Biocatalyst Options

There are a several ways to classify multienzyme systems [5,11], but perhaps one of the most helpful is to divide the possibilities according to the action of the enzymes in the reaction. There are two main groups  

For a reaction network to be classified as multienzyme, two or more enzymes must be involved. Hence, schemes can be envisaged with 

Selecting the correct type of the biocatalyst is one of the more interesting challenges raised by the use of multiple enzymes in a single reaction scheme and the last few years have seen several new developments. From a conceptual 

0 C(3 and represents the primary enzymes that are involved in the direct 

X is a secondary enzyme, which is involved in the inhibitory co-product removal. For the reaction A, C, and G represent substrates, B intermediate, and F, Q, and I co-products. 

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A very simple and elegant alternative to the use of ion-exchange columns or extraction to separate the mixture of D-amino add amide and the L-amino add has been elaborated. Addition of one equivalent of benzaldehyde (with respect to die D-amino add amide) to the enzymic hydrolysate results in the formation of a Schiff base with die D-amino add amide, which is insoluble in water and, therefore, can be easily separated. Add hydrolysis (H2SQ4, HX, HNO3, etc.) results in the formation of die D-amino add (without racemizadon). Alternatively the D-amino add amide can be hydrolysed by cell-preparations of Rhodococcus erythropolis. This biocatalyst lacks stereoselectivity. This option is very useful for amino adds which are highly soluble in die neutralised reaction mixture obtained after acid hydrolysis of the amide. 

A biosorption method for the separation of sulfur compounds from fossil fuels, by using a sulfur-biosorption agent and followed by the oxidation of the biosorbed complex. The oxidation is carried out in an aqueous phase containing an effective amount of oxygen and, optionally a biocatalyst, in which case an incubating stage is incorporated for the reaction to take place. 

Clearly, most of the products in Table 4.1 are chiral compounds. None of the products is racemic, and only a few are achiral. The biocatalysts are (combinations) of enzymes oi cells. If the key enzyme has been indicated it may be used pure, partly purified in a cell-free extract, or in a whole cell. For each option, the biocatalyst may be used free oi immobilized. If the name of a microorganism has been indicated, usually several of its enzymes are active in the catalysis. The entries that are displayed in bold are treated in the case studies further on in this chapter, in the same order as in Table 4.1. 

Immobilization onto a solid support, either by surface attachment or lattice entrapment, is the more widely used approach to overcome enzyme inactivation, particularly interfacial inactivation. The support provides a protective microenvironment which often increases biocatalyst stability, although a decrease in biocata-lytic activity may occur, particularly when immobilization is by covalent bonding. Nevertheless, this approach presents drawbacks, since the complexity (and cost) of the system is increased, and mass transfer resistances and partition effects are enhanced [24]. For those applications where enzyme immobilization is not an option, wrapping up the enzyme with a protective cover has proved promising [21]. 

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. 

Mode of immobilization. Immobilization can be effected either chemically, by covalent bonding of the biocatalyst on a surface (Figure 5.6, option 1), by adsorption, or by ionic interactions between catalyst and surface (option 2), as well as by cross-linking of biocatalyst molecules for the purpose of enlargement (option 3), or physically by encapsulation in matrices or by embedding in a membrane (option 4). 

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. 

A membrane can be generated by polymerization around a few biocatalyst molecules which surround a space of a few hundred micrometers (microencapsulation Figure 5.6, option 5), or it can be of macroscopic dimensions (Figure 5.6, option 6). In the latter case, membrane reactors can be classified according to (i) driving force, (ii) pore structure and (iii) pore size. 

Choice of process options for the successful integration of biocatalytic and chemical synthesis steps is heavily dependent on the type of chemical reaction used prior to or subsequent to the biocatalytic step and also on the robustness of the biocatalyst to inactivation by components of the chemical step. The use of organic solvent or highly reactive chemical reagents before subsequent conversion using a biocatalyst is usually expected to require the complete purification of the product of the chemical step. There are many examples in the literature where such an approach has been applied,... 

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. 

Cao et al. [60-62] examined a fractionation option that used corn cob and aspen woodchip as the substrates. In this biomass fractionation scheme (Fig. 6), the majority of lignin, alkaline extractives, and acetate were solubilized and separated from cellulose and hemicellulose fractions by alkaline treatment. Hemicellulose was then hydrolyzed to its sugar constituents with dilute acid (0.3 M HCl). Hemicellulose carbohydrates were then fermented to ethanol by a xylose-fermenting yeast strain (Fig. 7). The cellulose fraction, after separation from lignin and hemicellulose, was used as the substrate in the SSF process for ethanol production using a thermotolerant yeast strain as the biocatalyst (Fig. 8). 

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