Enzymes immobilised

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

Malic add has a limited use in the food industry as an addifying agent where it is an alternative to dtric add. In nature, only L(-) malic add is found whereas the relatively cheap, chemical synthetic methods yield D/L mixtures. The favoured industrial way to produce the L(-) add is by enzymic transformation from fumaric add. Either whole cells or isolated and immobilised enzymes can be used, with high conversion effidendes. 

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. 

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 ... 

The introduction of immobilised enzymes has several advantages over the chemical de-acylation of fl-lactam. List as many as you can. (You may need to refer back to Figures 6.13-6.16 and the associated text... 

Another feature of this particular exopolysaccharide is that gel strength depends upon the temperature used. It is constant between 60-80°C, increasing in strength from 80-100°C and finally changing structure from a single to a triple stranded helix at temperatures over 120°C. This makes it particularly well suited for use as a molecular sieve, immobilised enzyme support and a binding agent. 

As well as being active, the immobilised enzyme also needs to be stable (active for a long period) and the support must promote this. The support must also have appropriate mechanical characteristics it should not disintegrate if used in a stirred tank reactor it should produce even flow (without channelling) in a packed bed reactor. Hie cost of the support is also important. 

In this case study, an enzymatic hydrolysis reaction, the racemic ibuprofen ester, i.e. (R)-and (S)-ibuprofen esters in equimolar mixture, undergoes a kinetic resolution in a biphasic enzymatic membrane reactor (EMR). In kinetic resolution, the two enantiomers react at different rates lipase originated from Candida rugosa shows a greater stereopreference towards the (S)-enantiomer. The membrane module consisted of multiple bundles of polymeric hydrophilic hollow fibre. The membrane separated the two immiscible phases, i.e. organic in the shell side and aqueous in the lumen. Racemic substrate in the organic phase reacted with immobilised enzyme on the membrane where the hydrolysis reaction took place, and the product (S)-ibuprofen acid was extracted into the aqueous phase. 

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... 

It is well known that pine enzymes change then behaviour and stability when they are immobilised. In the past two decades the immobilisation of microorganisms, cells and parts of cells has gradually been introduced into microbiology and biotechnology. The cell immobilisation techniques are modifications of the techniques developed for enzymes. However, the larger size of microbes has influenced the techniques. As for immobilised enzymes, two broad types of method have been used to immobilise microorganisms attachment to a support and entrapment. 

In many biological processes, e.g. the fermentation of cells and sensitive microorganisms or bioconversion with immobilised enzymes, low shear stress is of crucial importance for the optimal coimse of processes. 

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. 

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). 

As a measure of particle stress the particle diameter, or the enzyme activity in the case of immobilised enzymes, can be used. 

Since in the case of using immobilised enzymes (acylase enzyme resin [27,45, 47]), normally only a small decrease in the particle diameter leads to a distinct decrease in the enzyme activity, the more sensitive enzyme activity has to be taken as the measure of stress. 

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. 

For bio-transformation processes, immobilised enzymes are often used because their activity persists over a longer period of time than that of free enzymes. The reduction of enzyme activity in enzymatic reactors is a consequence of energy dissipation by sparging and stirring, which is required for instance for oxygen transport or realisation of constant reaction conditions as regards temperature and pH. In the other hand low and high pH-values leads also to a decrease of enzyme activity and increase the stress sensitivity. 

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). 

Carr P.W., Bowers, L.D., Immobilised Enzymes in Analytical and Clinical Chemistry. Fundamentals and Applications, Wiley-Interscience, New York (USA), 1980. 

G.L. Turdean, I.C. Popescu, L. Oniciu, and D.R. Thevenot, Sensitive detection of organophosphorus... Thiocholine electrochemistry and immobilised enzyme inhibition. J. Enzyme Inhi. Medicinal Chem. 17, 107-115 (2002). 

Monsan P (1982) Les methodes immobilisation enzymes. In Durand G, Monsan P (eds) Les enzymes, productions utilizations industrielles. Gauthier-Villards, Paris, pp 81-118... 

For some recent reviews, see Pfenosil, J.E., Kut, O.M., Dunn, I.J. and Heinzle, E., Immobilized biocatalysts. In Ullman s Biotechnology and Biochemical Engineering, vol. 2. Wiley-VCH, Weinheim, 2007, pp. 683-734 Sheldon, R. A., Enzyme immobilization the quest for optimum performance. Adv. Synth. Catal., 2007, 349, 1289-1307 End, N. and Schoning, K.-U., Immobilized biocatalysts in industrial research and production. Topics Curr. Chem., 2004, 242, 273-317 Bornscheuer, U.T., Immobilizing enz3mies how to create more suitable biocatalysts. Angew. Chem. Int. Ed., 2003,42, 3336-3337 Cao, L. Immobilised enzymes science or art Curr. Opin. Chem. Biol., 2005, 9, 217-226. 

Particulate fluidization, where the fluidizing medium is usually a liquid, is characterised by a smooth expansion of fhe bed. Liquid-solid fluidized beds are used in continuous crystallisers, as bioreactors in which immobilised enzyme beads are fluidized by the reactant solution and in physical operations such as the washing and preparation of vegetables. The empirical Richardson-Zaki equation (Richardson... 

The reaction in a homogeneous solution with a polar organic solvent in which the enzymes and substrates are both soluble, occurs often at the expense of the enzyme stability [4, 5]. Besides immobilised enzymes in organic solvents [6], emulsion reactors, especially enzyme-membrane-reactors coupled with a product separation by membrane based extractive processes [7-9] and two-phase membrane reactors [10-12], are already established on a production scale. 

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