Cofactor Regeneration of both NAD+ from NADH and NADP+ from NADPH:NADH Oxidase from Lactobacillus sanfranciscensis (original) (raw)
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Engineering an Enzymatic Regeneration System for NAD(P)H Oxidation
Journal of Molecular Catalysis B: Enzymatic, 2015
A recently proposed coenzyme regeneration system employing laccase and a number of 27 various redox mediators for the oxidation of NAD(P)H was studied in detail by kinetic 28 characterization of individual reaction steps. Reaction engineering by modeling was used to 29 optimize the employed enzyme, coenzyme as well as redox mediator concentrations. Glucose 30 dehydrogenase from Bacillus sp. served as a convenient model of synthetic enzymes that 31 depend either on NAD + or NADP + . The suitability of laccase from Trametes pubescens in 32 combination with acetosyringone or syringaldazine as redox mediator was tested for the 33 regeneration (oxidation) of both coenzymes. In a first step, pH profiles and catalytic constants 34 of laccase for the redox mediators were determined. Then, second-order rate constants for the 35 oxidation of NAD(P)H by the redox mediators were measured. In a third step, the rate 36 equation for the entire enzymatic process was derived and used to build a MATLAB model. 37 After verifying the agreement of predicted vs. experimental data, the model was used to 38 calculate different scenarios employing varying concentrations of regeneration system 39 components. The modeled processes were experimentally tested and the results compared to 40 the predictions. It was found that the regeneration of NADH to its oxidized form was 41 performed very efficiently, but that an excess of laccase activity leads to a high concentration 42 of the oxidized form of the redox mediator -a phenoxy radical -which initiates coupling 43 (dimerization or polymerization) and enzyme deactivation. 44 45 Page 3 of 32 A c c e p t e d M a n u s c r i p t 3 45 65 interfere with the isolation of the product of interest or with enzyme stability should be 66 employed, (iv) high turnover numbers should be obtained, (v) the total turnover number of 67 the coenzyme should be at least between 10 2 and 10 4 , and (vi) an overall equilibrium for the 68 coupled enzyme system favorable to product formation should be reached. These criteria 69 Page 4 of 32 A c c e p t e d M a n u s c r i p t 4 have been already partially met for NAD + -reducing enzymes such as alcohol dehydrogenase, 70 lactate dehydrogenase and glutamate dehydrogenase [7,9,10]. However, the enzymatic 71 oxidation of NAD(P)H is not satisfactorily developed to date. The use of laccase for 72 NAD(P)H oxidation seems to fulfil most of the postulated criteria: (i) Laccases are technical 73 enzymes employed for decolorization or delignification processes, which can be produced 74 recombinantly and inexpensively. (ii) Laccase, a member of the blue multicopper oxidase 75 family, has a high specific activity for various substrates, which can reach up to several 76 hundred per second. (iii) Most of the investigated redox mediators, which typically are used 77 in low concentrations, are inexpensive, but more work needs to be done on their removal 78 from the product. Oxygen, the second substrate of laccase, can be easily provided to a 79 biocatalytic process, and since water is produced by its reaction no purification of a by-80 product is required. (iv) It should be possible to obtain high turnover numbers for the 81 coenzyme in a biocatalytic process when considering both the reported high stability and high 82 specific activity of laccases, and (v) based on this high stability / high activity high total 83 turnover numbers for the enzyme (laccase) should be achievable as well. (vi) The high redox 84 potential of laccase of up to 800 mV vs. SHE allows to oxidize even redox mediators with 85 high potentials [11,12]. The high thermodynamic driving force of oxygen reduction makes 86 processes irreversible and drives coenzyme-dependent reactions towards completion [2]. The 87
Three different coupled enzymatic systems for in situ regeneration of NADPH
Biotechnology Techniques, 1999
Three different coupled enzymatic systems used in the reduction of sulcatone by alcohol dehydrogenase from Thermoanaerobium brockii (TBADH), were kinetically compared. The first one involved the use of TBADH for both the principal and recycling reactions and 2-propanol 20%, v/v as the recycling substrate. The other two were based on the use of a different enzyme, glucose- or glucose-6-phosphate dehydrogenases,
Developing an ethanol utilisation pathway based NADH regeneration system in Escherichia coli
2021
Many industrially relevant biotransformation in whole-cells are dependent on cofactors such as NADH or NADPH. Cofactor regeneration is an established approach for providing a cheap source of cofactors in support of the main biotransformation reaction in biocatalysis. In essence, cofactor regeneration uses a sacrificial substrate to help regenerate a cofactor consumed by the main biotransformation reaction. Enzymatic in nature, alternative cofactor regeneration systems with high efficiency and which utilises low cost sacrificial substrate are of interest. Glucose dehydrogenase system has been dominant in NADH regeneration. But, in its current incarnation, glucose dehydrogenase system is relatively inefficient in regenerating NADH with theoretical yield of one NADH per glucose molecule. This work sought to explore the utility of a two-gene ethanol utilisation pathway in NADH regeneration. Comprising the first step that takes ethanol to acetaldehyde, and a second step that converts ace...
NADPH is an essential cofactor for the biosynthesis of several high-value chemicals, including isoprenoids, fatty acid-based fuels, and biopolymers. Tunable control over all potentially rate-limiting steps, including the NADPH regeneration rate, is crucial to maximizing production titers. We have rationally engineered a synthetic version of the Entner–Doudoroff pathway from Zymomonas mobilis that increased the NADPH regeneration rate in Escherichia coli MG1655 by 25-fold. To do this, we combined systematic design rules, biophysical models, and computational optimization to design synthetic bacterial operons expressing the 5-enzyme pathway, while eliminating undesired genetic elements for maximum expression control. NADPH regeneration rates from genome-integrated pathways were estimated using a NADPH-binding fluorescent reporter and by the productivity of a NADPH-dependent terpenoid biosynthesis pathway. We designed and constructed improved pathway variants by employing the RBS Library Calculator to efficiently search the 5-dimensional enzyme expression space and by performing 40 cycles of MAGE for site-directed genome mutagenesis. 624 pathway variants were screened using a NADPH-dependent blue fluorescent protein, and 22 were further characterized to determine the relationship between enzyme expression levels and NADPH regeneration rates. The best variant exhibited 25-fold higher normalized mBFP levels when compared to wild-type strain. Combining the synthetic Entner–Doudoroff pathway with an optimized terpenoid pathway further increased the terpenoid titer by 97%.
Advanced Synthesis & Catalysis, 2012
An enzymatic regeneration system consisting of laccase and the 2H + /2e À redox mediator Meldolas blue (MB) was developed for the efficient oxidation of reduced nicotinamide adenine dinucleotide (NADH) cofactor and employed in the gram-scale oxidation of cholic acid (1) to its 7-keto derivative (1a) by 7a-hydroxysteroid dehydrogenase (7a-HSDH) in an aqueous, buffered reaction system. The regenerating enzyme, laccase, reduces molecular oxygen as terminal 4H + /4e À acceptor to water and concomitantly reoxidizes NADH via MB at high turnover rates. The regeneration system was successfully applied to quantitatively convert 1 (50 mM, 20.4 g) into 1a with a space-time yield of 5.8 mmol L À1 h À1 . High total turnover numbers were achieved for 7a-HSDH (4.2 10 5 ) and laccase (1.1 10 6 ). Alternatively, the regeneration system was employed on the conversion of the methyl ester derivative of cholic acid (2, 200 mM, 5.9 g) dissolved in isopropyl acetate as organic solvent in a biphasic system. Due to the high concentration of 2 and the excellent performance of the enzymatic cascade reaction even under the harsh process conditions, space-time yields of up to 20 mmol L À1 h À1 (8.5 g L À1 h À1 ) of the produced 7-keto derivative 2a were obtained. The irreversibility and high driving force of the regenerating reaction allowed quantitative conversions and easy product recovery.
Guidelines for the Application of NAD(P)H Regenerating Glucose Dehydrogenase in Synthetic Processes
Advanced Synthesis & Catalysis, 2013
Glucose dehydrogenase (GDH) is frequently used for the reduction of NAD + and NADP + in bench-and industrial-scale syntheses because the coenzyme regenerating system GDH is easy to apply, robust and relatively inexpensive. To optimize the application of this long known coenzyme regeneration system we investigated the commonly applied Bacillus GDH and characterized this enzyme by its kinetic features in the presence of substrates and products at pH 6.4 and 8.0. Three substrates/products were found to inhibit GDH considerably: (i) the reaction product glucono-1,5lactone, (ii) the reduced coenzyme NAD(P)H and (iii) the oxidized coenzyme NAD(P) +. The inhibition of GDH under several process conditions was modeled using the determined kinetic constants. It was found that the GDH regeneration system is strongly inhibited by the usually applied conditions. This study provides the rate equation of the GDH reaction and simulations of this coenzyme regenerating system leading to an improved prediction and, thus, to a faster scale-up and increased efficiency of NAD(P)H-dependent synthetic processes.
BMC Biotechnology, 2011
Background The number of biotransformations that use nicotinamide recycling systems is exponentially growing. For this reason one of the current challenges in biocatalysis is to develop and optimize more simple and efficient cofactor recycling systems. One promising approach to regenerate NAD+ pools is the use of NADH-oxidases that reduce oxygen to hydrogen peroxide while oxidizing NADH to NAD+. This class of enzymes may be applied to asymmetric reduction of prochiral substrates in order to obtain enantiopure compounds. Results The NADH-oxidase (NOX) presented here is a flavoenzyme which needs exogenous FAD or FMN to reach its maximum velocity. Interestingly, this enzyme is 6-fold hyperactivated by incubation at high temperatures (80°C) under limiting concentrations of flavin cofactor, a change that remains stable even at low temperatures (37°C). The hyperactivated form presented a high specific activity (37.5 U/mg) at low temperatures despite isolation from a thermophile source. Im...
NADPH-Auxotrophic E. coli: A Sensor Strain for Testing in Vivo Regeneration of NADPH
ACS Synthetic Biology, 2018
Insufficient rate of NADPH regeneration often limits the activity of biosynthetic pathways. Expression of NADPH-regenerating enzymes is commonly used to address this problem and increase cofactor availability. Here, we construct an Escherichia coli NADPH-auxotroph strain, which is deleted in all reactions that produce NADPH with the exception of 6-phosphogluconate dehydrogenase. This strain grows on a minimal medium only if gluconate is added as NADPH source. When gluconate is omitted, the strain serves as a "biosensor" for the capability of enzymes to regenerate NADPH in vivo. We show that the NADPH-auxotroph strain can be used to quantitatively assess different NADPH-regenerating enzymes and provide essential information on expression levels and concentrations of reduced substrates required to support optimal NADPH production rate. The NADPH-auxotroph strain thus serves as an effective metabolic platform for evaluating NADPH regeneration within the cellular context.