Immobilised enzyme systems

When the reaction product is soluble in water, enzyme regeneration is difficult to achieve, since the enzyme is often lost during isolation of the product. One way to overcome this problem is application of immobilised enzyme systems. The enzyme is either covalently or ionically attached to an insoluble carrier material or is entrapped in a gel. Depending on the size of the particles used, a simple filtration and washing procedure can be used to separate the immobilised enzyme from the dissolved product A well-known example of this technique is the industrial production of 6-APA. 

The combination of the two models previously discussed provides a powerful tool in understanding the behaviour of the immobilised enzyme system. This enables the optimisation of the enzyme characteristics to be carried out so as to produce a commercially attractive glucose isomerisation system which is based upon the use of flocculated bacterial cells. 

The 2-ethoxyethanol was a by-product, as shown in Figure 5.13. The formation rate of 2-ethoxyethanol was the same as the conversion rate of the (S)- or (R)-ibuprofen ester one mole of 2-ethoxyethanol was formed when one mole of ester was catalysed. A known concentration of 2-ethoxyethanol was added in the organic phase before the start of the reaction for product inhibition. The plots of the kinetics for the free lipase system are presented in Figure 5.17 and immobilised enzyme (EMR) in Figure 5.18, respectively. The Kw value was 337.94 mmoFl 1 for the free lipase batch system and 354.20 mmoll 1 for immobilised... 

The stress acting on particles is of high importance for many technical processes. As well as dispersion processes in two-phase systems (liquid/liquid or gas/Hquid), where the disintegration of particles is desirable, there are also a number of processes that may be adversely affected by particle disintegration. These include precipitation, agglomeration, crystalHsation processes, and also bio conversion with immobilised enzymes and the fermentation of sensitive microorganisms and animal and plant cells. 

The equilibrium particle diameter in the case of non agglomerate particle systems or the enzyme activity of immobilised enzymes after a certain exposure of time is entirely due to the reactor-specific comminution process, and conclusions can therefore be drawn regarding the maximum intensity of hydrodynamic stress. 

Another possibility is to immobilise enzymes either on the sensor element itself or in the vicinity of the sensing element. The operation principle is in most cases a semi-continuous spectral difference measurement in combination with a kinetic data evaluation. A sample containing the analyte of interest is recorded by the sensor immediately after contact with the sample and again after a certain time. Provided that no other changes in the composition of the sample occur over time, the spectral differences between the two measurements are characteristic for the analyte (and the metabolic products of the enzymatic reaction) and can quantitatively evaluated. Provided that suitable enzymes are available that can be immobilised, this may be a viable option to build a sensor, in particular when the enzymatic reaction can not (easily) be monitored otherwise, e.g. by production or consumption of oxygen or a change of pH. In any case, the specific properties and stumbling blocks related to enzymatic systems must be observed (see chapter 16). 

In both media water and w/o-microemulsions the enzymes denaturate with time. The complete immobilisation of the enzyme system in a continuous process leads to a limitation caused by the denaturation of the enzymes only. In order to obtain a complete immobilisation of the enzyme the transmembrane pressure of the ultrafiltration unit should often not exceed 1 bar [120]. 

The enzyme acts stereoselectively to produce only the required L-isomer (Figure 4.10). Originally a fermentation process for the production of L-aspartic acid was established. This was modified into an immobilised enzyme process, but since the extracted enzyme is not very stable, an efficient continuous process was not possible. Therefore an immobilised cell system was developed with a very long operational lifetime. Another raw material for L-aspartic acid is maleic anhydride, which is first converted... 

In multiphase systems, biological reactions are always carried out in the presence of water. This is true even if the presence of water is almost negligible. The biocatalyst maybe present as a solid phase, for example as immobilised enzymes or cells, or as an individual cell the substrate may also constitute a solid phase. When necessary, gas is sparged into reactors to supply oxygen or a gaseous substrate and to remove carbon dioxide. Thus, heterogeneous systems with four phases involved are very typical cases. 

In some systems, for example, the immobilised enzyme is sandwiched between an external cellulose acetate membrane that blocks the passage of heavy molecules and an internal polycarbonate membrane that allows passage of the transformed products to the electrode. 

The production of H202 from this step can be monitored spectro-photometrically by the formation of a dye in the presence of peroxidase [49] disadvantages of this system include the use of carcinogenic dyes, lengthy incubation times and the need for laboratory-based equipment. As discussed in the previous section, SPCEs can be modified with mediators to produce effective H202 transducers when combined with immobilised enzymes, disposable biosensors ideal for de-centralised clinical analysis can be fabricated. The cholesterol molecule is nonpolar and thus provides an additional challenge to the development of... 

Phosphate is another anion which is of biological significance as well as being biologically active. Numerous phosphate biosensors have been developed, with one based on a multi-enzyme system immobilised on a cellulose membrane on a Pt electrode being able to detect levels down to 10 8 M [104]. Simpler methods, however, use enzymes such as polyphenol oxidase combined with alkaline phosphatase bound within an electropolymerised layer based on a substituted pyrrole [ 105]. This was reported to give a sensor with a detection... 

When choosing a support for an immobilised enzyme, what otirer factors (apart from activity and access to tire substrate) do you think need to be omside (Think about the cost of producing the imrrrobilised system and how it will be used). 

An enzyme-based Pz biosensor for the detection of organophosphorous and carbamate pesticides has also appeared [176]. The assay was based in the inhibitory effect of the pesticides on immobilised acetylcholinesterase. After exposure of the enzyme to the pesticides, the substrate 3-indoyl acetate was added. This was enzymatically converted to an insoluble product, the concentration of which was determined by the resulting frequency change. The rate and amount of conversion was due to the concentration of the active immobilised enzyme, which was dependent on the amount of pesticide present. The system was very similar to that used by Ebersole and Ward in their AMISA [66]. The authors measured paroxon to 5 x 10 M and carbaryl to 1 X 10- M. 

A further development of the wall-jet system is to insert a packed-bed electrode upstream of the jet to make a packed-bed wall-jet electrode (PBWJE) [5-7]. The packed bed can be used to generate reactant, to generate a fresh electrode surface or, if the bed has immobilised enzyme, to carry out an enzymatic reaction. The packed-bed wall-jet electrode is illustrated in Fig. 3. 

Another case of heterogeneous systems refers to immobilized enzymes. The kinetic behaviour of a bound enzyme can differ significantly from that of the same enzyme in free solution. The properties of an enzyme can be modified by suitable choice of the immobilisation protocol, whereas the same method may have appreciably different effects on different enzymes. These changes may be due to conformational alterations within the enzyme, immobilisation procedure, the presence and nature of the immobilisation support. The advantages of immobilised enzymes are for instance in reusability and possibility to use continuous mode. 

High-Pressine liquid Chromatography.- Reviews on the biomedical applications of h.p.l.c.-m.s., and immobilised enzyme reactors in h.p.l.c. detector systems, have included carbohydrate applications. 

Identify which of the following statements are true for immobilised biocatalysts, when compared to free enzyme or free cell systems. 

Polysaccharides can also be used to immobilise cells or enzymes, permitting the re-use of the catalyst and continuous flow systems. Alginates have the advantage that gel formation occurs under mild conditions, therefore cells remain viable and enzymes are not denatured but calcium gradually leaches out and the gel dissolves. Gellan or other combinations may prove superior for this application. 

Table 5.1 presents the intrinsic kinetic parameters (Km and Vln lx) for the free lipase system and apparent kinetic parameters (K and V ) for the immobilised lipase in the EMR using fixed 2g-l 1 lipase concentration. The immobilised lipase showed higher maximum apparent reaction rate and greater enzyme-substrate (ES) affinity compared with free lipase. 

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