Effective Cofactor Recycling

From the very beginning, scientists active in the field of biocatalysis wanted to promote the use of enzymes as powerful homogeneous catalysts for organic 

In 1991, the group of Willetts [13] published one of the first smart combinations of two redox enzymes for the oxidation of a secondary alcohol mediated by an alcohol dehydrogenase (ADH) from Thermoanaerobium brockii and the subsequent Baeyer-Villiger oxidation mediated by the cyclohexanone monooxygenase from Acinetobacter calcoaceticus NCIMB 9871) of the intermediate ketone 

In summary, many different cofactor recycling systems have been estabhshed. In vitro as well as in vivo systems display remarkable performance and they range from simple approach based on two single enzymatic to fusion-protein strategies. 

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Formate dehydrogenase in conjunction with polyethyleneglycol-immobilized nicotinamide adenine dinudeotide has been used to good effect as a cofactor recycle system (39). The alcohol dehydrogenase from Thermoanaerobium hrockii catalyzed the reduction of ketones independently when driven by the cooxidation of isopropanol (40,41). 

We have also investigated the use of artificial cells containing tyrosinase to carry out some metabolic functions of the liver [31]. Results in animals show that tyrosinase artificial cells, retained in extracorporeal shunts perfused by blood, can effectively lower the systemic blood tyrosine levels of liver failure rats. Artificial cells, to carry out other metabolic functions of the liver, are also being studied. For instance, using artificial cells containing multienzyme systems with cofactor recycling [32], we have studied the in-vltro conversion of ammonia sequentially into different types of amino acids. This way, ammonia... 

A further compHcation remains in the control of enzyme activities which to a large extent is dependent upon expression during the fermentation. One potential solution is to add supplementary enzymes to the cell-free extract. Such a precedent has already been set by a few reported cases where whole cells were mixed with the isolated enzyme for ex vivo cofactor recycle. E>espite these problems, there is no doubt that as genetic engineering for expression of the desired enzyme is improved, more systems wiU be tested in the cell-free environment [26]. At the very least it is clear that cell-free extracts, combined with network topology analysis can provide an excellent basis for effective analysis and targeting of the network so as to insulate the desired pathway from undesired enzymatic reactions [27]. 

Alternatively, 41 can be formed from glycerol by successive phosphorylation and oxidation effected by a combination of glycerol kinase and glycerol phosphate dehydrogenase, tvith an integrated double ATP/NAD+ cofactor recycling system [148]. 

For synthetic purposes hydroxypyruvate 119 can effectively replace the natural donor components [258]. Its covalent activation occurs at a reduced rate of about 4% relative to xylulose 5-phosphate (121) but is accompanied by spontaneous decarboxylation [262]. Thus, loss of carbon dioxide renders synthetic reactions irreversible whereas alternative donors, for example l-erythrulose, require coupling to cofactor recycling to shift the overall equilibrium [263]. The thermodynamic driving force from decarboxylation of 119 is particularly useful with equilibrating multi-enzyme systems such as that used in the gram-scale synthesis of two equivalents of 121 from 42 (Figure 5.54) [264]. 

For practical implementation the use of parallel cascades is of great value for cofactor recycle. For example, van Hecke and coworkers [45] report the synthesis of lactobi-onic acid from lactose using cellobiose dehydrogenase. The enzyme requires an electron acceptor, and using ABTS and laccase in a parallel cascade, such a system was effectively operated and modeled. 

Therefore, for preparative applications of redox enzymes, effective and simple methods for the continuous recycling of the active cofactors have to be available. In addition, such systems must be stable over long time periods and the separation of the product must be simple to render technical processes economically feasible. Until now, this problem has generally been solved by the application of a second enzymatic reaction (enzyme-coupled regeneration, Fig. 2). 

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