Multienzymatic processes

Synthesis of poly(catechol) was demonstrated by multienzymatic processes (Fig. 1) [13]. Aromatic compounds were converted to catechol derivatives by the catalytic action of toluene dioxygenase and toluene cis-dihydrodiol dehydrogenase, followed by the peroxidase-catalyzed polymerization to give the polymer with molecular weight of several thousands. 

Recently, there have been new technologies described for multienzymatic processes and tools to evaluate them [71,72]. These approaches should also be incorporated in the evaluation of multienzymatic cascades, as used for cofactor regeneration in many isolated enzyme reductions. 

Multienzymatic processes, which involve the use of two or more enzymes in a defined reaction pathway, are becoming very attractive for the production of many compounds at an industrial level [1-3]. 

Scheme n.l Representative multienzymatic processes, (a) Linear (or sequential) reactions. 

In the case of linear multienzymatic processes, this approach can be chosen to improve the process productivity, for example, by driving reversible reactions to completion or reducing the accumulation of unstable intermediates. 

Instead, the orthogonal multienzymatic reactions are always cascade processes by definition. However, this type of multienzymatic processes has been largely investigated in the past especially for the development of enzymecofactor regeneration systems. These studies not only allowed the wide exploitation of cofactor-dependent enzymes, such as NAD(P)H-dependent dehydrogenases, by making their reactions economically feasible but were also useful in identifying relevant process options for the development of effective multienzymatic reaction systems (3). 

Therefore, this chapter will focus on multienzymatic processes where at least one of the coupled reactions is a biocatalyzed reduction, apart from the necessary cofactor regeneration reactions. 

However, when biocatalysts showing a sufficient cofactor specificity are not naturally available, possible undesired interferences between the cofactor regeneration systems can be circumvented by different approaches still maintaining the one-pot fashion of the multienzymatic process. For example, in the biocatalyzed synthesis of 12-ketoursodeoxycholic acid, the performances of the investigated cascade system, in which five enzymes were involved in concurrent oxidation and reduction reactions at different sites of the starting substrate cholic acid, were significantly improved by simple compartmentalization of the oxidative and reductive enzymes in two membrane reactors (Scheme 11.4b) [11]. 

Several multienzymatic processes employing transaminases have been developed for the production of enantiopure amines and amino acids. 

For example, a sequential one-pot multienzymatic process was recently exploited for the preparation of optically pure (3-hydroxy carboxylic acids (Scheme 11.16) [38]. 

After incubation of P-halo ketones with these genetically modified cells, addition of sodium azide resulted in most cases in the full conversion of the starting materials into the expected azido alcohols, isolated yields being comparable with those obtained with isolated enzymes. Moreover, this multienzymatic process was combined with a subsequent click reaction, that is, Cu(I)-catalyzed [2 + 3]-dipolar cycloaddition, allowing the one-pot preparation of the corresponding enantiopure P-hydroxytriazoles (Scheme 11.17b). 

Compartmentalization is a concept that can be used widely in multienzymatic processes whereby different parts of the reactor operate imder different conditions (e.g., two-liquid phase biocatalysis) or catalysts are separated (e.g., by immobilization). The two compartments may be separated by a phase boundary (most likely solid-liquid or liquid-liquid). The compartments will selectively contain enzymes and reaction components such that not all enzymes and components are present in all parts of the reactor at the same concentration at a given time. This has benefits not only for the reaction itself (e.g., reducing product inhibition) but also downstream processing (e.g., separation of enzymes). 

TABLE 20.2 Conditions Required for Different Degrees of Integration for Multienzymatic Processes ... 

Inspired by its natural function, RibA has been applied in a multienzymatic commercial process for the production of different purine or pyrimidine containing deoxyribonucleosides such as (115) in good yield (Figure 10.42) [184,185]. 

The development of this multienzymatic system for the production of D-amino acids from any D,L-5-monosubstituted hydantoin allows the hydantoinase process to produce not only two amino acids, such as D-phenylglycine and D-p-hydroxy-phenylglycine (as explained at the beginning of the chapter), but also many non-natural D-amino acids that could be components of potential pharmaceuticals. 

Figure 1.8 shows the synthesis of fatty acids. This complex process is catalysed by the multienzymatic complex, fatty acid synthetase. This enzyme uses as substrates acetyl-coA and malonyl-coA to produce palmitic acid. Afterwards, palmitic acid, a saturated fatty acid of 16 carbon atoms, can be used to produce other fatty acids (Ratledge and Evans 1989). Fatty acids with more carbon units, such as estearic acid, are obtained by elongation of palmitic acid. 

CLEAs are made by the traditional protein methods of precipitation (e.g., solvents, salting out, etc.), but not full purification is required.The main features of CLEAs are the combination of easy procedures, low cost of protein processing, and robustness of the biocatalyst, which is required for the development of a biocatalyzed industrial process. This strategy has been demonstrated useful for multimeric enzymes. An interesting approach is to develop an enzymatic cascade based on many steps, i.e.,multienzymatic biotransformation process in just single CLEAs, or in noncascade type, which are named as combi-CLEAs. The vast potential of CLEAs at industrial level allows to explore this technolc for many appHcations in the food industries such as vinification, or citric juice clarification, or for medical purposes Hke scar debriding or cystic fibrosis appHcations. ... 

In plants, phenolics are mainly synthesized in close relationship with the ER through metabolic channeling processes. The phenolic skeleton is built by multienzymatic complexes... 

Nature created multienzymatic systems to accomplish extremely efficient one-pot tandem catalysis. As in an assembly line, tens of enzymes are well organised to transform simple materials to complex molecules with perfect control of selectivity hy a series of coupled reactions in the cell. It has long been chemists endeavor to extend such coordinated catalytic action to artificial processes to make synthetic chemistry more sustainable. Nowadays, owing to the resource-intensive nature of the current synthetic industry, the development of tandem one-pot reactions, avoiding the use of costly and time-consuming protection-deprotection processes as well as purification procedures of intermediates, has become especially important and valuable because society is confronted with bottle-neck problems such as energy and time shortage and environmental pollution. 

The fact that no growth is observed under these conditions enables us to conclude that an intermediate hydroxylation is very unlikely. However, this hypothesis cannot be completely excluded since the conversion of dethiobiotin into biotin, which is certainly a multistep process, could involve a multienzymatic complex carrying out the whole transformation or part of it without releasing the intermediates (or incorporating a free intermediate from the medium). This point is of course very difficult to check. 

Multienzymatic one-pot processes can be designed in different configurations (Scheme 11.1). The most common systems are those constituted by linear (or sequential) reactions, where a starting substrate is converted into a desired product via one or more intermediates by action of different enzymatic activities, and... 

One-pot processes based on linear multienzymatic reactions are in most cases more easily developed than those based on the orthogonal ones. In fact, they can also be carried out by sequential addition of reagents and/or biocatalysts to the reaction mixture, as long as the process is performed in the same reaction vessel and without isolation of the intermediate products. 

Other Examples of Multienzymatic Cascade Processes, Including Bioreductive Reactions... 

A great effort has also been made for the development of multienzymatic cascade processes finalized to the preparation of enantiopure epoxides, important chiral synthones for the synthesis of drugs and biologically active compounds [2]. 

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