose the carbon was coated with an inert, electrochemically obtained protein sheath (bovine serum albumin, BSA) a few micrometers thick. Then the sheath around the fiber was impregnated with lactate dehydrogenase (LDH), which could be immobilized onto the electrode and resulted in an electrode sensitive to pyruvate. 

One can distinguish between physical and chemical methods of immobilization (Fig. 42.2). The former makes use of weak interactions between the metal... 

The first two methods have the advantage that no modification of the homogeneous catalyst is needed. Surface hydrogen-bonded catalysts are limited to cationic complexes, while physical entrapment is more widely applicable. However, both methods are very sensitive to the solvent properties of the reaction medium. The chemical methods of immobilization require modification of the ligand, and this may be quite laborious. In the case of irreversible catalyst deacti-... 

Noncovalent attachment is a popular method of immobilization, and numerous different support materials have been employed, ranging from organic supports, like cellulose. 

Entrapment involves the physical confinement of an enzyme in a semipermeable matrix, in much the same manner as nature handles soluble enzymes. " This should represent an extremely mild method of immobilization, as the enzyme remains free, albeit confined to a small space. Two techniques, which at first sight appear unrelated, have been well utilized ... 

We turn now to discussion of the specific types of electroactive NPs that have been described. The NPs are grouped according to the composition of the electroactive NP rather than the method of immobilization. In most cases, metal-oxide/ hydroxide/oxyhy dr oxides materials are grouped together using a formulation such as MO , to indicate the different compositions and redox states of the metals that may be described. 

Electrochemical impedance measurements of the physical adsorption of ssDNA and dsDNA yields useful information about the kinetics and mobihty of the adsorption process. Physical adsorption of DNA is a simple and inexpensive method of immobilization. The ability to detect differences between ssDNA and dsDNA by impedance could be applicable to DNA biosensor technology. EIS measurements were made of the electrical double layer of a hanging drop mercury electrode for both ssDNA and dsDNA [34]. The impedance profiles were modeled by the Debye equivalent circuit for the adsorption and desorption of both ssDNA and dsDNA. Desorption of denatured ssDNA demonstrated greater dielectric loss than desorption of dsDNA. The greater flexibility of the ssDNA compared to dsDNA was proposed to account for this difference. 

The method of immobilization of the SO, the nature of the matrix (organic polymer, silica gel, pore size distribution) and hence the flexibility and accessibility of coordination positions for SA molecules to the > Cu( II) SO moiety are crucial. 

In an attempt to preserve the unique dual-nature selectivity of ILs while producing a stationary phase that is resilient to flowing at high temperatures, a method of immobilizing ILs as thin films in WCOT columns has been developed [42]. Figure 4.3 illustrates the steps used to form the immobilized IL stationary phase. 

Tuerker and Mavituna immobilized Trichoderma reesei within the open porous networks of reticulated polyurethane foam matrices. Growth pattern, glucose consumption, and cellulase production were compared with those of freely suspended cells. The method of immobilization was simple and had no detrimental effect on cell activity. Hundreds of similar projects could be cited. Not all rated the use of polymethane as the preferred technique. If a statistical analysis were conducted on aU the immobilization literature, we are sure that no single technique would be dominant. However, the combination of ease of immobilization, cost of materials, flow-through properties, control of flux rate through the immobilizing membrane, high surface-to-volume ratio, and other factors make polymethane a viable substratum for the continuous production of proteins. 

Table 11. Amount of Immobilized heparin as a function of the method of immobilization 981...

Method of immobilization Type of the bond Amount of heparin, mg/cm2... 

The fact that thromboresistance of HCP is dependent on the method of immobilization of heparin, together with a rather low activity of covalently immobilized heparin, makes the idea of long-term enhancement of thromboresistance of polymers on their heparinization doubtful 54,70,71J. Naturally, the answer can be given only after a detailed analysis of the interaction of HCP with blood and its components and clarification of the mechanism of the effect of the immobilized heparin on the blood clotting system and relying on the results of in vivo tests of these materials are necessary. 

---