Compatibility of the heterogeneous and enzyme catalyst

Initial control reactions using GOX to generate H 2 O 2 demonstrated that cyclohexane hydroxylation reactions to produce cyclohexanol could be carried out either using shaking or stirring to facilitate mixing. In addition, these reactions showed that the 0.5 wt% Au–0.5 wt% Pd/TiO 2 (1 mg ml−1) catalyst had no negative effect on the PaDa-I reaction when GOX was used to produce H 2 O 2 (Fig. 2a); demonstrating that the PaDa-I could still operate in the presence of the metal catalyst. Considering H 2 O 2 production requires H 2 and O 2 mixtures, which are combustible between H 2 concentrations of 5 and 75%, H 2 O 2 synthesis experiments were carried out in an open system by flowing 50 ml min−1 of gas (2%, 80%, 90% H 2 in air) through an aqueous solution of metal catalyst (0.1 mg ml−1). With 80% H 2 in air, a peak concentration of 25 ppm H 2 O 2 (0.74 mM) was produced (Supplementary Fig. 1). The effect of the catalyst concentration was also investigated between 0.1 and 0.6 mg ml−1 (Fig. 2b). At higher catalyst loadings, the maximum amount of H 2 O 2 generated decreased while at lower loadings, the peak concentration was higher and the system was capable of producing a sustained concentration of H 2 O 2 . These results were promising, given that Molina-Espeja et al. had reported the Michaelis constant (K m ) of PaDa-I for H 2 O 2 at pH 7 in 100 mM potassium phosphate buffer as 0.49 mM (17 ppm) when it was produced by Saccharomyces cerevisiae27 and 1.53 mM (53 ppm) when produced by Pichia pastoris28. Thus, concentrations of 1 or 2 mM H 2 O 2 (34 or 68 ppm) were used successfully in enzyme activity experiments27,28. These concentrations compared with our results suggested that the AuPd catalyst would be active enough at ambient conditions to supply H 2 O 2 in order for the PaDa-I to carry out the oxidation of cyclohexane at significantly lower precious metal catalyst loadings than previous studies19.

Fig. 2 Feasibility of Coupling Catalytic Systems. a Cyclohexanol production in control reactions to determine the effect of the metal catalyst on unspecific peroxygenase activity when H 2 O 2 is generated using GOX. b H 2 O 2 synthesis with various catalyst concentrations under flowing (30 ml min−1) 80% H 2 in air. Reaction Conditions: Solvent (H 2 O) 50 ml, ambient temperature and pressure, gas flow 30 ml min−1, 500 rpm stirring. Errors associated in the measurement of H 2 O 2 by titration were ± 3 ppm. c Cyclohexane conversion to cyclohexanol using PaDa-I (15 U ml RM −1) in potassium phosphate buffer (100 mM, pH 6) and H 2 O 2 generated in situ by metal catalysts. Efficiency of different AuPd catalysts (0.5 mg ml−1) in the presence of 77% H 2 in air. d Time course results for cyclohexanol formation by PaDa-I (15 U ml RM −1) was coupled with in situ H 2 O 2 generation by 2.5% Au-2.5% Pd/TiO 2 (0.1 mg ml RM −1) using a gas mixture of 77% H 2 in air (solid diamonds) or by GOX (0.2 U ml RM −1) using 200 mM glucose and air (circles). The GOX reactions were carried out using the same experimental setup as the metal catalyst reactions. Typically analysis of products was ± 0.2 mM as determined by GC (on duplicate experiments) Full size image

Four different AuPd catalysts, namely 2.5 wt% Au–2.5 wt% Pd/TiO 2 29, 0.5 wt% Au–0.5 wt% Pd/TiO 2 23, 2.5 wt% Au–2.5 wt% Pd on carbon and 5 wt% Au on carbon30 (0.5 mg ml RM −1) were tested for their ability to generate H 2 O 2 for PaDa-I conversion of cyclohexane with 77% H 2 in air (15 U ml RM −1, enzyme activities were determined using ABTS and H 2 O 2 as substrate with one unit defined as the amount of enzyme needed to converted 1 µmol in 1 min) (Fig. 2c). All of the AuPd catalysts gave promising results with between 0.6 and 1.3 mmol cyclohexanol l RM −1 produced after 2 h with these catalysts generating H 2 O 2 . In the absence of metal catalyst, negligible cyclohexanol (less than 0.03 mM) was detected when 77% H 2 in air was used (Supplementary Table 1). In all cases the presence of cyclohexanone resulting from overoxidation of cyclohexanol was observed only in negligible amounts in the gas chromatograms after 2 and 4 h, indicating that presence of the metal catalyst does not result in significant overoxidation (Supplementary Fig. 2).

A set of reactions with a maximum reaction time of 8 h indicated that the tandem reaction system was relatively stable with a maximum of 5.7 mmol cyclohexanol l RM −1 produced in 8 h (Fig. 2d). The reactions in which 0.2 U ml RM −1 GOX was used to produce H 2 O 2 gave comparable cyclohexanol production after 2 h and 4 h, but then leveled off to give only 3.4 mmol cyclohexanol l RM −1 after 8 h. This time course most likely resulted from the operational instability of GOX caused by inactivation by H 2 O 2 as well as auto-inactivation but demonstrates that the initial rate of the tandem process is the same for both systems31. The total turnover number (TTN) of 16,000 calculated for the 8 h reactions with the 2.5 wt% Au–2.5 wt% Pd/TiO 2 catalyst supplying H 2 O 2 is in the same order as the TTN of 17 900 reported for cyclohexane hydroxylation by wild-type AaeUPO with H 2 O 2 generated by photocatalysis (reaction time 5 h)17. Assays of residual activity remaining in the enzyme after 4 h of reaction in the GOX and metal catalyst systems showed that in both cases the enzyme retained 50–55% of the initial activity. Hence, the H 2 O 2 producing metal catalyst is not accelerating the deactivation of the PaDa-I during extended reaction times.

System optimisation and substrate scope

Due to the volatile nature of substrates such as cyclohexane, a flowing system with gas bubbling is not optimal and relies on constant substrate addition to prevent complete depletion of the substrate. Following the feasibility experiments showing that combining the two catalytic systems was possible, reactions were carried out in sealed pressurized glassware with a range of substrates. Into the reactor was placed the substrate (10 mM), and the tandem catalyst system formed by the heterogeneous catalyst and the biocatalyst in buffered reaction media. All reactions were carried out with the same initial substrate and enzyme concentration limiting the TTN to ~30,000 in these studies. The system was pressurized to 2 bar under a pre-mixed gas mixture of 80% H 2 and 20% air and stirred at 250 rpm. Under these conditions H 2 O 2 was produced over time (Fig. 3a) in a similar range 15–25 ppm to the flowing system over extended reaction times despite the lack of gas bubbling. The results in Fig. 3a demonstrate that under these conditions cyclohexane hydroxylation can be carried out to produce cyclohexanol with high yields (87%) and TTN of 25,300 with negligible overoxidation to cyclohexanone indicating that the metal H 2 O 2 producing catalyst is not facilitating further overoxidation reactions as observed in our previous experiments using the gas flow reactor.

Fig. 3 Hydroxylation reactions using tandem catalysis system. Time on line hydroxylation reactions of a H 2 O 2 production (black squares) (in the absence of enzyme) and cyclohexane (10 mM) oxidation to cyclohexanol and cyclohexanone b Ethylbenzene (10 mM) oxidation to 1-phenylethanol c Isophorone (30 mM) oxidation to 4 and 7-hydroxyisophorone when PaDa-I (15 U ml RM −1) was coupled with in situ H 2 O 2 generation by 0.5% Au-0.5% Pd/TiO 2 (0.1 mg ml RM −1) using a gas mixture of 80% H 2 in air in a sealed system under 2 bar total pressure Full size image

The ability of biocatalysts to carry out chiral transformations is a major advantage over traditional heterogeneous systems. The hydroxylation of ethylbenzene was carried out over extended times with near full conversion being reached at 16 h (Fig. 3b). While in this case some overoxidation was observed when the reaction had consumed the majority of ethylbenzene, again a high TTN was observed (25,900) at 12 h with near complete selectivity to 1-phenylethanol. Chiral analysis of the 1-phenylethanol produced showed an e.e. of ~98% towards R-1-phenylethanol at 16 h, which is comparable to reported values concerning the peroxygenase alone indicating that the presence of the metal catalyst does not affect the stereoselectivity of the PaDa-I hydroxylation reactions (Supplementary Fig. 3)19. To demonstrate the possibility of achieving higher TTN a reaction was carried out with 20 mM starting concentration for 16 h resulting in 13.1 mM of 1-phenyl ethanol and 2.9 mM of acetophenone at a TTN of 51,400. To test the system at extended reaction times an additional reaction was carried out with an initial concentration of 30 mM ethylbenzene at the same metal and enzyme catalyst loadings. After an initial 16 h of reaction, a further 30 mM of substrate was added and gas re-charged and this was repeated after another 24 h - resulting in 90 mM of substrate addition over 64 h. The system was able to produce 46 mM of 1-phenyl ethanol and 14 mM of acetophenone - achieving a TTN of 201,000 showing that this is among the most efficient tandem heterogeneous H 2 O 2 delivery—UPO systems reported to date.

The selective functionalization of isophorone provides a route to synthetic precursors of both fragrance molecules and the building blocks of carotenoids and tocopherols (Vitamin E)32,33. α-Isophorone is produced industrially on a large scale and selective hydroxylation to 4-hydroxyisophorone is a target transformation to produce ketoisophorone without the industrially executed detour of first isomerizing to ß-isophorone34,35,36. The direct transformations have been reported using both homogeneous metal chlorides as catalysts at 80 °C in the presence of radical initiators and biocatalytic transformations based on P450s requiring cofactors37,38. As a proof of principle using our tandem catalyst system we were able to drive the hydroxylation of isophorone (30 mM) to produce a mixture of 4-hydxoyisophorone and 7-hydroxyisophorone in roughly equal amounts over a 6 h period at a combined yield of 75% (Fig. 3c). The unselective nature of the hydroxylation is inherent to the selectivity of the enzyme but acts as a demonstration that this transformation could be possible with a more selective peroxygenase, which could be engineered by new rounds of laboratory evolution. In all cases cyclohexane, ethylbenzene and isophorone reactions containing the metal catalyst producing H 2 O 2 without the presence of the PaDa-I did not produce any hydroxylated products resulting from C–H activation as a result of the heterogeneous catalyst or the presence of H 2 O 2 alone (Supplementary Fig. 4). In addition, the activity of the metal catalyst producing H 2 O 2 alone towards the oxidation of cyclohexanol and 1-phenylethanol to the respective ketones was minimal (Supplementary Fig. 5).

The substrate scope was increased to include a wide range of C–H functionalization and is shown in Table 1 for a series of unoptimized reactions. Again, in the case of tetralin and propylbenzene negligible C-H activation was observed in the presence of the metal catalyst generating H 2 O 2 without the PaDa-I (Supplementary Fig. 6). Oxygenated products were produced by the tandem catalyst system for this wide range of substrates and with high enantiomeric excess (>99%), (Supplementary Fig. 7) typical of the reaction previously carried out with the PaDa-I alone, demonstrating that the system does not change the specificity of the biocatalyst but simply provides a continuous feed of low-level H 2 O 2 to facilitate clean hydroxylation. Reactions of styrene with the tandem catalysis system act as a demonstration of more complicated cascades that can be built by combing the two catalyst systems (Supplementary Fig. 8a). The AuPd catalyst used has no activity towards styrene epoxidation but is able to reduce styrene to ethylbenzene at these conditions (Supplementary Fig. 8b) leading to R-1-phenylethanol production with high enantiomeric excess (Supplementary Fig. 8c) through enzymatic hydroxylation along with styrene oxide. This reaction network of coupled C=C bond reduction and subsequent hydroxylation could be exploited to produce chiral alcohols from alkenes or mixtures of epoxides and chiral alcohols with proportions controlled by the design of the heterogeneous catalyst or reaction conditions. As with many process involving H 2 O 2 in situ reactions H 2 selectivity is challenging. The reported cyclohexane hydroxylation H 2 to cyclohexanol selectivity is of the order of 10% (Supplementary Discussion 1); however, we have previously shown that high selectivity can be engineered through effective catalyst design30,39 and detailed understanding of the reaction kinetics and will be a key feature of any process intensification.

Table 1 Substrate scope of tandem heterogeneous and biocatalytic system Full size table