Enzyme-catalyzed organic synthesis: NADH regeneration by using formate dehydrogenase

Shaked, Ze'ev; Whitesides, George M.

doi:10.1021/ja00543a038

Cited by 181 publications

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“…The enzymecoupled approach is characterized by the use of a second enzyme, which catalyzes the oxidation of a cosubstrate to regenerate the reduced form of the cofactor. Formate dehydrogenase (Ernst et al, 2005;Hummel and Kula, 1989;Shaked and Whitesides, 1980;Tishkov et al, 1999) or glucose dehydrogenase (Kizaki et al, 2001;Weckbecker and Hummel, 2004) can be applied as the second enzyme for cofactor regeneration. In the substrate-coupled approach the alcohol dehydrogenase (ADH) used for the reduction reaction also catalyzes the cofactor regenerating reaction by oxidation of a second substrate.…”

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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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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“…Thus, numerous efforts have been made over the past decades to accomplish in situ cofactor regeneration from their oxidized counterpart. [6][7][8][9] For example, researchers found that NAD(P)H can be successfully regenerated by introducing secondary enzymes [10][11][12] that reduce its oxidized counterpart (i.e., NAD(P) + ) or electrodes [13][14][15] with an external power supply into reaction media. However, these approaches present intrinsic drawbacks (e.g., by-product formation and requirement of secondary enzymes for biocatalytic regeneration, as well as extremely low yield and high overpotential for electrochemical regeneration) that hindered their practical application beyond the laboratory scale.…”

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confidence: 99%

Rational Design and Engineering of Quantum‐Dot‐Sensitized TiO₂ Nanotube Arrays for Artificial Photosynthesis

et al. 2011

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Redox enzymes can catalyze complex synthesis reactions under mild conditions but conventional catalysts rarely accomplish this task. Despite the high potential of redox enzymes for the synthesis of valuable compounds (e.g., chiral alcohols and drug intermediates), [1][2][3][4][5] their application is hampered by the high cost of enzyme-specifi c cofactors that are required as a redox equivalent, such as nicotinamide adenine dinucleotide (NAD(P) H) and fl avin adenine dinucleotide (FADH). Thus, numerous efforts have been made over the past decades to accomplish in situ cofactor regeneration from their oxidized counterpart. [6][7][8][9] For example, researchers found that NAD(P)H can be successfully regenerated by introducing secondary enzymes [10][11][12] that reduce its oxidized counterpart (i.e., NAD(P) + ) or electrodes [13][14][15] with an external power supply into reaction media. However, these approaches present intrinsic drawbacks (e.g., by-product formation and requirement of secondary enzymes for biocatalytic regeneration, as well as extremely low yield and high overpotential for electrochemical regeneration) that hindered their practical application beyond the laboratory scale. [6][7][8] Herein, we report on the development of quantum-dotsensitized TiO 2 nanotube arrays for redox enzymatic synthesis coupled with the photoregeneration of nicotinamide cofactors via inspiration from natural photosynthesis. In natural photosynthesis, [ 16 , 17 ] incident light electronically excites a membranebound protein-pigment complexes called a photosystem. The photogenerated electrons are rapidly delivered to reaction centers along the electron transport chain for regenerating NADPH cofactors. These cofactors drive redox enzymatic reactions to synthesize organic compounds in the Calvin cycle. Its unique features (e.g., environmental compatibility and nearunity quantum yield) have fascinated scientists and provided inspiration to improve the effi ciency of solar cells and photoelectrochemical hydrogen production systems. [18][19][20][21][22][23][24] In the present study, the photosystem for in situ NAD(P)H regeneration consisted of TiO 2 -CdS nanotubes as a photoelectrode, triethanolamine (TEOA) as an electron donor, and pentamethylcyclopentadienyl rhodium bipyridine ([Cp * Rh(bpy)(H 2 O)] 2 + ) as an electron mediator and a hydride transfer catalyst ( Figure S1, Supporting Information). As a photoelectrode for non-enzymatic regeneration of NAD(P)H, a nanotubular TiO 2 -CdS fi lm has many advantages that include easy synthesis and morphology control, [ 25 , 26 ] effi cient charge separation, [ 24 , 27 ] and better diffusion of reaction species through nanotube channels. Due to the small size and more negative position of the conduction band (CB) edge of CdS compared to TiO 2 (at least 0.2 V more negative), photogenerated electrons can be rapidly injected from CdS to TiO 2 in a thermodynamically favorable manner ( Figure S1, Supporting Information). This injection suppresses electron-hole recombination, which is more...

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“…This system has been successfully used for NADH regeneration in a membrane reactor for L-leucine synthesis (high conversion 90-99.7% maintained during more than 25 d) [5] or D-lactate synthesis (complete reaction in 7 d) [6]. The formate dehydrogenase generally used so far is specific to NAD + , but a new formate dehydrogenase has been produced [7] that is able to work with NADP + .…”

Section: Enzyme-catalyzed Regeneration Of the Cofactormentioning

confidence: 99%

“…3) was equipped with a platinum or a porous carbon felt working electrode (5). The solution circulated through this electrode, then through the membrane (4') until reaching the solution collector (6). In this case, the driving force is the pressure gradient.…”

Section: Description Of the Membrane Electrochemical Reactors (Mers)mentioning

confidence: 99%

Membrane electrochemical reactors (MER) for NADH regeneration in HLADH-catalysed synthesis: comparison of effectiveness

Délécouls-Servat

Bergel

Basseguy

2004

Bioprocess Biosyst Eng

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Two membrane electrochemical reactors (MER) were designed and applied to HLADH-catalysed reduction of cyclohexanone to cyclohexanol. The regeneration of the cofactor NADH was ensured electrochemically, using either methyl viologen or a rhodium complex as electrochemical mediator. A semipermeable membrane (dialysis or ultra-filtration) was integrated in the filter-press electrochemical reactor to confine the enzyme(s) as close as possible to the electrode surface. When methyl viologen was used, the transformation ratio of cyclohexanone varied from 0 to 65% depending on the internal arrangement of the reactor. Matching the reactor configuration to the reaction system was essential in this case. With the rhodium complex, the ultra-filtration MER was tested in continuous and recycling configurations. The best conditions led to 100% transformation of 0.1 L volume of 0.1 M cyclohexanone after 70 h with the recycling mode. Finally, the performances of the reactors are discussed with respect to different evaluations of the production yields.

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Enzyme-catalyzed organic synthesis: NADH regeneration by using formate dehydrogenase

Abstract: Existing methods for regeneration of NADH from NAD"1" for use in organic synthetic procedures based on NADH-dependent enzymes all have disadvantages.2,3 Here we describe the use of (1) Supported by the National Institutes of Health (Grant GM 26543).

Cited by 181 publications

References 0 publications

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

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

Rational Design and Engineering of Quantum‐Dot‐Sensitized TiO₂ Nanotube Arrays for Artificial Photosynthesis

Membrane electrochemical reactors (MER) for NADH regeneration in HLADH-catalysed synthesis: comparison of effectiveness

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Enzyme-catalyzed organic synthesis: NADH regeneration by using formate dehydrogenase

Abstract: Existing methods for regeneration of NADH from NAD"1" for use in organic synthetic procedures based on NADH-dependent enzymes all have disadvantages.2,3 Here we describe the use of (1) Supported by the National Institutes of Health (Grant GM 26543).

Cited by 181 publications

References 0 publications

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

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

Rational Design and Engineering of Quantum‐Dot‐Sensitized TiO2 Nanotube Arrays for Artificial Photosynthesis

Membrane electrochemical reactors (MER) for NADH regeneration in HLADH-catalysed synthesis: comparison of effectiveness

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Rational Design and Engineering of Quantum‐Dot‐Sensitized TiO₂ Nanotube Arrays for Artificial Photosynthesis