Multienzyme biocatalytic reactions

With a rapidly expanding biocatalytic toolkit that is capable of carrying out key chemical transformations in the discovery and development of pharmaceuticals (Table 16.1), combining multiple biocatalysts to carry out a series of transformations 

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Since enzymes generally function under the same or similar conditions, several biocatalytic reactions can be carried out in a reaction cascade in a single flask. Thus, sequential reactions are feasible by using multienzyme systems in order to simplify reaction processes, in particular if the isolation of an unstable intermediate can be omitted. Furthermore, an unfavorable equilibrium can be shifted towards the desired product by linking consecutive enzymatic steps. This unique potential of enzymes is increasingly being recognized as documented by the development of multienzyme systems, also denoted as artificial metabolism [15]. 

A biosynthetic multienzyme reaction of particular interest involves carbon dioxide fixation with the production of methanol [373, 374]. FDH catalyzes the reduction of carbon dioxide to formate, and methanol dehydrogenase (M DH) catalyzes the reduction of formate to methanol. Both of these enzymes require NAD+/NADH-cofactor, and in the presence of the reduced dimethyl viologen mediator (MV +), they can drive a sequence of enzymatic reactions. The cascade of biocatalytic reactions results in the reduction of GO2 to formate catalyzed by FDH, followed by the reduction of formate to methanol catalyzed by MDH. A more complex system composed of immobilized cells of Parococcus deni-trijicans has been demonstrated for the reduction of nitrate and nitrite [375]. 

Biocatalysis for drug discovery and development with an industrial perspective, and biocatytic cascade reactions with e integration of biocatalysts with one or more additional reaction steps, and multistep biocatalytic reaction sequences and multienzyme-catalyzed conversions, are presented. 

Multienzyme modular assemblies such as PKSs and NRPSs have flexible swinging tethers which channel covalently bound intermediates between successive active sites. Swinging arms plus specific protein-protein interactions offer mechanisms for the transfer of substrates between modules and offer concepts for the development of one-pot multi-reaction biocatalytic processes. 

Since the beginning of enzyme catalysis in microemulsions in the late 1970s, several biocatalytic transformations of various hydrophilic and hydrophobic substrates have been demonstrated. Examples include reverse hydrolytic reactions such as peptide synthesis [44], synthesis of esters through esterification and transesterification reactions [42,45-48], resolution of racemic amino acids [49], oxidation and reduction of steroids and terpenes [50,51], electron-transfer reactions, [52], production of hydrogen [53], and synthesis of phenolic and aromatic amine polymers [54]. Isolated enzymes including various hydrolytic enzymes (proteases, lipases, esterases, glucosidases), oxidoreductases, as well as multienzyme systems [52], were anployed. 

Microreactors are ideal for directing complex enzymatic synthesis, such as multienzyme catalysis and cascade reactions. There have been a grovdng number of studies [173-178] that represent the implementation of microreactions for multistep enzymatic catalysis and a list is presented in Table 10.5. Microfluidic biocatalytic... 

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