Enzymes immobilization approaches

Since many years, pectolytic enzymes have been widely used in industrial beverage processing to improve either the quality and the yields in fruit juice extraction or the characteristics of the final product [1,2]. To this purpose, complex enzymatic mixtures, containing several pectolytic enzymes and often also cellulose, hemicellulose and ligninolytic activities, are usually employed in the free form. The interactions among enzymes, substrates and other components of fruit juice make the system very difficult to be investigated and only few publications are devoted to the study of enzymatic pools [3-5], An effective alternative way to carry out the depectinisation process is represented by the use of immobilized enzymes. This approach allows for a facile and efficient enzymatic reaction control to be achieved. In fact, it is possible to avoid or at least to reduce the level of extraneous substances originating from the raw pectinases in the final product. In addition, continuous processes can be set up. 

The first belief in the possibility of enzyme stabilization on a silica matrix was stated by Dickey in 1955, but he did not give experimental evidence, only mentioning that his experiments were unsuccessful [65]. A sol-gel procedure for enzyme immobilization in silica was first developed by Johnson and Whateley in 1971 [66]. The entrapped trypsin retained about 34 % of its tryptic activity observed in solution before the encapsulation. Furthermore, the enzyme was not released from the silica matrix by washing, demonstrating the increased stability and working pH range. Unfortunately, the article did not attract attention, although their method contained all the details that may be found in the present-day common approach. This was probably due to its publication in a colloid journal that was not read by biochemists. 

Recently, we proposed an alternative process for encapsulating biomacromolecules within PE microcapsules. This approach involves using nanoporous particles as sacrificial templates for both enzyme immobilization and PE multilayer capsule formation (Figure 7.2, route (I)) [66,67]. Unlike previous LbL encapsulation strategies, this approach is not limited to species that undergo crystallization, and is not dependent upon adjustments in electrostatic interactions within PE microcapsules to alter shell permeability characteristics. The salient feature of this method is that it is applicable to a wide range of materials for encapsulation. 

Nanostructured silica and ordered mesoporous silicas have been envisaged as small enzyme immobilization supports [196]. The encapsulation approach is required either to further immobilize enzymes adsorbed in the channels by reducing the pore opening by further silylation or by encapsulating the enzyme directly [197]. 

Good accuracy and precision were obtained in connection to 100 pL blood samples. A wide range of amperometric enzyme electrodes, differing in the electrode design or material, membrane composition, or immobilization approach have since been described. 

The methodology of the proposed immobilization approach [164, 179] is, however, quite different from non-aqueous enzymology . Though during an immobilization procedure, enzymes have to be exposed to organic solvents, their activity is required only in aqueous solution in which resulting biosensors are operated. Hence, it is only important that the enzymes are able to retain their catalytic properties after exposure to organic solvents. 

Matinek K, Mozhaev W (1985) Immobilization of enzymes an approach to fundamental studies in biochemistry. Adv Enzymol 57 179-249 McLaughlin MJ, Smolders E, Merckx, R (1998) Soil-root interface physicochemical processes. In Huang PM, Adriano DC, Logan TJ, Checkai RT (eds) Soil Chemistry and Ecosystem Health. Soil Sci Soc Am, Madison, WI, USA, pp 233-277... 

In recent times the incorporation of enzymes into nanostructured materials is commonly referred to as nanobiocatalysis. Nanobiocatalysis has emerged as a rapidly growing research and development area. Lately, nanobiocatalytic approaches have evolved beyond simple enzyme immobilization strategies to include also topics like artificial enzymes and cells, nanofabrication, and nanopatterning [18]. A recent bibliometric analysis [19] of nanobiocatalysis publications shows a strong increase within the last decade (Fig. 14.1). The analysis has been compiled from... 

Immobilization onto a solid support, either by surface attachment or lattice entrapment, is the more widely used approach to overcome enzyme inactivation, particularly interfacial inactivation. The support provides a protective microenvironment which often increases biocatalyst stability, although a decrease in biocata-lytic activity may occur, particularly when immobilization is by covalent bonding. Nevertheless, this approach presents drawbacks, since the complexity (and cost) of the system is increased, and mass transfer resistances and partition effects are enhanced [24]. For those applications where enzyme immobilization is not an option, wrapping up the enzyme with a protective cover has proved promising [21]. 

A difficult problem in utilizing enzymes as catalysts for reactions in a non-cellular environment is their instability. Most enzymes readily denature and become inactive on heating, exposure to air, or in organic solvents. An expensive catalyst that can be used only for one batch is not likely to be economical in an industrial process. Ideally, a catalyst, be it an enzyme or other, should be easily separable from the reaction mixtures and indefinitely reusable. A promising approach to the separation problem is to use the technique of enzyme immobilization. This means that the enzyme is modified by making it insoluble in the reaction medium. If the enzyme is insoluble and still able to manifest its catalytic activity, it can be separated from the reaction medium with minimum loss and reused. Immobilization can be achieved by linking the enzyme covalently to a polymer matrix in the same general manner as is used in solid-phase peptide synthesis (Section 25-7D). 

The successful conversion of D-glucose into D-fructose on the industrial scale with immobilized D-glucose isomerase was a brilliant demonstration of the value of this kind of approach. Then followed a huge technical literature on enzyme immobilization, reviewed in Ref. 9 (page 353). We shall here restrict ourselves to the methods which have been utilized in the syntheses outlined in Tables II to X. We suggest to readers interested in theses techniques that they first use these methods. If they prove unsatisfactory, as there is a plethora of alternatives, other techniques, described in Refs. 8-10, may be tried a majority of readily available carbohydrate enzymes have been immobilized, often in several different ways. 

Different approaches have been reported for enzyme immobilization and membrane deposition including drop-on techniques [42, 51], ink-jet printing [52] and photolithographically patterned enzyme membranes [53,54]. 

Routinely, common chemical and enzymatic techniques are used to obtain protein fragments. Unfortunately, when enzymatic digestion techniques and nanograms quantities of proteins are used, the method become ineffective due to dilution and reduced enzymatic activity. An alternative approach to overcome this problem is the use of proteolytic enzymes immobilized to a solid support and a small-bore reactor column. Using trypsin immobilized to agarose, tryptic digests of less than 100 ng of protein can be reproducible obtained (49). 

Immobilized HRP and GOx on layered silicates using an avidin-biotin immobilization approach The immobilized enzymes retained high levels of activity compared to the native enzymes and showed improved thermal behavior. In addition, the immobilized GOx retained 65% of its initial activity at 58°C [30]... 

To test the reusability of the biocatalyst, five sequential reaction cycles with CPO immobilized on SBA-16 of different pore sizes were completed [6]. The authors found that immobilization on material with larger pore, 143 A, improved the reusability of the catalyst. Enzyme immobilized by covalent attachment to silica-based materials retained a higher residual activity after five reaction cycles than the physical approach. 

Despite its broad applicability, the confocal approach is restricted to the observation of one enzyme immobilized at a specific position. In contrast, wide-held detection schemes allow for the observation of an area of up to 1 mm and can therefore detect a number of enz unes in parallel. Moreover, the movement of individual molecules can be followed. This allows the study of processive enz5mies moving on their natural substrates, as has been elegantly demonstrated for some DNA interacting enz unes [12,30,31]. 

In this section, we discuss about the screen printed electrode (SPE) based AChE sensors for the selective determination of OP and CA pesticides. In the past decades, several attempts were made by the researchers to develop SPE based pesticide sensors, where the enzyme AChE was immobilized either directly onto the electrode or above other matrices incorporated SPE surfaces. Both approaches resulted in the good, rapid detection of OP and CA pesticides. Earlier, Hart et al. employed AChE/SPE to detect OP and CA pesticides [21], They measured the enzyme activity from the rate of hydrolysis of acetylthiocholine iodide. Three polymers such as hydroxyethyl cellulose, dimethylaminoethyl methacrylate, and polyethyleneimine were used as enzyme immobilization matrices. Initially, electrodes were exposed to drops of water or pesticide solution, dried and their activity was screened after 24 h. They found that, when the enzyme matrix was hydroxyethyl cellulose, electrode activity inhibited both by water as well as by pesticides. While with co-polymer matrix, a significant response towards pesticides alone was observed. Further, the long-term storage stability of electrodes was highest when the enzyme matrix consisted of the co-polymer. The electrodes retained their activity for nearly one year. In contrast, the electrodes made of hydroxyethyl cellulose or polyethyleneimine possess less stability. 

In addition to the analytical applications, there was sporadic work on the employment of flow calorimetry for the investigation of enzyme kinetics [23,24]. In 1985 Owusu et al. [25] published the first report on the use of flow microcalorimetry for the study of immobilized enzyme kinetics approaching... 

A variety of approaches have been adopted for the immobilization of enzymes with the help of antibodies. These range from simple complexing of enzymes with their antibodies to form insoluble complexes to enzyme immobilization on matrices with oriented antibodies. 

The amount of enzyme immobilized on the solid phase is inversely proportional to the amount of free antigen present in the incubation mixture. This approach has been used both in the equilibrium and sequential technique (Tijssen and Kurstak, 1981). The technical procedures are similar to those in Table 14.7 and its quasi-equilibrium variant with the modifications that Ag is coated on the solid phase and Ab E is used. 

Performing multiple reactions simultaneously in a single step offers possibilities for reduced waste and increased safety as well as manipulation of equilibria. This approach was inspired by the action of enzymes, which constitute interesting examples of multifunctional catalysts as they can promote multi-step reactions. In fact, enzymes immobilize mutually incompatible functional groups in a manner that maintains their independent functionality and, as such, are able to carry out multi-step reaction sequences with functionalities that would not be tolerated together in solution. 

So far SECM applications have been considered where enzymes immobilized at a surface catalyze redox reactions of low molecular weight compounds. The reaction products are detected at the ultramicroelectrode tip under diffusion-controlled conditions. This approach requires that the biochemically active layer continuously generate or consume redox active (for amperometric detection) or charged (for potentiometric detection) species. Since the tip signal depends on the diffusion coefficients and/or convective effects as well as the local concentration, it is possible to image localized mass transport phenomena instead of localized chemical fluxes (Chapter 9). In a general sense it is a process that is recorded with lateral resolution. 

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