Controlling product selectivity with nanoparticle composition in tandem chemo-biocatalytic styrene oxidation

Article abstract of DOI:10.1039/d0gc04320f

The combination of heterogeneous catalysis and biocatalysis into one-pot reaction cascades is a potential approach to integrate enzymatic transformations into existing chemical infrastructure. Peroxygenases, which can achieve clean C-H activation, are ideal candidates for incorporation into such tandem systems, however a constant supply of low-level hydrogen peroxide (H₂O₂) is required. The use of such enzymes at industrial scale will likely necessitate thein situgeneration of the oxidant from cheap and widely available reactants. We show that combing heterogeneous catalysts (Au_xPd_y/TiO₂) to produce H₂O₂in situfrom H₂and air, in the presence of an evolved unspecific peroxygenase fromAgrocybe aegerita(PaDa-I variant) yields a highly active cascade process capable of oxidizing alkyl and alkenyl substrates. In addition, the tandem process operates under mild reaction conditions and utilizes water as the only solvent. When alkenes such as styrene are subjected to this tandem oxidation process, divergent reaction pathways are observed due to the competing hydrogenation of the alkene by palladium rich nanoparticles in the presence of H₂. Each pathway presents opportunities for value added products. Product selectivity was highly sensitive to the rate of reduction compared to hydrogen peroxide delivery. Here we show that some control over product selectivity may be exerted by careful selection of nanoparticle composition.

Full text of DOI:10.1039/d0gc04320f

Green Chemistry
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PAPER
Controlling product selectivity with nanoparticle
composition in tandem chemo-biocatalytic
styrene oxidation†
Cite this: Green Chem., 2021, 23,
4170
Derik Wilbers,a,b Joseph Brehm,c Richard J. Lewis, c Jacqueline van Marwijk,d
a,d
Thomas E. Davies,c David J. Morgan, c Diederik J. Opperman,
a,b
Martha S. Smit, a,d Miguel Alcalde,e Athanasios Kotsiopoulos,
Susan T. L. Harrison,*a,b Graham J. Hutchings *c and Simon J. Freakley
*
f
The combination of heterogeneous catalysis and biocatalysis into one-pot reaction cascades is a potential
approach to integrate enzymatic transformations into existing chemical infrastructure. Peroxygenases,
which can achieve clean C–H activation, are ideal candidates for incorporation into such tandem systems,
however a constant supply of low-level hydrogen peroxide (H2O2) is required. The use of such enzymes
at industrial scale will likely necessitate the in situ generation of the oxidant from cheap and widely avail-
able reactants. We show that combing heterogeneous catalysts (AuxPdy/TiO2) to produce H2O2 in situ
from H2 and air, in the presence of an evolved unspecific peroxygenase from Agrocybe aegerita (PaDa-I
variant) yields a highly active cascade process capable of oxidizing alkyl and alkenyl substrates. In addition,
the tandem process operates under mild reaction conditions and utilizes water as the only solvent. When
alkenes such as styrene are subjected to this tandem oxidation process, divergent reaction pathways are
observed due to the competing hydrogenation of the alkene by palladium rich nanoparticles in the pres-
ence of H2. Each pathway presents opportunities for value added products. Product selectivity was highly
sensitive to the rate of reduction compared to hydrogen peroxide delivery. Here we show that some
control over product selectivity may be exerted by careful selection of nanoparticle composition.
Received 22nd December 2020,
Accepted 7th May 2021
DOI: 10.1039/d0gc04320f
rsc.li/greenchem
omic importance.1–4 Oxygenates produced by such processes
may be sold as value-added products or as platform chemicals
Introduction
The activation of hydrocarbons while preventing deep oxi- in diverse supply chains that include the pharmaceutical or
dation to CO2 remains a challenge in heterogeneous catalysis. fine chemical industries.1 Traditional chemocatalytic hydro-
The development of new processes to install oxygen functional- carbon oxidation routes are often inefficient due to operation
ity selectively into C–H bonds while operating under energy under harsh reaction conditions, limited product selectivity
efficient and green process conditions is of significant econ- and the use of environmentally unfriendly oxidants, particu-
larly at lab scale, such as tert-butyl hydroperoxide (TBHP),5
benzoylperoxide
and
meta-chloroperoxybenzoic
acid
(mCPBA).6,7 Enzymatic processes are
a
highly attractive
aSouth African DST-NRF Centre of Excellence in Catalysis, C*Change, University of
Cape Town, Private Bag, Rondebosch, 7701, Cape Town, South Africa.
E-mail: Sue.Harrison@uct.ac.za
bCentre for Bioprocess Engineering Research (CeBER), Department of Chemical
Engineering, University of Cape Town, Private Bag X3, Rondebosch, 7701, Cape
Town, South Africa
cCardiff Catalysis Institute, School of Chemistry, Cardiff University, Main Building,
Park Place, Cardiff, CF10 3AT, UK. E-mail: Hutch@Cardiff.ac.uk
dDepartment of Microbial, Biochemical and Food Biotechnology, University of the
Free State, Bloemfontein, South Africa
eInstitute of Catalysis, ICP-CSIC, Cantoblanco, 28049 Madrid, Spain
fDepartment of Chemistry, University of Bath, Claverton Down, Bath, BA2 7AY, UK.
E-mail: s.freakley@bath.ac.uk
approach for use in hydrocarbon oxidation reactions due to
the typically high stereo- and chemoselectivity of certain
classes of biocatalysts. A number of heme-containing enzymes
such as the cytochrome P450 family are highly active and selec-
tive biocatalysts for substrate specific C–H activations and
epoxidations, but their dependency on sacrificial redox cofac-
tors and auxiliary flavoproteins when using O2 can increase
both the complexity as well as the cost of a potential oxidative
process.8–12 Unspecific peroxygenases (UPOs) are extracellular
heme-thiolate enzymes of fungal origins, capable of various 1
and 2-electron oxidative transformations on a wide range of
substrates.13,14 UPOs can utilize H2O2 as both electron accep-
†Electronic supplementary information (ESI) available. See DOI: 10.1039/
d0gc04320f
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tor and oxygen donor to functionalize C–H bonds without the result in the production of primary or secondary alcohols from
need of a complex electron transport chain or co-factors that a terminal alkene substrate.
need to be regenerated.15 Due to this benefit, UPOs have been
identified as potential industrial biocatalysts for a variety of
oxidative transformations.16
Results and discussion
UPOs are however highly sensitive towards H2O2 concen-
tration. A large excess of H2O2 can result in oxidative damage Styrene was selected as model alkene substrate as it has an
to the enzyme itself, leading to a loss of activity. In order to cir- activated double bond due to the presence of an aromatic ring
cumvent this deactivation, a number of H2O2 delivery methods while possessing no allylic carbons as sites for direct hydroxy-
to UPOs have been reported. These include the use of enzyme lation by the PaDa-I enzyme. Industrially the production of
cascades (glucose oxidase/peroxygenase17,18 and formate styrene oxide from styrene is commonly carried out by either
oxidase/peroxygenase systems19,20) oxidase and peroxygenase chlorohydrin-based routes involving sequential chlorination–
fusions,21 and a number of heterogeneous chemo- and dehydrochlorination cycles or reactions involving benzoyl
photocatalysts.22–25 Among heterogenous catalysts, AuxPdy bi- peroxide.28–30 These reactions are undesirable from a green
metallic nanoparticles on various supports have been reported chemistry point of view as the chlorination–dechlorination
as highly active catalysts for H2O2 direct synthesis.23,26,27 We route produces a large amount of hazardous waste. Attempts
reasoned that such AuxPdy bimetallic nanoparticles might find to facilitate oxidation at milder conditions using radical
further utility in a chemo-bio tandem system when employing initiators such as TBHP and H2O2 are typically carried out at
peroxygenases to upgrade alkenes.
elevated temperature and can often result in the isomerisation
We recently reported on such a tandem catalytic system com- of styrene oxide to benzaldehyde. Styrene oxide, resulting from
posed of metal nanoparticles and the laboratory-evolved UPO the epoxidation of styrene, is an important industrial chemical
from Agrocybe aegerita (PaDa-I variant) for selective oxidation of while 2-phenylethanol resulting from ring opening of the
C–H bonds.22 In this work, we designed a one-pot system in styrene oxide finds application in the fragrance, cosmetics and
which metal nanoparticles catalyzed the in situ formation of food industry.31 These are both potential products of the reac-
H2O2 from a H2/air mixture at a rate and concentration appro- tion cascade if the styrene undergoes an epoxidation reaction
priate for subsequent enzymatic peroxygenase activity. This catalyzed by PaDa-I using the in situ produced H2O2 followed
combination could remove the need to transport, store and by hydrogenative ring opening in the presence of the hetero-
dilute concentrated H2O2 solutions on translation to a large geneous metal catalyst and gas phase H2 (Scheme 1, Pathway
scale. One-pot reaction setups can be attractive from an indus- 1). The demonstration of a possible one pot route to the
trial point of view due to fewer expensive separation and purifi- fragrance molecule 2-phenylethanol from styrene has potential
cation steps typically being required. However, process con- to replace sequential oxidative and reductive reactions which
ditions are often confined to narrow operating windows to require multiple stage catalytic processes. Alternatively, the
ensure compatibility between the different catalytic processes. styrene may be reduced by the Pd rich catalysts in the presence
In this case, we were able to operate the catalytic system in one- of H2 to form ethylbenzene. The formed ethylbenzene can
pot, at ambient temperature and relatively low pressures while then undergo C–H activation, catalyzed by PaDa-I using H2O2
employing buffered aqueous solutions as the solvent. Under as oxidant, to form 1-phenylethanol. PaDa-I selectively pro-
these reaction conditions a range of different alkane substrates duces the secondary alcohol from ethylbenzene hydroxylation
including cyclohexane and ethylbenzene were successfully oxi- (Scheme 1, Pathway 2) in high enantiomeric excess.22 By con-
dized to various oxygen-containing products with high chemo trolling the reaction rates of the first catalytic transformation
selectivity, retaining the advantage of high enantiomeric excess of styrene, it is in principle possible to produce either the
associated with biocatalytic transformations and achieving primary or secondary alcohol from the terminal alkene reac-
some of the highest turnover numbers yet reported.22
tant. The realization of a tandem hetero-bio reaction system
When employing alkenes as reaction substrate in our one- would lead to the demonstration of a route to process intensi-
pot system, the reduction of the alkenyl moiety by the Pd con- fication removing the need to produce and store concentrated
taining catalysts in the presence of H2 becomes a competing H2O2 solutions. In contrast to current industrial technologies
reaction and much more complex product distributions are (described above), the activation of C–H and CvC bonds at
observed. This requires a greater understanding of the reaction mild conditions (aqueous solutions, ambient temperatures,
networks as the heterogeneous catalyst not only dictates the near ambient pressure) and producing only water as a by-
amount of H2O2 produced but also catalyzes primary reactions product is an attractive green technology.
with the substrate that compete with enzymatic oxidation. In
Our initial studies established the efficacy of a series of 1%
this manner a greater number of potential products could be AuxPdy/TiO2 catalysts, of varying Au : Pd ratio, towards the
obtained by cascading bio and heterogeneous catalytic reac- direct synthesis and subsequent degradation of H2O2 under
tions and process selectivity may thus be controlled by vari- conditions previously optimized for H2O2 formation
ation of chemocatalyst composition. In this paper we aim to (Table S1†). Consistent with previous studies,32 we observe
demonstrate that control of the reaction cascade, in terms of that an enhancement in catalytic performance can be achieved
the order of enzymatic and heterogeneous catalytic steps can through the combination of Au and Pd, with H2O2 synthesis
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Scheme 1 Divergent reaction pathways observed for the tandem oxidation of styrene.
h
−1) far Both H2 and O2 are known to easily dissociate over Pd0 sites,
−1
rates of the Au50Pd50/TiO2 catalyst (94 molH O kgcat
2
2
greater than that observed with the corresponding monometal- whereas O2 is stable over PdO surfaces.36–38 As such the pres-
lic analogues when tested at elevated pressures. Measurement ence of Pd–PdO domains is likely a key factor responsible for
of the mean nanoparticle size of the various catalysts (Table 1) the enhanced activity observed for the optimal Au50Pd50/TiO2,
as determined by bright field transmission electron promoting the dissociation of adsorbed H2, while preventing
microscopy (BF-TEM) (Fig. S1†) reveals that the Au50Pd50/TiO2 the cleavage of the O–O bond, which would result in the unse-
catalyst displays the smallest mean particle size (4.2 1.8 nm) lective production of water.
amongst the catalysts with a comparatively narrow size distri-
Building on these studies we next evaluated the perform-
bution. In contrast the other bimetallic nanoparticles have ance of the supported 1% AuxPdy/TiO2 catalysts towards H2O2
much wider size distributions and, in some cases, large par- synthesis, under conditions identical to those that had been
ticles around 40 nm are observed. Similar nanoparticle size previously utilized for the UPO with the addition of gaseous
distributions have been previously reported by us for analo- reactants (2 bar, 80% H2, 20% air) (Fig. 1). The results demon-
gous systems with detailed studies indicating that such large strate that even at conditions that are significantly milder than
particles are typically very rich in Au with little to no alloying those optimized for H2O2 synthesis (high pressure, alcohol/
to Pd and as a result are much less active for H2O2 water solvent systems, high agitation) it is possible to produce
synthesis.32,33 Further analysis of the as-prepared AuxPdy cata- H2O2 concentrations of 5–20 ppm with all catalyst systems that
lysts via X-ray photoelectron spectroscopy (Fig. S2 and contain Pd. The Au100/TiO2 catalyst did not produce measur-
Table S2†) indicates that the introduction of Au into a mono- able concentrations of H2O2 during the test. The Pd100/TiO2
metallic Pd catalyst significantly modifies Pd-oxidation state, had the highest initial rate for H2O2 synthesis; however, over
with a shift towards Pd2+ upon Au incorporation. The presence time the concentration of H2O2 in solution decreased due to
of domains of mixed Pd oxidation state has been well reported the higher rates of subsequent H2O2 degradation that occur on
to improve catalytic performance towards H2O2 synthesis.34,35 monometallic Pd catalysts. In contrast, the Au50Pd50/TiO2 cata-
lyst showed comparable high initial rates and was able to
sustain a consistent concentration of H2O2 (15 ppm) through-
Table 1 Mean particle size of 1% AuxPdy/TiO2 catalysts, prepared via an out the test due to a reduced rate of H2O2 degradation which
has been reported for this class of catalyst.32 This H2O2 con-
excess chloride impregnation methodology, as determined by trans-
mission electron microscopy
centration is near optimal for sustained operation of the UPO
PaDa-I according to previous studies.16,39
Catalyst
Mean particle size/nm (standard deviation)
We next evaluated the performance of the supported
AuxPdy/TiO2 catalysts when used in tandem with PaDa-I, for
the oxidation of styrene (Table 2) in a one-pot system. The
Au100/TiO2 catalyst exhibited very low levels of product for-
mation in the tandem system, within the errors of our analysis,
which is unsurprising given the limited activity of monometal-
lic Au catalysts towards both the direct synthesis of H2O2 and
Au100/TiO2
24.9 (7.9)
13.1 (8.3)
4.2 (1.8)
6.5 (8.5)
n.d.a
Au75Pd25/TiO2
Au50Pd50/TiO2
Au25Pd75/TiO2
Pd100/TiO2
a Unable to determine due to particle sizes being below TEM limits of
detection.
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Fig. 1 (a) Net H2O2 productivity by various Au–Pd nanoparticles as a function of Au : Pd (b) concentration of H2O2 observed in solution as a func-
tion of Au : Pd. Key: Au25Pd75/TiO2 (purple triangles), Au50Pd50/TiO2 (black squares), Au75Pd25/TiO2 (green inverted triangles), Pd100/TiO2 (red
circles). H2O2 direct synthesis reaction conditions: Catalyst (2 mg), phosphate buffer (10 mL, pH 6.0), using a gas mixture of 80% H2/Air (1.6 bar H2,
0.4 bar air, 2 bar), 200 rpm.
Table 2 Obtained product selectivities and activity for the tandem oxidation/reduction of styrene employing various Au–Pd nanoparticles sup-
ported on TiO2
Product selectivity (%)
Pathway 1
Pathway 2
Catalyst
Styrene oxide 2-Phenylethanol Ethylbenzene 1-Phenylethanol Styrene conversion (%) Total turnover numbera (×103)
Au100/TiO2
Au75Pd25/TiO2 27
Au50Pd50/TiO2 40
Au25Pd75/TiO2
Pd100/TiO2
0
0
2
0
4
1
100
68
58
82
92
0
3
2
6
1
<1
39
37
66
48
0b
6.8
8.6
6.7
2.0
7
6
Reaction conditions: Substrate concentration – 20 mM, heterogeneous catalyst (2 mg), PaDa-I (15 U mL−1), phosphate buffer (10 mL, pH 6.0),
stirred (200 rpm) for 1 hour at 25 °C, 2 bar (80% H2, 20% air). a Total enzymatic turnovers to oxidised products. b No oxidation products detected.
alkene hydrogenation, under these reaction conditions.40,41 By amounts of oxygenated products derived from the epoxidation
comparison, use of the Pd-only catalyst resulted in far greater of styrene by the PaDa-I. In particular the Au50Pd50/TiO2
activity (48% styrene conversion), with high selectivity (92%) sample which produced the highest consistent H2O2 concen-
towards ethylbenzene, again this may be expected given that trations could obtain 40% styrene oxide selectivity under these
Pd is known to be a highly active hydrogenation catalyst.
reaction conditions. This suggests that if the nanoparticles can
These observations alone may lead to the conclusion that also effectively catalyze the direct H2O2 synthesis, the driving
increasing Pd content would typically favor the fast hydrogen- force behind Pathway 1, the balance between the rates of H2O2
ation of styrene followed by hydroxylation of the alkyl C–H synthesis and styrene hydrogenation will dictate the observed
bond (Pathway 2 as shown in Scheme 1). A set of experiments reaction selectivity and this is dependent on the hetero-
was performed in the absence of the enzyme in order to test geneous catalyst composition. A comparison of the relative
the catalytic styrene hydrogenation activity of the AuxPdy/TiO2 selectivity to Pathway 1 and 2 combined with H2O2 synthesis
catalysts. All ratios of AuxPdy/TiO2 (apart from Au100/TiO2) activity as a function of heterogeneous catalyst composition is
exhibited quantitative conversion of the styrene to ethylben- shown in Fig. 2.
zene within 1 hour, under our standard reaction conditions
Considering the two extremes in selectivity for each
and 1.6 bar of H2. Despite this, catalysts containing AuPd bi- pathway, the Pd100/TiO2 catalyst rapidly produced H2O2 in the
metallic nanoparticles were able to also produce significant initial stages of the reaction after which a drop in H2O2 con-
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Fig. 2 Pathway selectivity with increasing Pd content from Table 2 (left axis) superimposed on steady state H2O2 concentration determined from
4 h tests in Fig. 1b (right axis). Combined pathway selectivity calculated as ∑(styrene oxide, 2-phenylethanol) and ∑(ethylbenzene, 1-phenylethanol)
as a percentage of total reaction products. Conversion values can be found in Table 2.
centration is observed (Fig. 1b). However, only low concen- 20 min of reaction. After this, subsequent enzyme deactivation
trations of Pathway 1 products were observed in our tandem test appears to be reduced with 50% and 21% residual enzyme
reactions. Although H2O2 is rapidly produced in the early stages activities observed after 1 and 24 h respectively. This initial
of the reaction, the Pd100/TiO2 catalyst is likely the most active enzyme deactivation is likely taking place due to the sudden
catalyst for styrene hydrogenation and degradation of the increase in the concentration of organic substrate and H2O2 at
formed H2O2. This coupled with the high rate of CvC hydro- the start of the reaction.
genation relative to the enzymatic oxidation reaction, can
Based on the high selectivity towards Pathway 1 products,
explain the observed preference for Pathway 2 over Pathway 1 in the Au50Pd50/TiO2 catalyst was selected for further experimen-
this system. The Au50Pd50/TiO2 catalyst on the other hand tation. A study to determine the product distribution with
exhibited similarly high activity for H2O2 production but also respect to time online was then performed (Fig. 3 – split into
produced products derived from a primary interaction of the (i) primary reaction products (ii) ethylbenzene derived pro-
substrate with the enzyme in high selectivity (40%). The ducts (iii) styrene oxide derived products).
decreased amounts of Pd in this catalyst, resulting in lower
A maximum of styrene oxide yield (Pathway 1) is achieved
styrene hydrogenation activity relative to the Pd100/TiO2 catalyst after 1 hour of reaction time. The formed styrene oxide is then
with similarly high reactivity in the synthesis of H2O2 likely converted to 2-phenylethanol by hydrogenative ring opening
explains the high Pathway 1 selectivity achieved. Alternatively, between 1 and 4 hours. After 2 hours of reaction the starting
the PaDa-I enzyme might be inhibited by the high H2O2 concen- material has been mostly consumed by a combination of
trations in the initial stages of the reaction when using the epoxidation and hydrogenation reactions and no further
Pd100/TiO2 (shown in Fig. 1b). This would cause the styrene styrene oxide can form. The hydrogenation reaction is domi-
hydrogenation reaction to dominate and low selectivity for pro- nant during the early stages of the reaction and a high concen-
ducts along Pathway 1 (Table 2). The above hypothesis was eval- tration of ethylbenzene is produced even at relatively short
uated by monitoring residual enzyme activity with increasing reaction times. The formed ethylbenzene is then converted to
reaction time under our conditions. It was found that enzyme 1-phenylethanol, the major reaction product at long reaction
deactivation occurs rapidly during the initial stages of the reac- times, via enzymatic oxidation by the PaDa-I/H2O2 system
tion with residual enzyme activity of 56% observed after only which we have previously shown to retain the high ee (98%)
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Fig. 3 Reaction yield as a function of time obtained for a Au50Pd50/TiO2 catalyzed reaction. Reaction conditions: Substrate concentration – 20 mM,
heterogeneous catalyst (2 mg), PaDa-I (15 U mL−1), phosphate buffer (10 mL, pH 6.0), stirred (200 rpm) at 25 °C, 2 bar (80% H2, 20% air).
associated with enzymatic hydroxylation of ethyl benzene carbons during C–H activation reactions.22 Cumene does not
derived from styrene hydrogenation.22 The over-oxidation of possess a seconday carbon for activation, thus the lower
1-phenylethanol to acetophenone is also observed at extended activity for the production of 2-phenylpropan-2-ol is to be
reaction times, in excess of 15 h. At the end of the reaction the expected.
total enzymatic turnover number was 39 700.
In an attempt to circumvent the fast reduction of styrene
The tandem catalytic system proved to be highly active in and therefore obtain a clearer picture of enzyme specificity, an
the transformation of styrene. However, if styrene is replaced alternative method of in situ H2O2 generation was investigated.
by α-methylstyrene as substrate, a sharp decrease in catalytic The metal catalyst was replaced with glucose oxidase (GOX)
activity is observed. Under the same reaction conditions and glucose as a H2O2 delivery system. A competition experi-
(employing the Au50Pd50/TiO2 as heterogeneous catalyst) as ment was then performed by generating H2O2 in situ with GOX
reported above for styrene, only traces of oxygenated products (removing the Pd catalyzed hydrogenation of styrene) while in
and the reduced compound, cumene, were observed after the presence of styrene and ethylbenzene in equimolar
1 hour of reaction. Increasing the reaction time to 4 hours amounts to determine the enzyme preference for styrene
yielded a substrate conversion of 28%. Under these conditions epoxidation vs. C–H hydroxylation of ethylbenzene, (10 mM of
2-phenyl-propane-1,2-diol (determined by GC-MS), likely each component was used for comparison to standard sub-
formed by hydrolysis of the epoxide, was the major product of strate loading of 20 mM styrene only). Under these metal free
the reaction (78%) while other Pathway 1 (epoxidation first) conditions PaDa-I preferentially epoxidized styrene to styrene
products were observed in a combined selectivity of 5%. The oxide (78% selectivity), with comparatively much less oxidation
heterogenous catalyst (Au50Pd50TiO2) was much less active for of the ethyl benzene to 1-phenylethanol (22% selectivity)
the reduction of α-methylstyrene than styrene, with cumene observed. Relative to the GOX/PaDa-I system, the AuxPdy/TiO2
forming in relatively low selectivity (18%). After 24 h, near catalysts (with exception of the Au50Pd50/TiO2) exhibited poor
quantitative conversion of α-methylstyrene is achieved. High selectivity towards products along Pathway 1, due to the high
selectivity towards products along Pathway 1 is maintained catalytic activity exhibited for the styrene hydrogenation
(81% combined selectivity). At such extended reaction times, reaction.
2-phenylpropan-2-ol (6%), formed by the enzymatic oxidation
Based on this observation and in a further attempt to sup-
of cumene (13%) is also observed. Previous studies have press alkene hydrogenation in this system, the use of Lindlar’s
shown that PaDa-I is highly selective towards secondary catalyst (Alfa Aesar) was investigated as a means to inhibit the
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alkene hydrogenation reaction, which initiates reaction Under their process conditions deoxygenation and reduction
Pathway 2. Lindlar’s catalyst is reported to selectively reduce to ethylbenzene was also observed and lead to the formation
alkynes to alkenes but not alkenes to alkanes.42 Lindlar’s cata- of styrene and ethylbenzene as side products. Various sup-
lyst consists of Pd nanoparticles (typically large and poorly ported palladium catalysts have also been reported for this
defined) supported on CaCO3 and poisoned with lead.43 To process, some of which are highly selective towards the for-
the best of our knowledge this catalyst has never been tested mation of 2-phenylethanol.50,51
for H2O2 synthesis; however, we show that under our opti-
Under our reaction conditions, the Lindlar catalyst was rela-
mized conditions (Table S1†) the catalyst has very high levels tively inactive for the hydrogenation of styrene oxide, as might
of H2O2 degradation activity. Under our standard H2O2 syn- be expected from the low 2-phenylethanol selectivities reported
thesis conditions, the Lindlar catalyst performed poorly com- in Table 3 (short reaction times). In contrast, AuxPdy/TiO2 cata-
pared to the AuxPdy/TiO2 catalysts, however, small amounts of lysts were much more active, with the Au50Pd50/TiO2 catalyst
H2O2 were formed under our tandem reaction test conditions. converting more than 60% of the starting material after 1 h
Initial results of the Lindlar/PaDa-I tandem system (Table 3, (Fig. 4). This observation demonstrates that if the initial reac-
entry 1) were promising with the Lindlar catalyst achieving tion selectivity can be steered towards the production of
relatively high selectivity towards styrene oxide although at styrene oxide using AuxPdy catalysts, high yields of 2-pheny-
very low substrate conversion. This selectivity was maintained lethanol can be achieved. Under our reaction conditions using
for up to four hours, however, at longer reaction times (24 h) the AuxPdy/TiO2 catalysts, 2-phenylethanol was produced selec-
the selectivity towards Pathway 1 products is lost.
tively with no other products detected via GC analysis.
This could be an indication that the Lindlar catalyst is de-
activating the enzyme over the course of the reaction, with very
little enzyme activity left after 24 hours. This would also
explain the low 1-phenylethanol selectivity observed after
24 hours even though a high concentration of ethylbenzene is
formed. The deactivation of various different enzymes by
heavy metals in solution have previously been reported.44–46
Lead, present in small quantities within the Lindlar catalyst is
known to bind to disulfide bonds and cysteine residues poten-
tially precipitating and deactivating enzymes.47 Such moieties
are also present in PaDa-I, but to the best of our knowledge
such deactivation studies have not yet been performed on
PaDa-I.39,48 It was hoped that by employing the Lindlar catalyst
no alkene reduction would be observed. Unfortunately, the
double bond in styrene is likely too active and reduction of
this moiety is observed, particularly at longer reaction times.
While the Lindlar catalyst/PaDa-I system exhibited high
selectivity towards styrene oxide at short reaction times, no
2-phenylethanol, expected to form during the ring opening of
styrene oxide, was detected. This observation lead us to further
study the catalytic ring opening of styrene oxide using the
AuxPdy systems. Sasu et al. reported on supported Co and Ni
catalysts for the hydrogenation of styrene oxide to 2-pheny-
lethanol in a cascade process.49 Other authors have investi-
gated the production of 2-phenylethanol from styrene oxide.
Fig. 4 Hydrogenation of styrene oxide to 2-phenylethanol. Reaction
conditions: Substrate concentration – 20 mM, heterogeneous catalyst
(2 mg containing 1wt% metal, apart from Lindlar’s catalyst (5wt%)).
20 mM styrene oxide, phosphate buffer (10 mL, pH 6.0) and stirred for
1 hour at 25 °C, 2 bar (80% H2, 20% air).
Table 3 The application of Lindlar’s catalyst in the tandem oxidation of styrene
Product selectivity (%)/(yield%)
Pathway 1
Pathway 2
Time (h)
Styrene oxide
2-Phenylethanol
Ethylbenzene
1-Phenylethanol
Styrene conversion (%)
1
4
24
45 (2)
46 (10)
0 (0)
0 (0)
0 (0)
7 (7)
55 (2)
54 (11)
83 (83)
0 (0)
0 (0)
10 (10)
4
21
100
Reaction conditions: Substrate concentration 20 mM, heterogeneous catalyst (2 mg), PaDa-I (15 U ml−1), phosphate buffer (10 mL, pH 6.0),
stirred for 1 h at 25 °C, 2 bar (80% H2, 20% air).
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The stability of the PaDa-I enzyme in tandem systems is of consequently a much less atom efficient process for oxidation
paramount importance. Production and purification of the reactions.
enzyme is a time-consuming and expensive process. The feasi-
bility of this tandem process depends on the ability to recycle
the chemo- and biocatalysts. The TiO2 supported nanoparticle
Conclusion
catalysts have been shown to be recyclable up to three times
without significant loss in activity.33 Our previous studies
We have demonstrated the combination of a biocatalyst with
various chemocatalysts into a tandem catalytic process for oxi-
dation of alkenes to alcohols. In contrast to current industrial
technologies, the tandem process is able to operate in aqueous
solutions, under mild reaction conditions while producing
water as the byproduct. A heterogeneous catalyst produces
H2O2 in situ from abundantly available and cheap resources at
levels appropriate for biocatalytic hydroxylation demonstrating
an advance in green chemistry and a route to process intensifi-
cation removing the need to produce and store concentrated
H2O2 solutions. When alkenes are used as substrates in this
process, two competing reaction pathways arise. The first is
due to the production of H2O2 and subsequent enzymatic oxi-
dation to produce an epoxide (which can subsequently be
hydrogenated to a primary alcohol under the same con-
ditions); the second is due to initial hydrogenation of the
alkene to an alkane, followed by C–H activation to produce a
secondary alcohol. The obtained selectivity of this process may
be modulated (to an extent) by careful selection of hetero-
geneous catalyst to control the relative H2O2 synthesis and
alkene hydrogenation rates. This result is very promising for
future studies on this system combined with further catalyst
development to allow production of high yields of either
primary or secondary alcohols from terminal alkene substrates
in a more facile process with high efficiency.
using this system for ethylbenzene hydroxylation (Pathway 2)
have demonstrated that much higher substrate concentrations
can potentially be tolerated compared to the 20 mM used for
the screening reactions in this study. We have previously
reported that on addition of 90 mM of substrate over 64 h (3 ×
30 mM addition with gas recharging) the tandem system was
able to produce 46 mM of 1-phenylethanol and 14 mM of acet-
ophenone.22 This represented a total enzymatic turnover
number of 201 000 with possible accumulation of reaction pro-
ducts limiting activity. To extend the lifetime of these tandem
systems further, future studies will explore the effects of
product inhibition in more complex reaction systems and the
possibility of continuous product extraction or continuous
flow processes using immobilized enzymes. Apart from
enzyme lifetime and catalyst recyclability, analysis of bypro-
ducts and the associated cost of their disposal can have a huge
impact on process feasibility.
In contrast to the previously described chlorination–dehy-
drochlorination process,28–30 enzymatic oxidation employing
H2O2 can occur with higher relative atom economy (AE) as
water is the only side product formed. Previous studies by
Notari have directly compared the atom economies for the
closely related oxidation of propene to propene oxide via the
chlorohydrin and H2O2 based epoxidation processes.52,53 Due
to the large amounts of inorganic halide salts formed by the
traditional chlorohydrin route, the atom economy is low (25%)
while the catalyzed hydrogen peroxide oxidation was consider-
ably higher atom economy of 76%. A similar trend is observed
for the oxidation of styrene to styrene oxide which occurs with
an atom economy of 87% when employing a catalyzed H2O2
oxidation process. A further benefit of using enzymatic H2O2/
alkene epoxidation systems is the ability to work at mild
aqueous conditions compared to the elevated temperatures
needed to activate H2O2 with transition metal salts. The
tandem system we report generates H2O2 in situ and therefore
removes the need to store concentrated H2O2 solutions which
contain stabilisers and will lead to significantly increased reac-
tion volumes due to dilution to appropriate levels for enzyme
tolerance. The direct synthesis of H2O2 from H2 and air can
potentially achieve 100% atom economy when compared to
the anthraquinone process which requires sequential oxi-
dation and reduction of large anthraquinone molecules fol-
lowed by energy intensive extraction from the organic to
Experimental
Unspecific peroxygenase preparation
Evolved AaeUPO (PaDa-I variant) designed in Saccharomyces
cerevisiae was over produced in Pichia pastoris in a bioreactor
and purified to homogeneity (Reinheitszahl value [Rz] [A418
/
A280] ∼ 2.4) according to previous reports.22 Enzyme activities
was determined in triplicate using ABTS as substrate. 20 μl
PaDa-I was added to 180 μl ABTS reaction mixture (100 mM
sodium citrate–phosphate pH 4.4 with 0.3 mM ABTS and
2 mM H2O2) and substrate conversion was followed by measur-
ing the absorption at 418 nm (ε418 = 36 000 M−1 cm−1). The
PaDa-I concentration was appropriately diluted to give rise to
linear enzyme kinetics. One unit is defined as the amount of
enzyme that convert 1 µmol of substrate in 1 min.
Catalyst preparation
aqueous phase. By comparison, the previously reported Mono- and bi-metallic 1 wt%AuxPdy/TiO2 catalysts were pre-
enzyme cascades used for H2O2 delivery utilize either glucose/ pared by a wet co-impregnation procedure which has been pre-
GOX or sulfites/SO to generate H2O2 in situ.17,19,25 The use of viously reported.33 To produce 0.5 wt%Au–0.5 wt%Pd/TiO2
a co-substrate in a coupled redox process results in the (2 g): Aqueous acidified PdCl2 solution (1.667 mL, 0.58 M HCl,
production of significantly more byproducts (D-gluconic acid 6 mg mL−1, Sigma Aldrich) and aqueous HAuCl4·3H2O solu-
or sulfates respectively) by weight than H2O2 and tion (0.826 mL, 12.25 mg mL−1, Strem Chemicals) were mixed
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at 1000 rpm with H2O (HPLC grade) to a fixed working volume products obtained during the tandem oxidation of
of 16 mL in a 50 mL round bottom flask and thereafter heated α-methylstyrene were identified by a combination of GC-MS
to 60 °C. Upon reaching 65 °C, TiO2 (1.98 g, Degussa, P25 (Fig. S3†) and comparison with reference standards by GC-FID
(TiO2) was added to the continuously agitated solution over if available. Results reported are the average of at least two
the course of 5 minutes. The resulting slurry was stirred at duplicate experiments. Product selectivity (%) is defined as
60 °C for a further 15 min. Following this the temperature was mol product per mol total products × 100.
raised to 95 °C for 16 h to allow for complete evaporation of
Enzyme deactivation studies
the water. The resulting solid was ground prior to heat treat-
ment in a 5% H2/Ar atmosphere for 4 h with a ramp rate of Residual peroxygenase activity was determined by using the sub-
10 °C min−1 to 400 °C.
strate 5-nitro-1,3-benzodioxide (NBD) as previously reported.22
Direct synthesis of H2O2
Catalyst characterization
Reactions were carried out in 50 mL gas tight round bottomed X-ray photoelectron spectroscopy (XPS) analyses were made on
flasks rated to 4 bar and stirred using a Radleys 6 Plus Carousel a Kratos Axis Ultra DLD spectrometer. Samples were mounted
equipped with a gas distribution system. Heterogeneous catalyst using double-sided adhesive tape and binding energies were
powder (2 mg) was weighed directly into the glass flasks. To this referenced to the C(1s) binding energy of adventitious carbon
was added 10 mL of 100 mM potassium phosphate buffer (pH contamination that was taken to be 284.8 eV. Monochromatic
6.0) prepared with NaH2PO4 and Na2HPO4 obtained from Sigma AlKα radiation was used for all measurements; an analyser
Aldrich. Subsequently, the flask was sealed and pressurized to pass energy of 160 eV was used for survey scans, while 40 eV
2.0 barg with H2 (1.6 bar) and air (0.4 bar) to give a reaction was employed for more detailed regional scans. The intensities
atmosphere containing 80% H2 and 20% air. The reaction mix- of the Au(4f) and Pd(3d) features were used to derive the Au/
tures were stirred (200 rpm) at 25 °C for 0.5 h, unless otherwise Pd surface composition ratios.
stated. After the desired reaction time the vessel was depressur-
Transmission electron microscopy (TEM) was performed on
ized and the H2O2 concentration determined by UV/Vis spec- a JEOL JEM-2100 operating at 200 kV. Samples were prepared
troscopy. To determine H2O2 concentration an aliquot (1.5 mL) by dispersion in ethanol by sonication and deposited on
of the post reaction solution was combined with potassium tita- 300 mesh copper grids coated with holey carbon film.
nium oxalate dihydrate solution acidified with 30% H2SO4 (0.02
M, 1.5 mL) resulting in the formation of an orange pertitanic
acid complex. This resulting solution was analysed spectropho- Author contributions
tometrically using an Agilent Cary 60 UV/Vis Spectrophotometer
Tandem chemo-bio reactions were carried out by D.W. and S.J.
F. under supervision of A.K. and S.T.L.H. Heterogeneous H2O2
synthesis studies were carried out by J.B. and R.J.L. Enzyme
production in Pichia pastoris was carried out by M.A. Enzyme
at 400 nm by comparison to a calibration curve by taking ali-
quots of H2O2 in the buffer solution (1.5 mL) and adding acidi-
fied potassium titanium oxalate dihydrate solution (1.5 mL).
Reaction conditions used within this study to evaluate catalytic
purification was carried out by J.vM. under the supervision of
performance towards H2O2 production operate outside of the
D.J.O. and M.S.S. Transmission electron microscopy was
flammability limits of gaseous mixtures of H2 and O2.
acquired and analyzed by T.E.D. and X-ray photoelectron spec-
Catalytic testing of the tandem chemo-bio system for
hydrocarbon oxidation
troscopy by D.J.M. The research was directed by S.T.L.H., A.K.,
M.S.S., G.J.H. and S.J.F. The manuscript was written by D.W.,
S.J.F. and R.J.L. with input from all authors.
Hydrocarbon oxidation reactions were performed in 50 mL gas
tight round-bottomed flasks and stirred using a Radleys 6 Plus
Carousel equipped with
a
gas distribution system.
Funding sources
Heterogeneous catalyst powder (2 mg unless stated otherwise)
was weighed directly into the glass flask. To this was added
10 mL of 100 mM potassium phosphate buffer (pH 6.0) fol-
lowed by the substrate (20 mM). PaDa-I enzyme was then
added at a concentration of 15 U mL−1. Subsequently, the flask
was sealed and sequentially pressurized to 2.0 bar with H2 (1.6
bar) and then with air (0.4 bar). The reaction mixtures were
stirred at 200 rpm at 25 °C for 1 h. After 1 h the reaction vessel
was placed in an ice bath for 2 minutes and the pressure
slowly released. Organic products were extracted using (2 ×
5 mL) of ethyl acetate containing 1-decanol (2 mM) as internal
standard. A sample was then withdrawn for GC analysis.
Components were identified by comparison of retention times
SJF acknowledges the award of a Prize Research Fellowship from
the University of Bath and a RSC Research Mobility Grant
(RM1802-7768). Financial support from the University of Cape
Town and Department of Science and Technology/National
Research Foundation Centre of Excellence in Catalysis (C*Change)
is also gratefully acknowledged (DW). Further funding and support
through the DST-NRF SARChI chair in Bioprocess Engineering
(UID 64788), held by STLH, is gratefully acknowledged.
Conflicts of interest
with pure reference standards authentic samples. Reaction There are no conflicts to declare.
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23 W. Zhang, B. O. Burek, E. Fernández-Fueyo, M. Alcalde,
J. Z. Bloh and F. Hollmann, Angew. Chem., Int. Ed., 2017,
56, 15451–15455.
References
1 Y. Ishii, S. Sakaguchi and T. Iwahama, Adv. Synth. Catal.,
2001, 343, 393–427.
2 B. S. Lane and K. Burgess, Chem. Rev., 2003, 103, 2457–
2473.
24 W. Zhang, E. Fernández-Fueyo, Y. Ni, M. Van Schie, J. Gacs,
R. Renirie, R. Wever, F. G. Mutti, D. Rother, M. Alcalde and
F. Hollmann, Nat. Catal., 2018, 1, 55–62.
3 E. M. McGarrigle and D. G. Gilheany, Chem. Rev., 2005, 25 M. M. C. H. Van Schie, W. Zhang, F. Tieves, D. S. Choi,
105, 1563–1602.
C. B. Park, B. O. Burek, J. Z. Bloh, I. W. C. E. Arends,
C. E. Paul, M. Alcalde and F. Hollmann, ACS Catal., 2019,
9, 7409–7417.
4 G. Shul’pin, Catalysts, 2016, 6, 50–90.
5 A. Jimtaisong and R. L. Luck, Inorg. Chem., 2006, 45,
10391–10402.
6 M. Mitra, H. Nimir, D. A. Hrovat, A. A. Shteinman,
M. G. Richmond, M. Costas and E. Nordlander, J. Mol.
Catal. A: Chem., 2017, 426, 350–356.
26 B. O. Burek, S. R. de Boer, F. Tieves, W. Zhang, M. van
Schie, S. Bormann, M. Alcalde, D. Holtmann, F. Hollmann,
D. W. Bahnemann and J. Z. Bloh, ChemCatChem, 2019, 11,
3093–3100.
7 K. A. Jorgensen, Chem. Rev., 1989, 89, 431–458.
8 T. Sakaki, Biol. Pharm. Bull., 2012, 35, 844–849.
27 J. K. Edwards and G. J. Hutchings, Angew. Chem., Int. Ed.,
2008, 47, 9192–9198.
9 P. Durairaj, J. S. Hur and H. Yun, Microb. Cell Fact., 2016, 28 J. Sebastian, K. M. Jinka and R. V. Jasra, J. Catal., 2006, 244,
15, 125. 208–218.
10 M. Sono, M. P. Roach, E. D. Coulter and J. H. Dawson, 29 G. Xu, Q. H. Xia, X. H. Lu, Q. Zhang and H. J. Zhan, J. Mol.
Chem. Rev., 1996, 96, 2841–2888. Catal. A: Chem., 2007, 266, 180–187.
11 H. Lin, J. Y. Liu, H. B. Wang, A. A. Q. Ahmed and Z. L. Wu, 30 M. S. Batra, R. Dwivedi and R. Prasad, ChemistrySelect,
J. Mol. Catal. B: Enzym., 2011, 72, 77–89. 2019, 4, 11636–11673.
12 J. B. Y. H. Behrendorff, W. Huang and E. M. J. Gillam, 31 D. Hua and P. Xu, Biotechnol. Adv., 2011, 29, 654–660.
Biochem. J., 2015, 467, 1–15.
13 M. Hofrichter and R. Ullrich, Curr. Opin. Chem. Biol., 2014,
19, 116–125.
32 A. Santos, R. J. Lewis, G. Malta, A. G. R. Howe,
D. J. Morgan, E. Hampton, P. Gaskin and G. J. Hutchings,
Ind. Eng. Chem. Res., 2019, 58, 12623–12631.
14 M. Faiza, S. Huang, D. Lan and Y. Wang, BMC Evol. Biol., 33 M. Sankar, Q. He, M. Morad, J. Pritchard, S. J. Freakley,
2019, 19, 76.
J. K. Edwards, S. H. Taylor, D. J. Morgan, A. F. Carley,
D. W. Knight, C. J. Kiely and G. J. Hutchings, ACS Nano,
2012, 6, 6600–6613.
15 M. M. C. H. Van Schie, W. Zhang, F. Tieves, D. S. Choi,
C. B. Park, B. O. Burek, J. Z. Bloh, I. W. C. E. Arends,
C. E. Paul, M. Alcalde and F. Hollmann, ACS Catal., 2019, 34 L. Ouyang, P. F. Tian, G. J. Da, X. C. Xu, C. Ao, T. Y. Chen,
9, 7409–7417. R. Si, J. Xu and Y. F. Han, J. Catal., 2015, 321, 70–80.
16 P. Molina-Espeja, S. Ma, D. M. Mate, R. Ludwig and 35 X. Gong, R. J. Lewis, S. Zhou, D. J. Morgan, T. E. Davies,
M. Alcalde, Enzyme Microb. Technol., 2015, 73–74, 29–
33.
X. Liu, C. J. Kiely, B. Zong and G. J. Hutchings, Catal. Sci.
Technol., 2020, 10, 4635–4644.
17 F. Van De Velde, N. D. Lourenço, M. Bakker, F. Van 36 R. Imbihl and J. E. Demuth, Surf. Sci., 1986, 173, 395–410.
Rantwijk and R. A. Sheldon, Biotechnol. Bioeng., 2000, 69, 37 X. Guo, A. Hoffman and J. T. Yates, J. Chem. Phys., 1989, 90,
286–291.
5787–5792.
18 R. Narayanan, G. Zhu and P. Wang, J. Biotechnol., 2007, 38 M. Salmeron, Top. Catal., 2005, 36, 55–63.
128, 86–92.
39 P. Molina-Espeja, E. Garcia-Ruiz, D. Gonzalez-Perez,
19 F. Tieves, S. J. P. Willot, M. M. C. H. van Schie,
M. C. R. Rauch, S. H. H. Younes, W. Zhang, J. J. Dong,
R. Ullrich, M. Hofrichter and M. Alcalde, Appl. Environ.
Microbiol., 2014, 80, 3496–3507.
P. Gomez de Santos, J. M. Robbins, B. Bommarius, 40 M. Piccinini, J. K. Edwards, J. A. Moulijn and
M. Alcalde, A. S. Bommarius and F. Hollmann, Angew.
Chem., Int. Ed., 2019, 58, 7873–7877.
20 M. M. C. H. van Schie, A. T. Kaczmarek, F. Tieves, P. Gomez
de Santos, C. E. Paul, I. W. C. E. Arends, M. Alcalde,
G. Schwarz and F. Hollmann, ChemCatChem, 2020, 12,
3186–3189.
G. J. Hutchings, Catal. Sci. Technol., 2012, 2, 1908–1913.
41 D. I. Enache, J. K. Edwards, P. Landon, B. Solsona-espriu,
A. F. Carley, A. A. Herzing, M. Watanabe, C. J. Kiely,
D. W. Knight and G. J. Hutchings, Science, 2006, 311, 362–
366.
42 H. Lindlar, Helv. Chim. Acta, 1952, 35, 446–450.
21 P. Gomez de Santos, S. Lazaro, J. Viña-Gonzalez, 43 A. O. King and I. Shinkai, in Encyclopedia of Reagents for
M. D. Hoang, I. Sánchez-Moreno, A. Glieder, F. Hollmann
Organic Synthesis, John Wiley & Sons, Ltd, Chichester, UK,
and M. Alcalde, ACS Catal., 2020, 13524–13534.
2001, p. 17.
22 S. J. Freakley, S. Kochius, J. van Marwijk, C. Fenner, 44 D. Compagnone, A. S. Lupu, A. Ciucu, V. Magearu,
R. J. Lewis, K. Baldenius, S. S. Marais, D. J. Opperman, C. Cremisini and G. Palleschi, Anal. Lett., 2001, 34, 17–27.
S. T. L. Harrison, M. Alcalde, M. S. Smit and 45 P. N. Nomngongo, J. C. Ngila, V. O. Nyamori, E. A. Songa
G. J. Hutchings, Nat. Commun., 2019, 10, 4178.
and E. I. Iwuoha, Anal. Lett., 2011, 44, 2031–2046.
This journal is © The Royal Society of Chemistry 2021
Green Chem., 2021, 23, 4170–4180 | 4179

View Article Online
Paper
Green Chemistry
46 M. E. Ghica, R. C. Carvalho, A. Amine and C. M. A. Brett, 49 A. Sasu, B. Dragoi, A. Ungureanu, S. Royer, E. Dumitriu and
Sens. Actuators, B, 2013, 178, 270–278. V. Hulea, Catal. Sci. Technol., 2016, 6, 468–478.
47 N. M. Giles, A. B. Watts, G. I. Giles, F. H. Fry, 50 M. M. Telkar, C. V. Rode, R. V. Chaudhari, S. S. Joshi and
J. A. Littlechild and C. Jacob, Chem. Biol., 2003, 10, 677–
693.
48 D. M. Mate, M. A. Palomino, P. Molina-Espeja, J. Martin-
A. M. Nalawade, Appl. Catal., A, 2004, 273, 11–19.
51 I. Kirm, F. Medina, X. Rodríguez, Y. Cesteros, P. Salagre and
J. E. Sueiras, J. Mol. Catal. A: Chem., 2005, 239, 215–221.
Diaz and M. Alcalde, Protein Eng., Des. Sel., 2017, 30, 191– 52 B. Notari, Stud. Surf. Sci. Catal., 1988, 37, 413–425.
198.
53 R. A. Sheldon, Chem. Soc. Rev., 2012, 41, 1437–1451.
4180 | Green Chem., 2021, 23, 4170–4180
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