Multienzymatic cascades

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. 

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

Along with the multienzymatic cascades presented in the previous sections, further systems using either ketoreductases or ADHs for the reductive step together with different enzymatic activities have been recently investigated and are worth mentioning. 

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]. 

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. ... 

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). 

Further investigations allowed the selection of ADHs with sufficiently high cofactor preference for performing the deracemization reactions toward enantio-pure (S)- or (R)-alcohols in cascade systems (Scheme 11.4a) [10]. The feasibility of this simultaneous multienzymatic transformations was demonstrated using a set of 10 different racemic secondary alcohols as substrates. 

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]. 

Another interesting example of a redox neutral cascade has been proposed for the multienzymatic synthesis of (JJ)-3-fluorolactic acid together with the resolution of racemic 3-fluoroalanine (Scheme 11.5b) [13]. Optically enriched (S)-3-fluoroalanine (88% ee) was recovered unreacted after the enantioselective oxidative deamination of the racemic substrate catalyzed by the L-alanine dehydrogenase (i-AlaDH) from Bacillus subtilis. This oxidative reaction, which is thermodynamically unfavorable, was driven by the coupled reduction reaction of the intermediate 3-fluoropyruvate catalyzed by rabbit muscle i-lactate dehydrogenase (L-LDH). Since both enzymes are NADH dependent, this coupled... 

Using these two groups of multienzymatic systems as a basis, tiiere have been several attempts to classify such cascade reaction schemes [19, 24]. Three basic cascade schemes can be distinguished, namely, (1) linear, (2) parallel and cyclic, and (3) orthogonal. The motivation for implementahon in each case is a little different ... 

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