Biocatalyst retention

In a biodesulfurization process, there are actually three phases. For a liquid mixture containing the three phases - liquid fossil fuel, water, and the biocatalyst, more than one filter would be required. One filter will preferentially collect either the liquid fossil fuel or aqueous phase as the filtrate. The retentate will then flow to the second filter, which will collect the component not removed before. The remaining retentate, containing the biocatalyst, can then, preferably, be recycled. The process can be used to resolve an emulsion or microemulsion of the liquid fossil fuel and aqueous phase resulting from a... 

US5525235 [44] separating a petroleum containing emulsion phase liquid mixture, using wet filters. The liquid mixture contains a fossil fuel, an aqueous phase and a biocatalyst. The first filter is wetted with an agent miscible with the fossil fuel but immiscible with the aqueous phase. The second filter is wetted with a wetting agent miscible with the aqueous phase but immiscible with the fossil fuel. The mixture is then passed sequentially for each filter. The fossil fuel is recovered from the final filtrate and the biocatalyst is retained in the aqueous phase of the final retentate. 

The main task in technical application of asymmetric catalysis is to maximize catalytic efficiency, which can be expressed as the ttn (total turnover number, moles of product produced per moles of catalyst consumed) or biocatalyst consumption (grams of product per gram biocatalyst consumed, referring either to wet cell weight (wcw) or alternatively to cell dry weight (cdw)) [2]. One method of reducing the amount of catalyst consumed is to decouple the residence times of reactants and catalysts by means of retention or recycling of the precious catalyst. This leads to an increased exploitation of the catalyst in the synthesis reaction. 

Membrane reactors allow a different option for the separation of biocatalysts from substrates and products and for retention in the reactor. Size-specific pores allow the substrate and product molecules, but not the enzyme molecules, to pass the membrane. Membrane reactors can be operated as CSTRs with dead-end filtration (Figure 5.5e) or as loop or recycle reactors (Figure 5.5f) with tangential (crossflow) filtration. 

Immobilization influences the activity and stability of biocatalysts much more than encapsulation by membranes however, enhanced activity (rarely) or stability (often) can be an important reason to pick immobilization for the retention of enzymes. 

Membrane reactors became an option for the retention of biocatalysts when the processing of membrane materials had progressed sufficiently to control thickness and pore structure and to manufacture a membrane that was defect-free. Besides its function as a retainer the membrane also serves other functions such as (i) to stabilize the phase boundary in case of multi-phase reactions (ii) as a consequence of (i), to transport dissolved 02 preferentially over gaseous 02 and (iii) to support purification and sterilization of air and other nutrients in fermentations. 

Conventional filters, such as a coffee filter, termed depth filters , consist of a network of fibers and retain solute molecules through a stochastic adsorption mechanism. In contrast, most membranes for the retention of biocatalysts feature holes or pores with a comparatively narrow pore size distribution and separate exclusively on the basis of size or shape of the solute such membranes are termed membrane filters . Only membrane filters are approved by the FDA for sterilization in connection with processes applied to pharmaceuticals. Table 5.3 lists advantages and disadvantages of depth and membrane filters. 

In the preceding section, we analyzed an immobilized enzyme process and calculated some important parameters such as productivity. In this section, we investigate another process configuration for retaining biocatalysts, the membrane reactor. The advantages and disadvantages of immobilization and membrane retention have already been discussed in Chapter 5. As in the case of immobilization, retention of catalyst by a membrane vastly improves biocatalyst productivity, a feature important on a processing scale but usually not on a laboratory scale. 

The membrane has the premier function in the process of biogenesis. It allows for individual ownership and retention of biocatalysts, and thereby for up to a million fold increases in catalytic activity. Substrate/enzyme ratios in cells may approach unity and thus enzymes can actually change the equilibrium of some reactions. Clearly, membranes are essential and the hurdle for nascent life is the need for a selectively permeable membrane... that means a membrane that contains, suspended in its lipid layers, the first communication proteins.13,14 The cell must breathe at once if there is to be any future and that again equalizes units from different clones. Is it surprising then that all life forms have membranes Shapeless wafting life is a thing of poor science fiction. Membrane formation is the moment when life became competitive, it... 

Asymmetric hollow fibers provide an interesting support for enzyme immobilization, in this case the membrane structure allows the retention of the enzyme into the sponge layer of the fibers by crossflow filtration. The amount of biocatalyst loaded, its distribution and activity through the support and its lifetime are very important parameters to properly orientate the development of such systems. The specific effect that the support has upon the enzyme, however, greatly depend upon both the support and the enzyme involved in the immobilization as well as the method of immobilization used. 

When looking for an economically feasible enzymatic system, retention and reuse of the biocatalyst should be taken into account as potential alternatives [98, 99]. Enzymatic membrane reactors (EMR) result from the coupling of a membrane separation process with an enzymatic reactor. They can be considered as reactors where separation of the enzyme from the reactants and products is performed by means of a semipermeable membrane that acts as a selective barrier [98]. A difference in chemical potential, pressure, or electric field is usually responsible from the movement of solutes across the membrane, by diffusion, convection, or electrophoretic migration. The selective membrane should ensure the complete retention of the enzyme in order to maintain the full activity inside the system. Furthermore, the technique may include the integration of a purification step in the process, as products can be easily separated from the reaction mixture by means of the selective membrane. 

Products synthesized by the parent plant, in a variety of cell types and throughout the development of the plant, are made in culture under a range of conditions and considerable scope exists for improving the productivity of such cultures. Development of stable plant cell lines of sufficiently high productive capacity on which to base commercial processes remains an important problem. Immobilization of plant cells within a support matrix appears to offer both bioengineering and biochemical advantages compared with free cells. These include ease of use in a continuous process with retention of biomass reuse of biocatalyst (cells), cofactors or precursors protection of cells from mechanical stresses and superior productivity and longevity of cells. 

Advances in genetic and chemical enz)me modifications, enzyme immobilisation and enzymatic reactions in organic solvents, have increased the actual use and potential of enzymes in the production of industrial chemicals. Enzyme immobilisation, in particular, has proved to be a valuable approach to the use of enz5mes in chemical synthesis. The term denotes eirzymes that are physically confined or localised in a defined region in space with retention of their catalytic activities. A detailed consideration of immobilisation techniques is beyond the scope of this chapter the subject is covered adequately in the BKDTOL text entitled Technological Applications of Biocatalysts. ... 

Biocatalyst costs are often an important factor in the overall cost of the product. One of the most important reasons to consider the immobilization of a biocatalyst is therefore the possibility of facilitated reuse or continuous utilization. This enables prolonged use of the biocatalyst and can thus significantly reduce the process costs. Furthermore, the stability of biocatalysts, in particular of enzymes and recombinant cells, can be improved dramatically in many cases by immobilization. Immobilization also creates the possibility of using the biocatalyst in a packed-bed or fluidized-bed reactor. Due to easy retention of the immobilized biocatalyst in the reactor, high volumetric activities can be realized and, in case of immobilized growing cells, the operation can be under wash-out conditions with respect to free cells. Obviously, the extra costs associated with the immobilization must be earned back by the possibility of a more efficient use. Many techniques are available for immobilizing biocatalysts [3] some of the more useful are discussed in Chapter 9. 

Another type of stability of immobilized biocatalysts is the retention of activity after periodic use in batch processes, as has been reported previously for penicillin acylase entrapped in polyacrylamide gel [40]. This option can be used to advantage for rapid monitoring of biocatalyst activity under conditions of industrial application. Apart from the measurement of activity as an indication of the necessity to replace the biocatalyst, the periodic analysis of the variation of kinetic properties permits greater insight into deviation from the optimal parameters. 

Both chemical and physical methods may be used to immobilize biocatalysts while retaining or modifying their activity, selectivity, or stability. Among the techniques used for immobilization of enzymes are physical adsorption, covalent bonding, ionic binding, chelation, cross-linking, physical entrapment, microencapsulation, and retention in permselective membrane reactors. The mode of immobilization employed for a particular application depends not only on the specific choice of enzyme and support, but also on the constraints imposed by the microenvironment associated with the application. 

Figure 8.47 Stereochemistry in Catalysis. Biocatalyst stereochemistry may be followed with chiral substrates to reveal stereochemical changes during catalysis. Substrates include, chiral acetylCoA and phosphate (that undergo inversion or retention of configuration with reaction), and chiral NADH(D) designed to demonstrate whether prochiral (proR or proS) are employed in reduction. mMDH uses the proR hydrogen as illustrated by deuterium (D) transfer to the product (bottom). D, deuterium T, tritium.

The preparative synthesis took place in stirred batch reactors of 200-500 mL volume, with a sintered plate at the bottom, allowing the retention of the immobilized enzyme after discharge of the reacted medium. The reactions were performed in organic solvent (ethyl acetate or acetonitrile) at controlled initial water activity (a = 0.1). For KCS reactions, only a very small amount of water is produced so that its concentration can be considered constant during the reaction. After addition of the biocatalyst (previously equilibrated) and substrates, the water content was... 

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