Photobiocatalytic alcohol oxidation using LED light sources

Article abstract of DOI:10.1039/c6gc02008a

The photocatalytic oxidation of NADH using a flavin photocatalyst and a simple blue LED light source is reported. This in situ NAD⁺ regeneration system can be used to promote biocatalytic, enantioselective oxidation reactions. Compared to the traditional use of white light bulbs this method enables very significant reductions in energy consumption and CO₂ emission.

Full text of DOI:10.1039/c6gc02008a

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Photobiocatalytic alcohol oxidation using LED light sources†
M. Rauch,a S. Schmidt,a I.W.C.E. Arends,a K. Oppelt,b S. Kara,c F. Hollmann*a
Received 00th January 20xx,
Accepted 00th January 20xx
DOI: 10.1039/x0xx00000x
www.rsc.org/
The photocatalytic oxidation of NADH using a flavin photocatalyst photoexcitation of the flavin catalyst. The oxidised flavin
and a simple blue LED light source is reported. This in situ NAD+ catalyst is regenerated spontaneously through aerobic
regeneration system can be used to promote biocatalytic, reoxidation yielding hydrogen peroxide (Scheme 1).14, 15
enantioselective oxidation reactions. Compared to the traditional
use of white light bulbs this method enables very significant
reductions in energy consumption and CO2 emission.
Oxidation reactions employing alcohol dehydrogenases are
experiencing an increased interest in the field of biocatalysis. 1-
4 While the toolbox of selective enzymes is growing constantly,
the choice of efficient in situ regeneration systems of the
oxidized nicotinamide cofactors to sustain the catalytic cycle is
still comparably limited.5 This is particularly true for
regeneration systems employing molecular oxygen as terminal
Scheme 1. Aerobic oxidation of reduced nicotinamide cofactors (NAD(P)H) to the
corresponding cofactors (NAD(P)+) using photoexcited flavin catalysts. Upon
oxidant, which is attractive from the high thermodynamic
driving force and the innoxious nature of the by-products.6-12 photoexcitation (λ=450 nm) the redox potential of the oxidised flavin catalyst increases
dramatically16 enabling fast hydride transfer from NAD(P)H to the flavin. The reduced
flavin reacts spontaneously in a dark reaction with molecular oxygen.14
As early as 1973 Jones and coworkers proposed using simple
flavins to catalyse the aerobic oxidation of reduced
nicotinamide cofactors.13 The slow reaction kinetics of the
During these proof-of-concept experiments we have utilised a
hydride transfer from NAD(P)H to the oxidized isoalloxazine
simple, commercially available light-white bulb. Despite its
moiety rendered the system impractical as significant molar
simplicity, this setup severely suffered from heat generated by
surpluses of the flavin ‘catalyst’ were necessary to attain full
the bulb. As a consequence not only a significant amount of
conversion within a reasonable time frame. More recently, we
electrical power used was deviated into heat generations but
have reported that this limitation (low reaction rates) can be
also additional cooling of the reaction mixture was necessary
overcome simply by applying visible light to the reaction
system.11 The overall rate-limiting hydride transfer step from
to maintain optimal reaction conditions for the enzyme
reaction.
NAD(P)H to the oxidised flavin could be accelerated by
With the advent of cheap light emitting diode (LED) light
sources we became interested in using those to promote
photobiocatalytic oxidation reactions. Compared to the use of
traditional light sources LEDs bear the promise of double
energy efficiency: First, LEDs enable
a more efficient
conversion of electrical power into light energy (only a narrow
range of wavelengths is emitted instead or an entire spectrum
containing ‘useless’ wavelengths) and, second, due to the
absence of thermal effects, also less thermostatting is needed.
As commercial LEDs come with three individual colours (blue,
green and red exhibiting wavelength maxima at 465, 519, and
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631 nm respectively, Figure 1A) we investigated each
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wavelength to promote the photochemical oxidation of NADH. In terms of light intensity, the LED-based photocatalytic system
It should be mentioned here that in accordance with the was well-behaved (Figure 2) and the overall rate of the system
mechanism proposed in Scheme 1, no conversion of NADH was correlated directly with the intensity of the LED light source.
observed the absence of either, the photocatalyst, a light
source or under anaerobic reaction conditions. As shown in
Figure 1B only blue light ( λmax of 465 nm) significantly
accelerated the overall reaction while green light (λmax of 519
nm) yielded significantly lower rates and red light (λmax of 631
nm) had almost no accelerating influence as compared to
‘dark’ conditions. In terms of turnover frequencies the flavin
catalysts performed <5, 30 and 680 catalytic cycles per hour
under red, green and blue light irradiation, respectively. This
trend is in agreement with the relative overlap of the LED
emission spectra and the FMN absorption spectrum Figure
1A). Interestingly, using blue LEDs enabled slightly higher
NADH oxidation rates as compared to bright white light bulbs
under otherwise identical conditions (Figure S8). This
observation, however, should not be over-interpreted as a
quantitative comparison of the light intensities is yet missing.
Figure 2. A: Influence of the intensity of the Blue LED in the oxidation of NADH in
presence of FMN. Conditions: 50 mM KPi Buffer (pH 7), T = 30 °C, [NADH]0 = 0.2 mM,
[FMN] = 2 µM; relative blue light intensity :1:ꢁ, 2:ꢀ, 3: ꢀ, 4:ꢀ, 5:ꢁ, 6:ꢂ; B:
Dependence of the rate of the FMN-catalysed NADH oxidation (expressed as TF of the
FMN catalyst, ꢁ) on the intensity of the blue LED (ꢁ). Conditions: KPi Buffer (pH 7, 50
mM), T = 30 °C, [NADH]0 = 0.2 mM, [FMN]0 = 2 µM
Another advantage of the LED system is the missing thermal effect
on the reaction system as compared to the white light bulb. While
with the first no significant temperature change was observed (in
the absence of external cooling), the latter lead to a temperature
increase of more than 30oC within one hour under the same
conditions(Figure S9).
Encouraged by these results we decided to apply the LED-based
photocatalytic NADH oxidation system to the horse liver alcohol
dehydrogenase (HLADH) catalysed oxidative lactonisation of α,ω-
diols such as meso-3-methyl-1,5-pentanediol (
Scheme 2
).17 In this system, HLADH catalyses the enantioselective
Figure 1. Emsision wavelengths of the LEDs used (A) and the influence of the LED
wavelength on the rate of the photocatalytic aerobic NADH oxidation (B). A: the
emission spectra of the blue, green and red LED are shown (in arbitrary units) together
with the absorption spectrum of the FMN photocatalyst, yellow). B: Time course of the
aerobic oxidation of NADH catalysed by FMN and red (ꢀ), green (ꢀ) or blue (ꢁ) light.
Conditions: 50 mM KPi Buffer (pH 7), [NADH]0 = 0.2 mM, [FMN] = 2 µM, T = 30 °C.
two-step oxidation of the diol starting material yielding enantiopure
(ee > 98%) (S)-4-methyltetrahydro-2H-pyran-2-one using the
oxidised nicotinamide cofactor (NAD+) as primary hydride acceptor.
In situ regeneration of NAD+ from NADH is achieved using the
photocatalytic system outlined in Scheme 1.
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Table 1. Estimation of the wastes generated in the photobiocatalytic lactonisation
reactions.
Contribution
Water
White light bulb
LED
877
6.6
Buffer (glycine)
NAD
0.6
FMN
0.04
0.25
1973
HLADH
Scheme 2. Oxidative lactonization of meso-3-methyl-1,5-pentanediol to (S)-4-
methyltetrahydro-2H-pyran-2-one using horse liver alcohol dehydrogenase (HLADH)
and photocatalytic, aerobic regeneration of NAD+.
Ethyl acetate [a]
CO2 from Cooling [b]
CO2 from Light source [b]
40.4
0
40780
3920
Pleasingly, smooth conversion of the starting material into the
enantiopure lactone product was observed (Figure 3).
[a] used as extraction solvent for GC analysis; [b] to estimate the CO2 emissions
due to electricity consumption, we used the average European CO2 emission
intensity (see SI for further details).
10
8
Though shocking at first sight, these numbers also guide us to
optimise the proposed photobiocatalytic reaction system en
route to higher sustainability. First of all, the overall substrate
loading (and the resulting product titers, respectively) must be
increased significantly. In these experiments, 10 mM product
accumulated resulting in an enormous amount of waste water.
This is obviously inacceptable, both, from an environmental
and an economical point-of-view. Raising the product titers to
industrially demanded 50-200 gL-1 will reduce the E-factor to
approx. 5-20. Similarly, also the contributions of buffers and
catalysts will be reduced. The very high E-factor contribution
of the extraction solvent should not be taken too serious, the
aim of the extraction procedure was to adequately determine
the reagent concentrations and not to establish an
environmentally friendly downstream processing method.
Nevertheless, the high solvent consumption again exemplifies
6
4
2
0
0
1
2
3
4
t [h]
20
the importance of DSP to the overall environmental impact.
Figure 3. Time course of the photoenzymatic oxidation of meso-3-methyl-1,5-
pentanediol (ꢃ) to (S)-4-methyltetrahydro-2H-pyran-2-one (ꢁ) via the intermediate
Finally, we made an attempt to quantify the ‘hidden E-factor’
contributions caused by the electrical power consumed. The
calculation of the CO2 emissions was straightforward using the
power consumptions provided by the manufacturers. Again,
the exorbitant values shown in Table 1 should not be taken too
serious as relatively large light sources were used for a rather
small reaction volume. Raising the product concentration
together with increasing the reaction volume can easily reduce
the E-factor by several orders of magnitude to acceptable
values. These experiments are currently underway.
Nevertheless, Table 1 also visualises the significant positive
contribution that LED technology can have on the
sustainability of photo(bio)catalytic reactions!
lactol (ꢁ). General conditions: 100 mM Glycine-NaOH buffer (pH 9), 100 mM. [diol]0
10 mM, [NADH]0 = 1 mM, [FMN] = 100 µM, [HLADH] = 7.4 µM, 5 drops of catalase, T =
30oC.
°C.
=
Within the first 2 hours of the reaction the mass balance of the
reaction was closed (sum of all reagents 10 ± 0.2 mM) and
decreased to 8 mM after 4 hours. This observation is most
likely explained by the slow, spontaneous hydrolysis of the
lactone product to the corresponding hydroxy acid.17 This
finding is also supported by the significant acidification of the
reaction mixture especially at higher substrate loadings (Figure
S10). Further optimisation of the reaction conditions to
minimise or prevent this undesired side reaction are currently
underway. For example, a tighter pH control and in situ
extraction of the lactone product are promising approaches to
circumvent the undesired hydrolysis.
The energy consumption caused by heating (or cooling) of the
reaction mixture is usually not included in E-factor
calculations. Table 1, however, demonstrates that this ‘hidden
contribution’ by no means is to be neglected as it easily
exceeds the contributions of the reagents.
The catalytic activity of the photocatalyst was 122 h-1 and
thereby significantly lower than determined before (Figure 2).
Partially this can be attributed to the higher pH value of the
reaction medium.11
To assess the environmental impact of the presented
photobiocatalytic oxidative lactonisation we performed an E-
factor analysis (Table 1).18, 19
Overall, the use of simple LEDs represents a simple, more
user-friendly alternative compared to bright white light bulbs.
More excitingly, LEDs also offer significant advantages in terms
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12.
13.
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of energy efficiency as futile wavelengths and heat generation
are omitted. Taking together the lower power consumption
with the increased catalytic activity of the LED system a more
than 90% decrease of energy consumption (and corresponding
CO2 emissions) can be achieved. Furthermore, the significant
thermal effect of the white light bulb also necessitates
additional cooling of the reaction mixture, which can be
reduced or even omitted in case of the LEDs.
,
14.
15.
16.
17.
Conclusions
Today, photocatalysis is in focus of catalysis research.21-23
Comparably few studies deal with photocatalysis to promote
biocatalytic reactions, a ‘combination of two intrinsically green
technologies’.24
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R. A. Sheldon, Chem. Comm., 2008, 3352-3365.
Y. Ni, D. Holtmann and F. Hollmann, ChemCatChem,
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Kroutil, N. d'Alessandro and F. Hollmann, Green Chem.,
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18.
19.
20.
However, neither biocatalysis nor photocatalysis are
environmentally sustainable per se. A (self-)critical evaluation
of the possible environmental impact is necessary to
substantiate ‘green claims’ and to identify bottlenecks as a
basis for improved reaction setups en route to truly
sustainable procedures.
21.
22.
23.
24.
R. N. Perutz and B. Procacci, Chem. Rev., 2016, 116
8506-8544.
J. A. Maciá-Agulló, A. Corma and H. Garcia, Chem. Eur.
J., 2015, 21, 10940–10959.
,
In this study, we have demonstrated that simple LEDs are well-
suited to substitute conventional light sources in flavin-based
NAD+ regeneration systems to promote dehydrogenase-
catalysed oxidation reactions. Particularly, the significantly
deceased energy demand of LED systems make it attractive
envisioning environmentally acceptable syntheses.
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