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Efficient NADH Regeneration by a Redox Polymer-Immobilized Enzymatic System Mengwei Yuan, Matthew Kummer, Ross D. Milton, Timothy Quah, and Shelley D. Minteer ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.9b00513 • Publication Date (Web): 06 May 2019 Downloaded from http://pubs.acs.org on May 7, 2019
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ACS Catalysis
Efficient NADH Regeneration by a Redox PolymerImmobilized Enzymatic System Mengwei Yuan,† Matthew J. Kummer,† Ross D. Milton,†‡ Timothy Quah,† Shelley D. Minteer*† † Department of Chemistry, University of Utah 315 S 1400 E, Salt Lake City, UT 84112, USA ‡ Present Address: Department of Civil and Environmental Engineering, Stanford University Clark Center E250, 318 Campus Drive, Stanford, CA 94305, USA
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ABSTRACT Due to the high costs and stoichiometric amounts of reduced nicotinamide adenine dinucleotide (NADH) required by the many oxidoreductases used for organic synthesis and the pharmaceutical industry, there is a need for the efficient reductive regeneration of NADH from its oxidized form, NAD+. Bioelectrocatalytic methods for NADH regeneration involving diaphorase and a redox mediator have shown promise; however, strong reductive mediators needed for this system are scarce, generally unstable, and require downstream separation. The immobilization of diaphorase in cobaltocene-modified poly(allylamine) redox polymer is presented which is capable of producing bioactive 1,4-NADH with yields between 97% and 100%, faradaic efficiencies between 78% and 99%, and turnover frequencies between 2091 h-1 and 3680 h-1 over the range of temperatures spanning 20 °C to 60 °C. By using this system, methanol and propanol production by an NADH-dependent alcohol dehydrogenase were enhanced 7.1- and 5.2-fold, respectively, compared to a negative control. Finally, the efficiency of this approach coupled with its high operational stability (91% of the maximum activity after five experimental cycles) renders it among the most promising means of NADH regeneration yet developed.
KEYWORDS NADH regeneration, cobaltocene, redox polymer, bioelectrosynthesis, alcohol synthesis
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1. INTRODUCTION Oxidoreductases, enzymes which catalyze electron transfer processes, possess highly desirable properties which encourage their use in a variety of applications.1-3 For the production of pharmaceutical compounds, the enantioselectivity of oxidoreductases is of critical importance. For example, the hypoglycemic compound saxagliptin used to treat type 2 diabetes can be produced using phenylalanine dehydrogenase.4 In organic synthesis, high throughput at mild conditions of temperature, pressure, and pH is achieved by these enzymes and serve to enhance the efficiency of chemical conversions.5-7 These merits are demonstrated in the production of (2S,3S)-2,3-butanediol (2,3-BD) by 2,3-BD dehydrogenase, a compound used as a chemical building block for molecules with two vicinal stereogenic centers.8 For environmental applications, difficult reaction pathways such as the reduction of atmospheric carbon dioxide in the production of fuels are achievable with relative ease by enzymes.9-11 The majority of oxidoreductases, including those described above, employ the highly metabolically interconnected redox cofactor, nicotinamide adenine dinucleotide (NADH) or its oxidized form, NAD+.12-13 Despite being a prolific redox cofactor, the high cost and stoichiometric amounts of NADH required by these enzymes make the consumption of this coenzyme economically infeasible.14-15 This issue has motivated researchers to explore the possibility of regenerating NADH from its oxidized form in situ. The reduction of NAD+ to NADH for this purpose has primarily been carried out using enzymatic,16-19 chemical,20-21 photochemical,22-24 and electrochemical25-27 means.28 Among all of the approaches investigated so far, electrochemical NADH regeneration has attracted significant attention due to the potential for lower costs and system simplicity;29 water serves as the sole proton source, and the use of electricity eliminates the need for a reducing agent. Furthermore, 3 ACS Paragon Plus Environment
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electrochemical NADH regeneration could provide a solution for renewable energy storage.30 While the direct reduction of NAD+ to biologically-active 1,4-NADH at an electrode is possible, the process requires a high overpotential and has low faradaic efficiency owing to the formation of biologically-inactive NAD2 dimers and 1,6-NADH.31-33 Electrode surface modifications have been carried out to combat these issues, namely the yield of bioactive 1,4-NADH, with moderate success. Baik and colleagues modified gold amalgam electrodes with cholesterol, observing 75% yield compared to 10% on an unmodified surface.34 High-porosity copper foam electrodes were utilized by Asadollahi and coworkers to improve bioactive yields to 80% compared to 54% on a copper foil electrode.35 Beyond these efforts to work within the context of direct NAD+ reduction at an electrode, indirect approaches have been developed. Homogeneous organometallic catalysts, typically containing ruthenium, rhodium, and iridium, have been utilized for the electrochemical reduction of NAD+ as well.36-37 This type of catalyst offers enhanced stability and prevents the formation of the inactive NAD2 dimer.38-39 Among those organometallic catalysts designed for this reduction, one of the most successful yet developed is [Rh(Cp*)(bpy)Cl]+ by Steckhan and coworkers.40-41 However, rhodium complexes are difficult to remove from solution and have much lower turnover frequencies (TOFs) than biological catalysts, preventing their applicability to many NADH-dependent enzymes.42-44 In addition, the relatively high concentrations of the rhodium complex needed to achieve the necessary NADH production rates cause mutual deactivation of the organometallic catalyst and the enzyme.41, 44-45 As a promising alternative to organometallic catalysts for NADH regeneration, biological catalysts generally have improved regioselectivity, lower overpotentials, milder catalytic conditions (temperature, pressure, pH), and higher TOFs.28, 46 For these reasons, enzymatic NADH regeneration has become the industrial standard. In enzymatic NADH regeneration, an auxiliary 4 ACS Paragon Plus Environment
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enzyme is combined with an auxiliary substrate to reduce NAD+ by a separate reaction as NADH is oxidized in the primary reaction. The three most common enzymes and substrates used for this coupled reaction scheme are phosphite dehydrogenase (PDH) / phosphite, formate dehydrogenase (FDH) / formate, and glucose dehydrogenase (GDH) / glucose. FDH, which oxidizes formate to carbon dioxide as NAD+ is reduced to NADH, is the traditional choice and is most commonly used at industrial scales. There are advantages to potentially using GDH or PDH in its place, however. GDH, which converts glucose to gluconolactone, is capable of versatile utilization of NAD+ or NADP+ and is highly active and stable in comparison to FDH. PDH, which converts phosphite to phosphate, requires no addition of acid or base to maintain pH balance as the auxiliary product is generated.19 These alternatives are currently being developed for use in industrial applications, whereas FDH has already found many applications in industrial NADH regeneration. Specifically, FDH is used to regenerate NADH for the production (tons/year) of L-tert-leucine using leucine dehydrogenase.47 In the synthesis of the antihypertensive drug Omapatrilat, the Bristol-Myers Squibb company uses phenylalanine dehydrogenase to produce the chiral intermediate in this process, and FDH is utilized to provide NADH regeneration.48 Asymmetric ketones are converted to polyols by enzymes for the production of homogeneous catalysts with specific optical activities, and the NADH consumed in this reaction is recycled by FDH; such processes are being developed for use in industrial production.49 The TOF of this prominent industrial biocatalyst, FDH from Candida boidinii, was reported for enzymatic NADH regeneration as 3900 h-1, more than three times the frequency of the fastest reported organometallic complex used for this purpose.42, 44 However, these enzymes require the continuous addition of substrate (e.g., formate) to the reaction mixture which is 5 ACS Paragon Plus Environment
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subsequently not fully consumed, contaminating the product solution and requiring downstream separation and purification.28, 50 These issues associated with enzymatic NADH regeneration can be addressed by the use of diaphorase, an enzyme which directly reduces NAD+ to NADH and can be turned over electrochemically without consuming any other substrates. Considering all of the merits discussed above, a system that has the ability to electrochemically regenerate NADH with diaphorase has the potential to replace existing techniques and has been rarely reported. A handful of enzymes are able to directly transfer electrons between their cofactors and electrode surfaces (direct electron transfer, DET).51 However, it has been proposed that redox cofactors within enzymes should be
