Accessible Published by De Gruyter October 31, 2018

Multienzymatic in situ hydrogen peroxide generation cascade for peroxygenase-catalysed oxyfunctionalisation reactions

Milja Pesic, Sébastien Jean-Paul Willot, Elena Fernández-Fueyo, Florian Tieves, Miguel Alcalde and Frank Hollmann

Abstract

There is an increasing interest in the application of peroxygenases in biocatalysis, because of their ability to catalyse the oxyfunctionalisation reaction in a stereoselective fashion and with high catalytic efficiencies, while using hydrogen peroxide or organic peroxides as oxidant. However, enzymes belonging to this class exhibit a very low stability in the presence of peroxides. With the aim of bypassing this fast and irreversible inactivation, we study the use of a gradual supply of hydrogen peroxide to maintain its concentration at stoichiometric levels. In this contribution, we report a multienzymatic cascade for in situ generation of hydrogen peroxide. In the first step, in the presence of NAD+ cofactor, formate dehydrogenase from Candida boidinii (FDH) catalysed the oxidation of formate yielding CO2. Reduced NADH was reoxidised by the reduction of the flavin mononucleotide cofactor bound to an old yellow enzyme homologue from Bacillus subtilis (YqjM), which subsequently reacts with molecular oxygen yielding hydrogen peroxide. Finally, this system was coupled to the hydroxylation of ethylbenzene reaction catalysed by an evolved peroxygenase from Agrocybe aegerita (rAaeUPO). Additionally, we studied the influence of different reaction parameters on the performance of the cascade with the aim of improving the turnover of the hydroxylation reaction.

Fungal peroxygenases (E.C. 1.11.2.4) are promising catalysts for the selective oxyfunctionalisation of non-activated C–H- and C=C-bonds [1], [2], [3], [4], [5]. Compared with the well-known P450 monooxygenases, peroxygenases excel by their simpler reaction mechanism and minor requirements, as they are secreted – extracellular – enzymes. Instead of reductively activating molecular oxygen at the expense of NAD(P)H and sometimes rather complex and vulnerable electron transport chains [6], peroxygenases utilise H2O2 directly through a H2O2-shunt pathway [7]. This approach, however, is challenged by the poor robustness of heme-dependent enzymes against the strong oxidant H2O2. To alleviate this, catalytic reduction of O2 is the most commonly used approach nowadays. As a source of reducing equivalents, the cathode [8], [9], [10] glucose and other small molecules have been reported [11], [12], [13], [14], [15], [16], [17].

Inspired by a contribution by Guisan et al. [18] we decided to evaluate formic acid (or its sodium salts) as stoichiometric reductant to promote peroxygenase-catalysed oxyfunctionalisation reactions. The system envisages the coupling of formate dehydrogenase (FDH)-catalysed [19], [20] oxidation of formic acid to the reduction of molecular oxygen to hydrogen peroxide, utilising the NAD(P)H oxidase activity of old yellow enzymes such as the one from Bacillus subtilis (YqjM) [21], [22]. In previous studies, it was demonstrated that YqjM, in the absence of a reducible conjugated C=C double bond, functions also as a NADH oxidase. Hence, the combination of FDH (formate consuming and NADH regenerating) with YqjM (NADH consuming and H2O2 generating) resulted in an artificial, bienzymatic formate oxidase system. Both reactions are being coupled via the natural redox cofactor (NAD+/NADH). As the model reaction to establish the system, the stereoselective hydroxylation of ethyl benzene to (R)-1-phenyl ethanol (Scheme 1) catalysed by the recombinant peroxygenase from Agrocybe aegerita (rAaeUPO, -PaDa-I mutant-), a variant evolved in the laboratory for functional expression in yeasts [23], [24], was used.

Scheme 1: Proposed in situ H2O2 generation system to promote the stereoselective hydroxylation of ethyl benzene to (R)-1-phenyl ethanol using the recombinant peroxygenase from A. aegerita (rAaeUPO). In situ H2O2 generation is achieved by reductive activation of O2 through a cascade of FDH-catalysed oxidation of formic acid and concomitant reduction of nicotinamide adenine dinucleotide (NAD+) to its reduced form (NADH). The latter is reoxidised by the old yellow enzyme homologue from B. subtilis (YqjM) to reduce O2 to H2O2.

Scheme 1:

Proposed in situ H2O2 generation system to promote the stereoselective hydroxylation of ethyl benzene to (R)-1-phenyl ethanol using the recombinant peroxygenase from A. aegerita (rAaeUPO). In situ H2O2 generation is achieved by reductive activation of O2 through a cascade of FDH-catalysed oxidation of formic acid and concomitant reduction of nicotinamide adenine dinucleotide (NAD+) to its reduced form (NADH). The latter is reoxidised by the old yellow enzyme homologue from B. subtilis (YqjM) to reduce O2 to H2O2.

The relevant enzymes for this study (i.e., FDH, YqjM and rAaeUPO) were recombinantly expressed in Escherichia coli (FDH and YqjM) and Pichia pastoris (rAaeUPO). Details for the expression conditions, yields and purification protocols can be found in the Supporting Information. In essence, it was most important to remove any catalase activity from the E. coli-derived enzyme preparations as this would interfere with the rAaeUPO-catalysed oxyfunctionalisation reaction.

Having the catalase-free enzyme preparations at hand, we further characterised the crucial parameters for FDH for formate-driven NADH regeneration and YqjM-catalysed aerobic reoxidation of NADH, yielding H2O2 as the desired product. In accordance with the literature [25], FDH exhibited a medium affinity towards formate (KM=13.5±1.1 mM, Figure S3 and Table S2). Therefore, to avoid rate limitations caused by low formate concentrations, we used at least 75 mM initial concentrations of formate. Both, YqjM and FDH showed sufficient activity at slightly alkaline pH values, which had previously been shown to be optimal for rAaeUPO for benzylic hydroxylation reactions (Table 1) [26].

Table 1:

Activity of YqjM and FDH at different pH values.

pH Aspec (YqjM), U/mga Aspec (FDH), U/mgb
7 0.51±0.02 1.58±0.02
7.5 0.64±0.01 1.98±0.06
8 0.65±0.01 2.21±0.04

    aGeneral conditions: 0.5 mM NAD+; 0.1 mg/mL FDH; 150 mM sodium formate; 50 mM KPi; 30 °C. bGeneral conditions: 0.15 mM NADH; 50 mM KPi; 30 °C.

To test our system, we combined the three enzymes and cofactor in one pot in the presence of ethyl benzene (Figure 1). It is worth mentioning that in all experiments reported here, the enantiomeric excess (ee) of the product ((R)-1-phenyl ethanol) was higher than 96% ee, thereby representing the normal enantioselectivity of rAaeUPO for this reaction. It is worth noting here that because of the high volatility of the starting material (ethyl benzene), comparably high initial concentrations were applied to compensate for the evaporation loss of the reagents. To attain such elevated concentrations, we used 0.8% (v/v) of methanol as a cosolvent. Control reactions in the absence of either of the catalysts (FDH, NAD, YqjM, or rAaeUPO) yielded no detectable product formation; the same is true for experiments performed in the absence of formate or under anaerobic conditions (Table S1).

Figure 1: Influence of the volume ratio of air to reaction mixture: ♦, 3:1; ■, 1:1; and ○, 1:4. Conditions: 3-(N-Morpholino)propanesulfonic acid (MOPS)-buffer (50 mM, pH 7), c(ethylbenzene)=50 mM, c(methanol)=200 mM, c(NAD+)=0.5 mM, c(NaHCO2)=75 mM, c(rAaeUPO)=50 nM, c(FDH)=2.5 μM, c(YqjM)=2.67 μM; T=30 °C; 600 rpm. For reasons of clarity, the ‘overoxidation product’ acetophenone as well as the remaining ethyl benzene were omitted.

Figure 1:

Influence of the volume ratio of air to reaction mixture: ♦, 3:1; ■, 1:1; and ○, 1:4. Conditions: 3-(N-Morpholino)propanesulfonic acid (MOPS)-buffer (50 mM, pH 7), c(ethylbenzene)=50 mM, c(methanol)=200 mM, c(NAD+)=0.5 mM, c(NaHCO2)=75 mM, c(rAaeUPO)=50 nM, c(FDH)=2.5 μM, c(YqjM)=2.67 μM; T=30 °C; 600 rpm. For reasons of clarity, the ‘overoxidation product’ acetophenone as well as the remaining ethyl benzene were omitted.

Under arbitrarily chosen conditions, enantioselective conversion of ethyl benzene into (R)-1-phenyl ethanol was observed. However, only 2.5 mM of the desired product was obtained, suggesting a limitation in O2 availability. Therefore, the headspace volume was increased (Figure 1, ♦ and ■). Using a phase ratio of 1:1 (ambient air to reaction mixture), approx. 9.6 mM of (R)-1-phenyl ethanol may be expected (calculated from the O2 availability in the gas phase plus O2 dissolved in buffer), which is also roughly the product concentration observed under these conditions (Figure 1, ■). However, further increases of the headspace volume did not result in the expected increases in product formation.

We suspected other factors, particularly enzyme stability, to account for the lower than expected performance. Therefore, we investigated the influence of the catalytic components of Scheme 1 more systematically (Table 2). In general, increasing the concentration of either catalyst (FDH, NAD, or YqjM) increased the initial rate of the overall reaction and consequently influenced the turnover number of the other catalysts. Compared with this, the initial rate of the overall reaction was rather insensitive towards changing rAaeUPO concentrations. Depending on the reaction, turnover numbers of 80,000–390,000 for rAaeUPO, 14,000–84,000 for FDH, 1600–6700 for YqjM and 7–69 for NAD were observed.

Table 2:

Influence of the different reaction parameters on initial reaction rates and turnover numbers.

Concentration, mM v0, mM/h Turnover number (mol product/mol catalyst)
FDH NAD+ YqjM rAaeUPO
[FDH], nMa  50 0.36 84,000 8.4 1600 80,000
 125 0.66 47,000 11.9 2200 120,000
 250 0.96 26,000 13.1 2500 130,000
[NAD+], mMb  0.1 0.52 28,000 68.8 2600 140,000
 0.25 0.76 30,000 30.3 2800 150,000
 0.5 0.96 26,000 13.1 2500 130,000
[YqjM], μMc  0.53 0.15 14,000 7.1 6700 70,000
 2.67 0.96 26,000 13.1 2500 130,000
 13.33 1.78 38,000 19.2 700 190,000
[rAaeUPO], nMd  25 0.83 39,000 19.3 3600 390,000
 50 0.96 26,000 13.1 2500 130,000
 100 0.78 34,000 17 3200 90,000

    Conditions: MOPS-buffer (50 mM, pH 7), [ethylbenzene]=10 mM, [methanol]=200 mM, [NaHCO2]=75 mM; T=30 °C; 600 rpm. a[NAD+]=0.5 mM, [rAaeUPO]=50 nM, [YqjM]=2.67 μM. b[FDH]=250 nM, [rAaeUPO]=50 nM, [YqjM]=2.67 μM. c[NAD+]=0.5 mM, [rAaeUPO]=50 nM, [FDH]=250 nM. d[NAD+]=0.5 mM, [FDH]=250 nM, [YqjM]=2.67 μM.

This observation was rather expected considering the very high catalytic activity of rAaeUPO under initial rate conditions of 410 s−1 [26]. Hence, the overall reaction appeared to be limited by the availability of H2O2. Its concentration, however, cannot be increased at will as too much H2O2 will also irreversibly inactivate rAaeUPO. Therefore, we followed the operational stability of the overall process in the presence of different FDH concentrations.

As shown in Figure 2, the product accumulation ceased earlier in the presence of higher FDH concentrations. Hence, FDH activity plays a crucial role for both the productivity and the robustness of the overall system. Future studies will have to be undertaken to optimise the relative concentrations of the catalysts used. A design of an experimental approach appears promising to find the optimal conditions of this highly interconnected reaction system. Also, it should be very interesting to identify the amino acid residues prone to oxidative modification and substitute them by less reactive ones, eventually yielding more robust FDH mutants.

Figure 2: Effect of FDH concentration on the robustness of the overall reaction. Reaction rates at different time intervals were normalised by corresponding initial reaction rates. Conditions: MOPS-buffer (50 mM, pH 7), c(ethylbenzene)=50 mM, c(methanol)=200 mM, c(NAD+)=0.5 mM, c(NaHCO2)=75 mM, c(rAaeUPO)=50 nM, c(YqjM)=2.67 μM; T=30 °C; 600 rpm. c(FDH)=0.25 μM (■), 0.63 μM (), 1.25 μM (), 1.87 μM () and 2.5 μM (□). The initial rates were 1.1, 1.8, 2.6, 3.0 and 2.6 mM/h, respectively.

Figure 2:

Effect of FDH concentration on the robustness of the overall reaction. Reaction rates at different time intervals were normalised by corresponding initial reaction rates. Conditions: MOPS-buffer (50 mM, pH 7), c(ethylbenzene)=50 mM, c(methanol)=200 mM, c(NAD+)=0.5 mM, c(NaHCO2)=75 mM, c(rAaeUPO)=50 nM, c(YqjM)=2.67 μM; T=30 °C; 600 rpm. c(FDH)=0.25 μM (■), 0.63 μM (

), 1.25 μM (
), 1.87 μM (
) and 2.5 μM (□). The initial rates were 1.1, 1.8, 2.6, 3.0 and 2.6 mM/h, respectively.

Overall, in this contribution, we have provided the proof-of-concept for a FDH/NAD/YqjM cascade to mediate the reductive activation of molecular oxygen to hydrogen peroxide. Promising turnover numbers for all catalytic components have been achieved, but significant improvements are necessary to turn this system truly practical. For this, the identification of the limiting factors as provided in this study will lay the foundation.

Acknowledgements

Financial support by the European Research Council (ERC Consolidator Grant No. 648026) is gratefully acknowledged.

References

1. Dong JJ, Fernández-Fueyo EE, Hollmann F, Paul CE, Pesic M, Schmid S, et al. Biocatalytic oxidation reactions: a chemist’s perspective. Angew Chem Int Ed 2018;57:9238–61. Search in Google Scholar

2. Martínez AT, Ruiz-Dueñas FJ, Camarero S, Serrano A, Linde D, Lund H, et al. Oxidoreductases on their way to industrial biotransformations. Biotechnol Adv 2017;35:815–31. Search in Google Scholar

3. Hofrichter M, Ullrich R. Oxidations catalyzed by fungal peroxygenases. Curr Opin Chem Biol 2014;19:116–25. Search in Google Scholar

4. Wang Y, Lan D, Durrani R, Hollmann F. Peroxygenases en route to becoming dream catalysts. What are the opportunities and challenges? Curr Opin Chem Biol 2017;37:1–9. Search in Google Scholar

5. Bormann S, Gomez Baraibar A, Ni Y, Holtmann D, Hollmann F. Specific oxyfunctionalisations catalysed by peroxygenases: opportunities, challenges and solutions. Catal Sci Technol 2015;5:2038–52. Search in Google Scholar

6. Holtmann D, Hollmann F. The oxygen dilemma: a severe challenge for the application of monooxygenases? ChemBioChem 2016;17:1391–8. Search in Google Scholar

7. Joo H, Lin ZL, Arnold FH. Laboratory evolution of peroxide-mediated cytochrome P450 hydroxylation. Nature 1999;399:670–3. Search in Google Scholar

8. Choi DS, Ni Y, Fernández-Fueyo E, Lee M, Hollmann F, Park CB. Photoelectroenzymatic oxyfunctionalization on flavin-hybridized carbon nanotube electrode platform. ACS Catal 2017;7:1563–7. Search in Google Scholar

9. Krieg T, Huttmann S, Mangold KM, Schrader J, Holtmann D. Self-sustained enzymatic cascade for the production of 2,5-furandicarboxylic acid from 5-methoxymethylfurfural. Green Chem 2011;13:2686–9. Search in Google Scholar

10. Lutz S, Steckhan E, Liese A. First asymmetric electroenzymatic oxidation catalyzed by a peroxidase. Electrochem Commun 2004;6:583–7. Search in Google Scholar

11. Zhang W, Fernández-Fueyo E, Ni Y, van Schie M, Gacs J, Renirie R, et al. Selective aerobic oxidation reactions using a combination of photocatalytic water oxidation and enzymatic oxyfunctionalizations. Nat Catal 2018;1:55–62. Search in Google Scholar

12. Zhang W, Burek BO, Fernández-Fueyo E, Alcalde M, Bloh JZ, Hollmann F. Selective activation of C−H bonds in a cascade process combining photochemistry and biocatalysis. Angew Chem 2017;129:15451–5. Search in Google Scholar

13. Ni Y, Fernández-Fueyo E, Baraibar AG, Ullrich R, Hofrichter M, Yanase H, et al. Peroxygenase-catalyzed oxyfunctionalization reactions promoted by the complete oxidation of methanol. Angew Chem Int Ed 2016;55:798–801. Search in Google Scholar

14. Paul CE, Churakova E, Maurits E, Girhard M, Urlacher VB, Hollmann F. In situ formation of H2O2 for P450 peroxygenases. Bioorg Med Chem 2014;22:5692–6. Search in Google Scholar

15. Churakova E, Arends IW, Hollmann F. Increasing the productivity of peroxidase-catalyzed oxyfunctionalization: a case study on the potential of two-liquid-phase systems. ChemCatChem 2013;5:565–8. Search in Google Scholar

16. Churakova E, Kluge M, Ullrich R, Arends I, Hofrichter M, Hollmann F. Specific photobiocatalytic oxyfunctionalization reactions. Angew Chem Int Ed 2011;50:10716–9. Search in Google Scholar

17. Perez DI, Mifsud Grau M, Arends IW, Hollmann F. Visible light-driven and chloroperoxidase-catalyzed oxygenation reactions. Chem Commun 2009;0:6848–50. Search in Google Scholar

18. Rocha-Martin J, Velasco-Lozano S, Guisan JM, Lopez-Gallego F. Oxidation of phenolic compounds catalyzed by immobilized multi-enzyme systems with integrated hydrogen peroxide production. Green Chem 2014;16:303–11. Search in Google Scholar

19. Popov VO, Lamzin VS. NAD+-dependent formate dehydrogenase. Biochemical J 1994;301:625–43. Search in Google Scholar

20. Shaked Z, Whitesides GM. Enzyme-catalyzed organic synthesis: NADH regeneration by using formate dehydrogenase. J Am Chem Soc 1980;102:7104–5. Search in Google Scholar

21. Fitzpatrick TB, Amrhein M, Macheroux P. Characterization of YqjM, an old yellow enzyme homolog from Bacillus subtilis involved in the oxidative stress response. J Biol Chem 2003;278:19891–7. Search in Google Scholar

22. Pesic M, Fernández-Fueyo E, Hollmann F. Characterization of the old yellow enzyme homolog from Bacillus subtilis (YqjM). ChemistrySelect 2017;2:3866–71. Search in Google Scholar

23. Molina-Espeja P, Garcia-Ruiz E, Gonzalez-Perez D, Ullrich R, Hofrichter M, Alcalde M. Directed evolution of unspecific peroxygenase from Agrocybe aegerita. Appl Environ Microbiol 2014;80:3496–507. Search in Google Scholar

24. Molina-Espeja P, Ma S, Mate DM, Ludwig R, Alcalde M. Tandem-yeast expression system for engineering and producing un specific peroxygenase. Enzyme Microb Technol 2015;73–74: 29–33. Search in Google Scholar

25. Tishkov VI, Popov VO. Catalytic mechanism and application of formate dehydrogenase. Biochem-Moscow 2004;69:1252–3. Search in Google Scholar

26. Kluge M, Ullrich R, Scheibner K, Hofrichter M. Stereoselective benzylic hydroxylation of alkylbenzenes and epoxidation of styrene derivatives catalyzed by the peroxygenase of Agrocybe aegerita. Green Chem 2012;14:440–6. Search in Google Scholar

Supplementary Material

The online version of this article offers supplementary material (https://doi.org/10.1515/znc-2018-0137).

Received: 2018-09-04
Revised: 2018-09-19
Accepted: 2018-10-04
Published Online: 2018-10-31
Published in Print: 2019-02-25

©2018 Walter de Gruyter GmbH, Berlin/Boston