Recent Developments in NAD(P)H Regeneration for Enzymatic Reductions in One- and Two-Phase Systems

Eckstein, Marrit; Daußmann, Thomas; Kragl, Udo

doi:10.1080/10242420410001692769

Cited by 57 publications

(29 citation statements)

References 36 publications

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“…Different methods can be applied to achive an efficient cofactor regeneration, even in organic sovents or biphasic systems. [29,30] Using Eq. (1), it is now possible to predict and compare equilibrium conversions X-A C H T U N G T R E N N U N G (DE 0 ) of processes with different regeneration methods and thus to choose a suitable regeneration method for the desired process to maximise XA C H T U N G T R E N N U N G (DE 0 ).…”

Section: Choice Of Cofactor Regeneration Methodsmentioning

confidence: 99%

Maximise Equilibrium Conversion in Biphasic Catalysed Reactions: How to Obtain Reliable Data for Equilibrium Constants?

et al. 2006

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For the prediction and optimisation of the equilibrium conversion in biphasic catalysed reactions, the equilibrium constant of the desired reaction and the partition coefficients of all reactants have to be known. Within this contribution we have examined the alcohol dehydrogenase-catalysed reduction of several linear and aromatic ketones in biphasic reaction media with respect to equilibrium conversion. In this example, the equilibrium constant can be expressed in terms of differences in oxidation-reduction potentials DE 0 . However, for a large variety of organic compounds, these data are quite rare in the literature. To overcome this lack of data, we have utilised methods of computational chemistry to calculate data for the Gibbs free energy DG R leading to the equilibrium constants of a homologous series of linear ketones. To obtain comparable data for the reduction of substituted acetophenone derivatives, the Hammett relation leads to the necessary equilibrium constants. Furthermore, we compare the equilibrium conversions of a set of cofactor regeneration methods for the alcohol dehydrogenase-catalysed reductions. These results lead to a time-saving experimental design for the enantioselective reduction of 2-octanone to (R)-2-octanol on a preparative scale utilising biphasic reaction conditions.

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Section: Choice Of Cofactor Regeneration Methodsmentioning

confidence: 99%

Maximise Equilibrium Conversion in Biphasic Catalysed Reactions: How to Obtain Reliable Data for Equilibrium Constants?

et al. 2006

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“…For efficient utilization of biocatalysts the consumed amount of cofactors has to be regenerated sufficiently during the biotransformation process (Wandrey, 2004). Different strategies for cofactor regeneration were developed for oxidoreductase catalyzed whole cell biotransformation processes making use of glucose metabolism or cofactor regeneration by applying a second enzymatic reaction (Eckstein et al, 2004;Faber, 2000;Goldberg et al, 2006Goldberg et al, , 2007aWichmann and Vasic-Racki, 2005). For the enzyme-mediated cofactor regeneration two different approaches are established: the enzyme-coupled and the substrate-coupled cofactor regeneration.…”

Section: Introductionmentioning

confidence: 99%

Metabolomics for biotransformations: Intracellular redox cofactor analysis and enzyme kinetics offer insight into whole cell processes

Schroer

Zelić

Oldiges

et al. 2009

Biotech & Bioengineering

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For redox reactions catalyzed by microbial cells the analysis of involved cofactors is of special interest since the availability of cofactors such as NADH or NADPH is often limiting and crucial for the biotransformation efficiency. The measurement of these cofactors has usually been carried out using spectrophotometric cycling assays. Today LC-MS/MS methods have become a valuable tool for the identification and quantification of intracellular metabolites. This technology has been adapted to measure all four nicotinamide cofactors (NAD, NADP, NADH, and NADPH) during a whole cell biotransformation process catalyzed by recombinant Escherichia coli cells. The cells overexpressing an alcohol dehydrogenase from Lactobacillus brevis were used for the reduction of methyl acetoacetate (MAA) with substrate-coupled cofactor regeneration by oxidation of 2-propanol. To test the reliability of the measurement the data were evaluated using a process model. This model was derived using the measured concentrations of reactants and cofactors for initiation as well as the kinetic constants from in vitro measurements of the isolated enzyme. This model proves to be highly effective in the process development for a whole cell redox biotransformation in predicting both the right concentrations of cofactors and reactants in a batch and in a CSTR process as well as the right in vivo expression level of the enzyme. Moreover, a sensitivity analysis identifies the cofactor regeneration reaction as the limiting step in case for the reduction of MAA to the corresponding product (R)-methyl 3-hydroxybutyrate. Using the combination of in vitro enzyme kinetic measurements, measurements of cofactors and reactants and an adequate model initiated by intracellular concentrations of all involved reactants and cofactors the whole cell biotransformation process can be understood quantitatively.

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“…These reactions often require a stoichiometric amount of the expensive cofactor NAD(P)H or NAD(P) ϩ , and thus practical applications require efficient recycling of the necessary cofactor (1,5,9,13,16,17,22,32). In general, cofactor recycling can be achieved by coupling a desired enzymatic reaction with an additional chemical, electrochemical, photocatalytic, or enzymatic reaction, and the enzymatic method is favored (1,5,9,13,16,17,22,32,39). Enzymatic cofactor recycling can be obtained by using "coupled-substrate" (8,12,30,31) and "coupled-enzyme" (14,15,18,19,20,21,29,33,34) approaches.…”

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confidence: 99%

Bioreduction with Efficient Recycling of NADPH by Coupled Permeabilized Microorganisms

Zhang

O’Connor

Wang

et al. 2009

Appl Environ Microbiol

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The glucose dehydrogenase (GDH) from Bacillus subtilis BGSC 1A1 was cloned and functionally expressed in Escherichia coli BL21(pGDH1) and XL-1 Blue(pGDH1). Controlled permeabilization of recombinant E. coli BL21 and XL-1 Blue with EDTA-toluene under optimized conditions resulted in permeabilized cells with specific activities of 61 and 14 U/g (dry weight) of cells, respectively, for the conversion of NADP ؉ to NADPH upon oxidation of glucose. The permeabilized recombinant strains were more active than permeabilized B. subtilis BGSC 1A1, did not exhibit NADPH/NADH oxidase activity, and were useful for regeneration of both NADH and NADPH. Coupling of permeabilized cells of Bacillus pumilus Phe-C3 containing an NADPHdependent ketoreductase and an E. coli recombinant expressing GDH as a novel biocatalytic system allowed enantioselective reduction of ethyl 3-keto-4,4,4-trifluorobutyrate with efficient recycling of NADPH; a total turnover number (TTN) of 4,200 mol/mol was obtained by using E. coli BL21(pGDH1) as the cofactorregenerating microorganism with initial addition of 0.005 mM NADP ؉ . The high TTN obtained is in the practical range for producing fine chemicals. Long-term stability of the permeabilized cell couple and a higher product concentration were demonstrated by 68 h of bioreduction of ethyl 3-keto-4,4,4-trifluorobutyrate with addition of 0.005 mM NADP ؉ three times; 50.5 mM (R)-ethyl 3-hydroxy-4,4,4-trifluorobutyrate was obtained with 95% enantiomeric excess, 84% conversion, and an overall TTN of 3,400 mol/mol. Our method results in practical synthesis of (R)-ethyl 3-hydroxy-4,4,4-trifluorobutyrate, and the principle described here is generally applicable to other microbial reductions with cofactor recycling.Biocatalytic oxidoreductions are important reactions in asymmetric synthesis, and they have great potential for industrial production of enantiopure chemicals and pharmaceuticals (4,11,23,24,26). These reactions often require a stoichiometric amount of the expensive cofactor NAD(P)H or NAD(P) ϩ , and thus practical applications require efficient recycling of the necessary cofactor (1,5,9,13,16,17,22,32). In general, cofactor recycling can be achieved by coupling a desired enzymatic reaction with an additional chemical, electrochemical, photocatalytic, or enzymatic reaction, and the enzymatic method is favored (1,5,9,13,16,17,22,32,39). Enzymatic cofactor recycling can be obtained by using "coupled-substrate" (8, 12, 30, 31) and "coupled-enzyme" (14,15,18,19,20,21,29,33,34) approaches. The latter is a more general approach and utilizes the first enzyme for the desired biotransformation and the second enzyme for cofactor regeneration. Formate dehydrogenase (15, 18) and glucose dehydrogenase (GDH) (33, 34) are well-known enzymes used for regeneration of NADH and NADPH, respectively. The "coupled-enzyme" approach has been successfully used with two isolated enzymes (15,18,29,33,34) or whole cells (14,20,21) of a microorganism coexpressing the two necessary enzymes. While the use of isolated enzymes is c...

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Recent Developments in NAD(P)H Regeneration for Enzymatic Reductions in One- and Two-Phase Systems

Cited by 57 publications

References 36 publications

Maximise Equilibrium Conversion in Biphasic Catalysed Reactions: How to Obtain Reliable Data for Equilibrium Constants?

Maximise Equilibrium Conversion in Biphasic Catalysed Reactions: How to Obtain Reliable Data for Equilibrium Constants?

Metabolomics for biotransformations: Intracellular redox cofactor analysis and enzyme kinetics offer insight into whole cell processes

Bioreduction with Efficient Recycling of NADPH by Coupled Permeabilized Microorganisms

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