Immobilisation enzyme

Process B Genetic instability Poor enzyme stability Cofactor requirement Product (non-polar) inhibition Biocatalyst Free enzyme Free cells Immobilised enzyme Immobilised cells... 

Here we will focus on the biochemical aspects. The techniques of isolating enzymes, the process of enzyme immobilisation and the behaviour of immobilised enzyme reactors are discussed in detail in the BIOTOL text Technological Applications of Biocatalysts", so will not deal with these aspects in detail here. In outline, however, once the desired enzyme is isolated, it is attached to a carrier material. In order to ascertain sufficient accessibility of the enzyme, a bifunctional spacer molecule is attached to the carrier ... 

Special reactors are required to conduct biochemical reactions for the transformation and production of chemical and biological substances involving the use of biocatalysts (enzymes, immobilised enzymes, microorganisms, plant and animal cells). These bioreactors have to be designed so that the enzymes or living organisms can be used under defined, optimal conditions. The bioreactors which are mainly used on laboratory scale and industrially are roller bottles, shake flasks, stirred tanks and bubble columns (see Table 1). 

Recommended model particle systems are enzymes immobilised on carriers ([27,44,45,47,49]), oil/water/surfactant or solvent/water/surfactant emulsions ([27, 44, 45] or [71, 72]) and a certain clay/polymer floccular system ([27, 42-52]), which have proved suitable in numerous tests. The enzyme resin described in [27,44,47] (acylase immobilised on an ion-exchanger) is used on an industrial scale for the cleavage of Penicillin G and is therefore also a biological material system. In Table 3 are given some data to model particle systems. 

An alternative strategy to obtain silica immobilised catalysts, pioneered by Panster [23], is via the polycondensation or co-condensation of ligand functionalised alkoxysilanes. This co-condensation, later also referred to as the sol-gel process [24], appeared to be a very mild technique to immobilise catalysts and is also used for enzyme immobilisation. Several novel functional polymeric materials have been reported that enable transition metal complexation. 3-Chloropropyltrialkoxysilanes were converted into functionalised propyltrialkoxysilanes such as diphenylphosphine propyltrialkoxysilane. These compounds can be used to prepare surface modified inorganic materials. Two different routes towards these functional polymers can be envisioned (Figure 3.4). One can first prepare the metal complex and then proceed with the co-condensation reaction (route I), or one can prepare the metal complex after the... 

Keywords. Reverse micelles. Microemulsion, Enzymes, Immobilisation... 

Immobilisation methods are treated in detail in chapter 6. Most enzyme immobilisation methods used in coimection with non-conventional media rely on noncovalent interactions between the support and the enzyme. The reason why this works well in many cases is that enzymes normally have a low tendency to dissolve in the reaction media used. Adsorption or deposition on porous supports are often used methods. It is important to remember that other substances (for example salts and other polar substances) are often immobilised on the support because they are present during the immobilisation procedure and not soluble in the reaction medium. Those substances influence the microenviromnent of the enzyme and thereby its catalytic activity. 

The first monolithic materials initially emerged in the 1960s, but it is during the last 20 years that monoliths have been intensively developed in a variety of fields and particularly in analytical chemistry for separation techniques. Nowadays, these macroporous materials are widely used and have found numerous applications in different chromatographic modes such as liquid chromatography (LC) or CEC, as indicated by several reviews [150, 151]. Less commonly, monolithic materials can also be applied, for example, to solid-phase extraction, combinatorial synthesis and for enzyme immobilisation. 

Despite these improvements, there are other important biosensor limitations related to stability and reproducibility that have to be addressed. In this context, enzyme immobilisation is a critical factor for optimal biosensor design. Typical immobilisation methods are direct adsorption of the catalytic protein on the electrode surface, or covalent binding. The first method leads to unstable sensors, and the second one presents the drawback of reducing enzyme activity to a great extent. A commonly used procedure, due to its simplicity and easy implementation, is the immobilisation of the enzyme on a membrane. The simplest way is to sandwich the enzyme between the membrane and the electrode. Higher activity and greater stability can be achieved if the enzyme is previously cross-linked with a bi-functional reagent. 

Ref. Matrix Enzyme/immobilisation method Electrode configuration/ applied potential Mediator... 

Ref. Analyte Matrix Enzyme/immobilisation Electrode configuration/ Mediator... 

M. Badea, A. Curulli and G. Palleschi, Oxidase enzyme immobilisation through electropolymerised films to assemble biosensors for batch and flow injection analysis, Biosens. Bioelectron., 18(5-6) (2003) 689-698. 

C. Malitesta and M.R. Guascito, Heavy metal determination by biosensors based on enzyme immobilised by electropolymerisation, Biosens. Bioelectron., 20 (2005) 1643-1647. 

Following immobilisation, the beads were dispersed in an aqueous solution of HEC and cast onto Pt electrodes. Activity tests showed that leaching of immobilised enzyme was 2.5 times slower than that of free enzyme dispersed in HEC. Comparisons of activity to acetylthiocholine after 72 h constant operation showed a large stability enhancement for enzymes immobilised on both silica and carbon when compared to dispersion in HEC [36]. 

G.S. Nunes, G. Jeanty and J.-L. Marty, Enzyme immobilisation procedures on screen-printed electrodes used for the detection of anticholinesterase pesticides. Compartive study, Anal. Chim. Acta, 523 (2004) 107-115. 

The enzyme immobilisation was carried out on 7,7,8,8-tetra-cyanoquinodimethane (TCNQ)-modified screen-printed electrodes. TCNQ allows the electrochemical oxidation of thiocholine, the product of the reaction between acetylthiocholine and the enzyme, at +100 mV (vs. Ag/AgCl) (Fig. 16.6). The enzymatic activity of the acetylcholinesterase can thus be monitored by electrochemical methods. 

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