Coupled enzyme approach

Figure 5.9 NAD+/NADH cofactor regeneration a the coupled enzyme approach b the coupled substrate approach c, d examples [42,43] of the two approaches.

Coupled-Enzyme Approach. The use of two independent enzymes is more advantageous (Scheme 2.112). In this case, the two parallel redox reactions - i.e., conversion of the main substrate plus cofactor recycling - are catalyzed by two different enzymes [721]. To achieve optimal results, both of the enzymes should have sufficiently different specificities for their respective substrates whereupon the two enzymatic reactions can proceed independently from each other and, as a consequence, both the substrate and the auxiliary substrate do not have to compete for the active site of a single enzyme, but are efficiently converted by the two biocatalysts independently. 

Recently [63], we studied the behavior of two-enzyme system catalyzing two consecutive reactions in a macroheterogeneous medium (modified Lewis cell). The system consisted of lipase-catalyzed hydrolysis of trilinolein and subsequent lipoxygenation of liberated fatty acids (Fig. 3). Our approach compared the kinetic behavior of coupled enzymes in the Lewis cell with the sequential study of separated phenomena presented before ... 

Biocatalytic approaches to cofactor regeneration can be divided into coupled-enzyme methods and coupled-substrate methods.In the coupled-enzyme method, the oxidized cofactors (NAD+ and NADP+) are recycled in situ by performing an oxidation reaction using a second enzyme and an inexpensive auxiliary substrate. This second enzyme must employ the same cofactor, but neither enzyme should be able to accept the same substrate. 

Yang and Schulz also formulated a treatment of coupled enzyme reaction kinetics that does not assume an irreversible first reaction. The validity of their theory is confirmed by a model system consisting of enoyl-CoA hydratase (EC 4.2.1.17) and 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) with 2,4-decadienoyl coenzyme A as a substrate. Unlike the conventional theory, their approach was found to be indispensible for coupled enzyme systems characterized by a first reaction with a small equilibrium constant and/or wherein the coupling enzyme concentration is higher than that of the intermediate. Equations based on their theory can allow one to calculate steady-state velocities of coupled enzyme reactions and to predict the time course of coupled enzyme reactions during the pre-steady state. 

Another approach of using fluorimetric assays for screening purposes is the use of coupled enzyme systems. McElroy et al. presented an assay for glutamate... 

In a different approach, the hydrolase-catalyzed kinetic resolution of chiral acetates was studied using a high-throughput ee assay also based on an enzyme-coupled test, the presence of a fluorogenic moiety not being necessary [16]. The assay is based on the idea that the acetic acid formed by hydrolysis of a chiral acetate can be transformed stoichiometrically into NADH in a series of coupled enzyme reactions using commercially available enzyme kits (Fig. 9.10). The NADH is then... 

Photosensitized regeneration of an oxidized cofactor, i.e. NAD(P)+ or a reduced cofactor NAD(P)H, could provide general routes for oxidative or reductive biocatalyzed photosynthetic transformations (see Sect. 3.2.3). Coupling of the regenerated NAD(P)+/NAD(P)H cofactors to enzymes that depend on these cofactors, opens a broad array of feasible photo-biocatalytic syntheses. An alternative approach to couple enzymes as catalysts for artificial photosynthetic... 

For the production of sulcatol (3, 6-methyl-5-hepten-2-ol). 71 brockii alcohol dehydrogenase has been used as a soluble enzyme, immobilized onto solid supports (PAN or Eupergit)26 113 237 and, recently, in a continuously working membrane reactor250. Generally the coupled substrate approach is used. 

The application of competition schemes realized by coupled enzyme reactions also provides access to analytes not determinable with usual enzyme electrodes. Here the analytical information is gained either from the competitive action of two enzymes, of which one produces the signal, on the same substrate or from the competition of the analyte with the substrate for the same (signal generating) enzyme, the latter approach resembling that of inhibitor determination. 

These two approaches require different apparatus. Steady-state kinetics usually requires only the apparatus of the routine biochemical laboratory. Most steady-state studies are spectrophotometric. If the reaction does not involve a convenient absorbance change, it may be necessary to use other methods of measurement, but often it is possible to obtain an absorbance change by using coupling enzymes or secondary reactants. Rapid-reaction studies require more sophisticated equipment for precise and rapid mixing of enzymes with reactant solutions and for synchronous rapid recording of the appropriate signal. 

The use of immobilised cells for industrial and analytical enzymic processes is prophetically advantageous, the problems of isolation of the enzyme(s) and separation of the enzyme(s) from the product being avoided. However, the majority of the reaction currently used for direct enzyme immobilisation would cause cell death if applied to cells. Our approach has been based on the ability of water-insoluble metal hydroxides to chelate and retain peptides, proteins, etc. including enzymes. From various studies it was concluded that gelatinous titanium and zirconium hydroxide matrices are effective matrices for enzyme etc. immobilisation. Their advantages include low cost, convenient preparation (which may be conducted in any location without specialised facilities), the absence of any need for pre-preparation, ability to couple enzyme at neutral pH, the high retentions of specific activity of the enzyme on immobilisation, and the ability of modification to exert microenvironmental effects on and thereby alter the characteristics of the immobilised enzyme. 

YADH and HLADH are less useful for the asymmetric reductirai of open-chain ketones, but this gap is efficiently covered by a range of alcohol dehydrogenases from mesophiUc bacteria, such as Rhodococcus (ADH-A) and Lactobacillus (LBADH, LKADH), and thermophilic Thermoanaerobacter [802] and Thermo-anaerobium (TBADH) strains (Scheme 2.119) [296, 803-806]. Some of these enzymes are remarkably thermostable (up to 85°C) and can tolerate the presence of organic solvents such as /sopropanol, which serves as hydrogen-donor for NADP-recycling in a coupled-substrate approach [807-809]. 

In an enzymatic assay, spectrophotometric or electrochemical determination of the reactant or the product is the preferred approach. When this is not applicable, the determination is performed by a coupled enzyme assay. The coupled reaction includes an auxiliary reaction in which the food constituent is the reactant to be converted to product, and an indicator reaction which involves an indicator enzyme and its reactant or product, the formation or breakdown of which can be readily followed analytically. In most cases, the indicator reaction follows the auxiliary reaction ... 

The development of kinetic schemes for sequences of enzyme reactions contributes to the resolution of two problems. The first of these, the more complex one, concerns the study of the control of metabolic pathways and has been of major interest to biochemists for some time. Two related approaches to the problem have been developed for the interpretation of the behaviour of large assemblies of coupled enzyme reactions. Models can be made which contain the differential equations for the progress of the reactions for all the enzymes of a system. The numerical solutions of this set of equations can be compared with the experimental data for the concentrations of intermediates and their rates of change. Iterative improvements of the model can then be made. Alternatively, if data are only available for a... 

ADH from Lactobacillus brevis (IBADH) was used for the synthesis of a statin side chain, used as an alternative key intermediate in the synthesis of atorvastatin (Figure 13.2). In this variant, the NADPH-dependent enzyme was highly regio-and stereoselective, reducing fert-butyl 6-chloro-3,5-dioxohexanoate to tert-butyl (S)-6-chloro-5-hydroxy-3-oxohexanoate in 72% yield and with >99.5% ee. The cofactor was recycled in a coupled-substrate approach [1], where isopropanol was concomitantly oxidized to acetone at the expense of NADP, thereby driving the reaction toward product formation. Crude cell extract of recombinant LBADH expressed in E. coli was used in a fed-batch system on 8 1 scale, and a TTN of 2x10 could be calculated for IBADH [13-15]. 

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