Biocatalyst immobilisation

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

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 ... 

True Hie concentration of immobilised biocatalyst in a reactor can be much greater than for systems using soluble of free cells. 

In fermentation for the production of acetic acid, ethyl alcohol is used in an aerobic process. In an ethanol oxidation process, the biocatalyst Acetobacter aceti was used to convert ethanol to acetic acid under aerobic conditions. A continuous fermentation for vinegar production was proposed for utilisation of non-viable A. aceti immobilised on the surface of alginate beads. 

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). 

This work deals with the preparation and study of a biocatalyst prepared by immobilising a commercial pectinase on a tailor made support, and its use in a... 

Several scouting experiments were performed to find the best pH conditions. Figure 3 reports the ratio between the PG specific activity measured after the purification procedure (ASf) and the initial PG specific activity (ASi). At pH 3.5, the microspheres are able to remove from the broth the major part of the protein without PG activity, thus providing a four time increase of the enzyme specific activity. The purified PG from Kluyveromyces marxianus was immobilised following the above procedure. Batch reactions in the packed bed reactor were done to evaluate the biocatalyst stability. After an initial loss, due to enzyme release, the residual PG activity reaches a plateau value corresponding to about 40% of the initial activity. Probably, some broth component interfered during the immobilisation reaction weakening the protein-carrier interactions. 

The pectinase was supported on y-alumina and the three enzymes present in the pectinase sample were found still active after the immobilisation. The supported biocatalyst was used in several reaction cycles to perform consecutive depectinisations of a cloudy apple juice with a negligible loss of biocatalytic activity. 

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. 

A single enzyme, L-aspartate ammonia lyase obtained from E. coli is used acting on ammonium fumarate substrate. Little cell activity was lost upon immobilisation. Initially polyacrylamide was used as the immobilisation medium, and later cross-linked K-carrageenan was used, as higher operational life-times for the biocatalyst were obtained. The immobilized cell activity is very stable with a half-life of 120 days, while achieving 95% conversion of substrate into product. 

Immobilisation of biocatalyst to allow its reuse or continuous use. L-aspartic acid, LIFCS, L-ferf-leucine... 

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. 

Fig. 5.49. Dimensionless plot of overall reaction rate against bulk substrate concentration for a surface immobilised biocatalyst...

The rate of reaction of a surface-immobilised biocatalyst may be determined graphically, as shown in Fig. 5.52. The curve marked is that relating the substrate concentration to the rate of reaction when there is no diffusional... 

In the case of gel entrapped biocatalysts, or where the biocatalyst has been immobilised in the pores of the carrier, then the reaction is unlikely to occur solely at the surface. Similarly, the consumption of substrate by a microbial film or floe would be expected to occur at some depth into the microbial mass. The situation is more complex than in the case of surface immobilisation since, in this case, transport and reaction occur in parallel. By analogy with the case of heterogeneous catalysis, which is discussed in Chapter 3, the flux of substrate is related to the rate of reaction by the use of an effectiveness factor rj. The rate of reaction is itself expressed in terms of the surface substrate concentration which in many instances will be very close to the bulk substrate concentration. In general, the flux of substrate will be given by ... 

Fig. 5.53. The overall rate or reaction for uniformly distributed immobilised biocatalyst...

Volume of first and second vessels Liquid volume Enzyme velocity constant Maximum enzyme velocity constant in unprotonated form Initial enzyme velocity constant Enzyme velocity constant based on unit volume of immobilised biocatalyst Maximum rate of reaction involving substance S Maximum rate of reaction involving substance P Specific rate of generation of biomass fraction Biomass concentration Initial or feed biomass concentration Average biomass concentration Concentration of prey Concentration of predator Biomass concentration at optimum dilution rate... 

From the previous paragraph it can be discerned that the half-life of a biocatalyst is determined not only by the deactivation properties of the free enzyme but also by the mass-transfer properties of an immobilisate. Figure 19.7 reveals the factor by which the dependence on temperature increases operating stability in the case of immobilized enzyme. 

Gekas VC (1986) Artificial membranes as carriers for the immobilisation of biocatalysts. Enzyme Microb Technol 8 450 -61... 

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