Highly Productive Oxidative Biocatalysis in Continuous Flow by Enhancing the Aqueous Equilibrium Solubility of Oxygen

Article abstract of DOI:10.1002/anie.201803675

We report a simple, mild, and synthetically clean approach to accelerate the rate of enzymatic oxidation reactions by a factor of up to 100 when compared to conventional batch gas/liquid systems. Biocatalytic decomposition of H₂O₂ is used to produce a soluble source of O₂ directly in reaction media, thereby enabling the concentration of aqueous O₂ to be increased beyond equilibrium solubility under safe and practical conditions. To best exploit this method, a novel flow reactor was developed to maximize productivity (g product L^?1 h^?1). This scalable benchtop method provides a distinct advantage over conventional bio-oxidation in that no pressurized gas or specialist equipment is employed. The method is general across different oxidase enzymes and compatible with a variety of functional groups. These results culminate in record space-time yields for bio-oxidation.

Full text of DOI:10.1002/anie.201803675

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Accepted Article
Title: Highly Productive Oxidative Biocatalysis in Continuous-Flow by
Surpassing the Aqueous Equilibrium Solubility of Oxygen
Authors: Michael R Chapman, Sebastien C Cosgrove, Nicholas J
Turner, Nikil Kapur, and Andrew John Blacker
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201803675
Angew. Chem. 10.1002/ange.201803675
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10.1002/anie.201803675
Angewandte Chemie International Edition
COMMUNICATION
Highly Productive Oxidative Biocatalysis in Continuous-Flow by
Surpassing the Aqueous Equilibrium Solubility of Oxygen
Michael R. Chapman,[a] Sebastian C. Cosgrove,[c] Nicholas J. Turner,*[c] Nikil Kapur*[b] and
A. John Blacker*[a]
Abstract: We report a simple, mild and synthetically clean approach
to accelerate the rate of enzymatic oxidation reactions by up to a
factor of 100 when compared to conventional batch gas/liquid
systems. Biocatalytic decomposition of H2O2 is used to produce a
soluble source of O2 directly in reaction media, enabling the ambient
solubility of aqueous O2 to be increased under safe and practical
conditions. To best exploit the method, a novel flow reactor was
developed to maximize productivity (g product/L/h). This scalable
benchtop protocol provides a distinct advantage over conventional
bio-oxidation, in that no pressurized gas or specialist equipment is
employed. The method is general across different oxidase enzymes
Figure 1. Generating soluble O2 in situ to overcome mass transfer restrictions.
and compatible with a variety of functional groups. These results
culminate in record space-time-yields for bio-oxidation.
An attractive approach to both improve mass transfer and
productivity is to translate bio-oxidation into continuous-flow mode,
whereby the interfacial surface-to-volume ratio may be maximized,
whilst overall reaction time may be minimized. This was well-
demonstrated by Kragl and Löb, who developed a continuous
falling-film microreactor (FFMR) which reaches an O2 saturation
of 8.9 mgL-1 in < 6 s, equating to a kLa value of ~ 20590 h-1
(170 gO2L-1h-1) – 40 × faster than observed in batch mode.[12]
When applied to the O2-dependent galactose oxidase (GOase)
catalyzed oxidation of glucose to gluconic acid, space-time-yield
(STY) was increased 300-fold in the FFMR compared with an
equivalent bubble column bioreactor. More recently, Woodley has
employed an agitated cell reactor (ACR) for the same
transformation.[13] The ACR was shown to double overall reaction
rate solely due to improved O2 mass transfer rates. Likewise,
Gasparini and Jones have independently shown the continuous
ACR model to facilitate the rate of biocatalytic oxidation
reactions,[14,15] whilst Ludwig and Berger have separately
developed aerated-membrane flow reactors for the same
purpose.[16,17] Sadowski has also shown that co-solvents can be
used in biological media to increase the rate of enzyme catalyzed
reactions.[18,19] Despite these recent contributions, no whole-cell
or enzyme flow oxidation process has been demonstrated for
commercial chemical manufacture. In every example described,
the enzyme underperforms due to O2 deprivation.
We envisioned that a different mode of access to soluble O2
is necessary which does not rely on interfacial mass transfer.
Bowers and Hofstetter have previously shown that the iodide-
catalyzed decomposition of aqueous H2O2 results in a maximum
O2 concentration of 0.12M, or ~100 times the equilibrium
solubility.[20] We hypothesized that prior to O2 evaporation, there
is an opportunity for an oxidative biocatalyst to function under 1
bar external pressure yet akin to conditions of a solution saturated
under 100 bar pressure. In doing so, the rate of bio-oxidation may
be increased significantly under mild, safe and general conditions,
avoiding specialist pressurized equipment (Figure 1). By
accelerating biocatalysis in this manner, it becomes feasible to
consider new continuous-flow routes for large-scale oxidation
which would be inoperable in batch mode.
Oxidation reactions represent the second largest process after
polymerization in the bulk chemical industry, contributing ~ 30%
of total production.[1,2] The pharmaceutical and fine chemical
industries generally conduct oxidation steps using metal catalysts,
organoperoxides, sulfoxides or amine oxides.[3–5] However, these
methods are often accompanied by harsh reaction conditions,
poor chemical selectivity and generate significant quantities of
waste.[6,7] In fact, a recent publication by the Green Chemistry
Institute (GCI), alongside six global pharmaceutical corporations,
highlighted oxidation as a reaction type which “companies employ
but would strongly prefer better reagents” – ranking 3rd of 7 priority
areas.[8] One option to address this problem involves use of
oxidative enzymes as natural biocatalysts, which operate under
mild, aqueous process conditions and, in principle, produce less
waste or product contamination than their chemocatalytic
counterparts.[9] The use of water as solvent also allows safer
handling of air or pure O2 as an oxidant when compared with
organic solvents, and equally fits well within the remit of “green
chemistry”.[10] However, O2 is poorly soluble in water (~ 8 mgL-1
from air), representing a fundamental bottleneck for large scale
implementation of enzymatic oxidation. This low driving force for
O2(g) → O2(aq) results in low biocatalytic turnover rates and places
an upper limit on volumetric productivity, as governed by the mass
transfer capability of selected equipment (e.g. an industrial scale
bioreactor has a maximum kLa ~ 500 h-1 for O2 transfer).[11]
[a]
Dr M. R. Chapman, Prof A. J. Blacker
School of Chemistry and School of Chemical and Process
Engineering, Institute of Process Research and Development,
University of Leeds, Woodhouse Lane, Leeds, LS2 9JT, UK
E-mail: j.blacker@leeds.ac.uk
[b]
[c]
Prof N. Kapur
School of Mechanical Engineering, University of Leeds
Woodhouse Lane, Leeds, LS2 9JT, UK
E-mail: n.kapur@leeds.ac.uk
Dr S. C. Cosgrove, Prof N. J. Turner
Manchester Institute of Biotechnology
131 Princess Street, Manchester, M1 7DN, UK
Supporting information for this article is given via a link at the end of
the document.
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For the purpose of our study, catalase was selected as an
enzyme capable of catalyzing the decomposition of H2O2 at near-
diffusive rate (kcat ~ 107 s-1), for in situ generation of O2
equivalents.[21] The irreversible oxidation of benzyl alcohol to
benzaldehyde via GOase M3-5, a mutant capable of oxidizing
benzylic alcohols, was chosen as benchmark reaction for study.
[22]
This choice was deliberate as benzyl alcohol represents a
volatile, poorly soluble and non-natural substrate for the enzyme,
which under traditional aerated conditions is notoriously slow to
oxidize (for time-point experiments, see ESI). The catalyst was
deployed in the form of the cell-free extract (CFE) throughout the
investigation, to maximize industrial relevance; consequently, a
Cu salt was also added to load the metal into the apoenzyme.
Horseradish peroxidase (HRP), a well-known GOase activator
enzyme, was used to maximize catalyst activity (Scheme 1). [23,24]
Table 1. Optimization of GOase-catalyzed bio-oxidation in continuous-flow.
Entry
GOase CFE
(mg.mL-1)[a]
[H2O2][b]
(# feeds)
CSTRs
(n)
τres
(min)
Conv. (%)[c]
/
STY (g.L-1d-1)
1
2
0.5
0.5
6.5
6.5
6.5
6.5
6.5
15
60 (1)
120 (1)
120 (1)
240 (1)
120 (2)
300 (3)
300 (4)
300 (4)
0(0)
1
1
1
1
2
3
4
4
4
4
11
11
11
22
11
13
26
13
26
26
5
9
3
40
4
43
5
64
6
84
7
95/41
92/168
18
8[d]
9
Scheme 1. Model bio-oxidation reaction for study. [E] = enzyme.
6.5
0
10
300 (4)
0
It is noteworthy that fed-batch addition of H2O2 to the
biocatalytic reaction mixture led to a rapid increase in [O2] (up to
40 mg.L-1). Under this condition, the rate of production of 2a was
at its highest (see ESI, Figure S7), though challenging to maintain
in fed-batch mode as all concentrations were diluted over time.
Using a miniaturized cascade CSTR recently developed in our
laboratory,[25] we began optimizing the proposed biocatalytic
cascade reaction with emphasis on the development of a scalable
continuous-flow protocol. An aqueous solution of GOase, HRP,
catalase and CuSO4 was flowed into the CSTR to meet a solution
of 1a (30 mM) and H2O2 – initiating production of both O2 and 2a
(Scheme 1, Table 1). Due to gas liberation, a back-pressure
regulator (40 psi) was used to balance flow-rate and an antifoam
agent employed to prevent blockage (0.01% w/w).
It is important to first note that despite a complex oxidation
mixture, omission of any component led to a reduction in reaction
rate, with no conversion when the GOase CFE was absent. Initial
optimization involved employment of 0.5 mg.mL-1 oxidase CFE
(1.9 U.mL-1), keeping all other enzyme concentrations fixed.
Equal flow-rate of a substrate stream containing 2 and 4
equivalents H2O2 gave 5 and 9% conversion after 11 minutes
residence time (τres), respectively (entries 1 and 2). Increasing
catalyst loading 13-fold allowed 40% conversion of 1a under
identical conditions (entry 3). At this point, supplying more H2O2
(8 equiv.) or doubling τres (22 min) did not show a notable
improvement in conversion (43%, entry 4). Based on these
observations, it was deemed likely that much of the oxidation is
occurring at the initial mixing zone of the reactor (i.e. injection
point), prior to O2 evaporation.
[a] Specific activity of CFE = 3.8 U.mg-1. [b] Total H2O2 concentration (mM)
relative to 1a. [c] Determined by HPLC analysis at steady-state.
[d] [1a] = 60 mM. τres = residence time, conv. = conversion.
By cascading a second CSTR in series, a sequential H2O2
feed was added to the geometry. Delivering 4 equivalents H2O2
over 2 feeds, 64% conversion of alcohol was observed in
equivalent τres (entry 5). Extending τres to 26 minutes, a 10-fold
excess of H2O2 was supplied across 4 sequential reactor inlets,
affording 2a in 95% conversion (41 g.L-1.d-1, entry 7). By
increasing catalyst loading further, a 60 mM substrate feed was
converted to 92% in half the τres (entry 8), demonstrating a space-
time-yield of 168 g.L-1.d-1 (for further optimization details, see ESI).
From our optimization study, we recognized that generating
soluble O2 at multiple positions across the length of the reactor is
essential for rapid oxidation of substrate. To do so more efficiently,
a multi-point injection flow reactor (MPIR) was designed and
constructed, capable of sequentially dosing H2O2 across the full
reactor manifold (Figure 2). Our original model consisted of a
winding PTFE flow channel (660 × 2 × 2 mm) positioned between
two Perspex blocks (100 × 100 × 20 mm). Eleven subchannels
(8 × 0.1 × 2 mm each) are connected between the master channel
and two distributor channels milled directly into the front-face of
the reactor. Each distributor channel acts as a reservoir for H2O2,
allowing fresh peroxide to be injected through multiple points of
the main reaction channel, maximizing the availability of soluble
O2 for the biocatalyst as a function of reactor position (for
computational fluid dynamic analysis, see ESI).
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H2O2 distributor channel
Feed
Product
H2O2 distributor channel
1.0 1.5
2.0
0
0.5
2.5
Scheme 2. Flow process diagram for bio-oxidation using MPIR. BPR = 40 psi.
Figure 2. Left: multi-point injection flow reactor (MPIR), 1 mL syringe for scale.
Right: flow velocities through channel (mm/s), generating O2 at 11 addition ports.
The total volume of the reactor is 2.6 mL, with a pressure capacity
of 5.2 bar. To our knowledge, no flow reactor of this type has been
reported before.
Experimental evaluation of the MPIR showed significant
improvement in performance over the CSTR, as demonstrated by
quantitative conversion of 1a (30 mM) in a τres of 8 minutes (Table
S7, entry 3). Under these unoptimized conditions, reducing [H2O2]
3-fold showed no significant loss in conversion (97%, entry 7).
Alternatively, a 3-fold increase in [1a] (90 mM) allowed 95%
conversion in a τres of 12 minutes (entry 14). Following this
productivity study, [1a] was increased 5-fold (150 mM) and
oxidized to 90% conversion (τres = 15 min), affording a space-time-
yield of 344 g.L-1 d-1 which is twice the maximum theoretical value
.
calculated by Woodley using a mass transfer coefficient for this
system.[11] It is of note however, that the CFE loading required for
this level of reactivity leads to increased viscosity of enzyme feed,
which may become problematic in flow mode. With this in mind,
and to maintain a practical biocatalyst yield (g product/g enzyme),
the conditions of entry 7 (Table S7) were carried forward for
further investigations.
A reaction scope survey indicated that the method is general
across a range of electronically differentiated aryl alcohols.
Literature precedent suggests that electron-poor benzyl alcohols
are necessary for complete oxidation, whilst electron-rich
versions rarely reach > 50% conversion.[26] Directly translating the
optimized flow conditions towards various 4-substituted benzyl
alcohols (1b – 1f, 1i, 1k), we observed ≥ 88% conversion for all
examples, with no apparent electronic requirement on the
aromatic ring with respect to rate of oxidation. Supporting this,
doubly activated aryl 1l was oxidized under the same conditions
to give aldehyde 2l in excellent yield. A broad range of functional
groups were well tolerated, highlighting the exceptionally mild
reaction conditions. Aromatic halides 1g – 1j underwent clean
oxidation to their aldehydes, showing no signs of dehalogenation
to represent an advantage over metal-catalyzed routes.
Furthermore, the method was extended towards N- and S-
heteroaryl alcohols 1n and 1o, producing carboxaldehydes 2n
and 2o in good yield. To exemplify industrial relevance, 5-
hydroxymethylfurfural (1p) was converted to 2,5-diformylfuran
(2p), a key platform chemical in the polymer and pharmaceutical
industries, in 86% at steady-state with full selectivity.
Figure 3. Aldehydes produced from substrate scope investigations. General
conditions are as follows. Enzyme feed: GOase M3-5 (6.5 mgmL-1 CFE), HRP
(0.1 mgmL-1), catalase (0.13 mgmL-1), CuSO4 (0.13 mgmL-1). Substrate feed:
alcohol substrate (30 mM), H2O2 (1 equiv.), antifoam 204 (0.01 wt. %). Peroxide
injection feeds (linked to the same pot): H2O2 (2 equiv.), antifoam 204 (0.01
wt. %). All solutions are made up of aqueous NaPi buffer at pH 7.4 at rt.
This symbolizes a productivity of ~ 144 g.L-1.d-1, which is > 5-fold
greater than the optimized preparative batch version.[27]
Importantly, over-oxidation to the acid was not detected in any
case.
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With a reliable flow protocol in hand, we turned our attention
toward other oxygen dependent biocatalysts. Monoamine
oxidases (MAO-N from Aspergillus niger) are emerging as a
popular class of biocatalyst for the deracemization of racemic
amines, via enantioselective amine oxidation to produce achiral
imine intermediates. These intermediates may then be
intercepted by a source of nucleophile,[28] or non-selectively
reduced back to the racemate for further rounds of
enantiopurification.[29-31] However, all preparative scale
employment of these biocatalysts involve reaction times in the
order of days due to slow oxidation of the covalently-bound FAD
cofactor, which is essential to the catalytic cycle.
We questioned whether our flow method could be useful in
accelerating the rate of oxidation of cyclic amines, a common
substructure to many bioactive molecules. As test candidate,
tetrahydroisoquinoline 3a (THIQ) was chosen to be oxidized to
the corresponding dihydroisoquinoline (DHIQ), 4a, via the D9
mutant of MAO-N. Administering the enzyme in the form of whole
In conclusion, we have exploited a biocatalytic cascade
reaction which utilizes catalase-mediated decomposition of H2O2
to increase the natural aqueous equilibrium solubility of O2 under
ambient conditions. Whilst under this regime, we have shown the
rate of enzymatic oxidation reactions to be enhanced significantly
without the requirement of pressurized gas. To capitalize on this,
a novel multi-point injection flow reactor was constructed which
enables in situ generation of O2 across the entire reaction space.
Using this reactor geometry, various alcohols and amines have
been oxidized to produce a range of aldehydes and imines,
respectively, in excellent yield with unprecedented productivities.
This methodology could be expanded to many O2-dependent
enzymatic reactions, thus unlocking continuous-flow bio-oxidation
more generally.
Acknowledgements
cells in the MPIR, 23% conversion to DHIQ was observed in τres
=
We would like to thank the ACS GCI Pharmaceutical Roundtable
for Green Chemistry Award (M.R.C., N.K., N.J.T., A.J.B.).
N.K. thanks GSK and RAEng for funding his research chair.
The authors would also like to thank Prozomix Ltd. for their
generous donation of biocatalysts.
8 minutes (Table 2, entry 1). It was recognized that diffusion of
substrate/product into/out of the cells may impede the overall rate
of oxidation. Consequently, glass beads (2 mm dia.) were packed
into the flow channel, reducing the reactor volume by 1 mL to
facilitate mixing (see ESI). With these in place, conversion to
DHIQ doubled under otherwise uniform conditions (49%, entry 3).
Optimum conditions were identified maintaining 3 equivalents
H2O2 and τres = 12 minutes, producing DHIQ in 97% conversion at
steady-state (entry 7), with a complete mass balance.
Keywords: biocatalysis • oxidation • flow reactor • continuous-
flow • space-time-yield
[1]
[2]
A. M. Thayer, Chem. Eng. News 1992, 70, 27–49.
(a) Z. Guo, B. Liu, Q. Zhang, W. Deng, Y. Wang, Y. Yang, H. Bengaard,
B. Hammer, Z. Sljivancanin, M. Mavrikakis, Y. Xu, S. Dahl, C. J. H.
Jacobsen, N. Nilius, H. J. Freund, H. Hagen, C. J. Kiely, G. J. Hutchings,
Chem. Soc. Rev. 2014, 43, 3480–3524; (b) W. Kroutil, H. Mang, K.
Edegger, K. Faber, Adv. Synth. Catal. 2004, 346, 125–142; (c) F.
Hollmann, I. W. C. E. Arends, K. Bühler, A. Schallmey, B. Bühler, Green
Chem. 2011, 13, 226–228.
[3]
G. P. Chiusoli, P. M. Maitlis in Metal-catalysis in industrial organic
processes, Vol. 1 (Eds.: G. Paolo Chiusoli, P. M. Maitlis), RSC Publishing,
Great Britain, 2008.
[4]
[5]
J. D. Blackmore, R. H. Crabtree, G. W. Brudvig, Chem. Rev. 2015, 115,
12974–13005.
Table 2. Benchmark MAO-catalyzed bio-oxidation in continuous-flow for study.
Y. Sawama, M. Takubo, S. Mori, Y. Monguchi, H. Sajiki, Eur. J. Org.
Chem. 2011, 3361–3367.
Entry
MAO-D9
(mgmL-1)[a]
[H2O2][b]
τres
(min)
Conv.[c] (%)
[6]
[7]
J. A. Labinger, J. Mol. Catal. A Chem. 2004, 220, 27–35.
B. L. Conley, W. J. Tenn, K. J. H. Young, S. K. Ganesh, S. K. Meier,
V. R. Ziatdinov, O. Mironov, J. Oxgaard, J. Gonzales, W. A. Goddard,
R. A. Periana, J. Mol. Catal. A Chem. 2006, 251, 8–23.
D. J. C. Constable, P. J. Dunn, J. D. Hayler, G. R. Humphrey, J. L. Leazer,
Jr., R. J. Linderman, K. Lorenz, J. Manley, B. A. Pearlman, A. Wells, A.
Zaks, T. Y. Zhang, P. J. Sheeran, C. E. Silverman, R. E. Waltermire, C.
C. Wood, Green Chem. 2007, 9, 411–420.
1
20
20
20
20
40
60
80
80
30
60
30
60
30
30
30
0
8
8
23
38
49
61
83
94
97
19
[8]
[9]
2
3[d]
4[d]
5[d]
6[d]
7[d]
8[d]
8
8
J.-P. Mahy, J.-D. Maréchal, R. Ricoux, N. Doucet, A. Lledós, J. D.
Marechal, A. Urvoas, J. P. Mahy, R. Ricoux, G. J. Davies, R. Mondiere,
A. Taglieber, Chem. Commun. 2015, 51, 2476–2494.
8
[10] C.-J. Li, B. M. Trost, Proc. Natl. Acad. Sci. U. S. A. 2008, 105, 13197–
13202.
8
[11] (a) A. Toftgaard Pedersen, W. R. Birmingham, G. Rehn, S. J. Charnock,
N J. Turner, J. M. Woodley, Org. Process Res. Dev. 2015, 19, 1580–
1589; (b) R. H. Ringborg, A. Toftgaard Pedersen, J. M Woodley,
ChemCatChem 2017, 9, 3285–3288.
12
12
[a] Used in the form of whole cells. [b] Total H2O2 concentration (mM) relative to
3a (10 mM input concentration). [c] Determined by HPLC analysis at steady-
state. [d] Glass beads employed. τres = residence time, conv. = conversion.
[12] S. Illner, C. Hofmann, P. Löb, U. Kragl, ChemCatChem 2014, 6, 1748–
1754.
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10.1002/anie.201803675
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COMMUNICATION
[13] A. Toftgaard Pedersen, T. M. de Carvalho, E. Sutherland, G. Rehn, R.
Ashe, J. M. Woodley, Biotechnol. Bioeng. 2017, 114, 1222–1230.
[14] G. Gasparini, I. Archer, E. Jones and R. Ashe, Org. Process Res. Dev.
2012, 16, 1013–1016.
[15] E. Jones, K. McClean, S. Housden, G. Gasparini, I. Archer, Chem. Eng.
Res. Des. 2012, 90, 726–731.
[16] W. Van Hecke, D. Haltrich, B. Frahm, H. Brod, J. Dewulf, H. Van
Langenhove, R. Ludwig, Journal Mol. Catal. B, Enzym. 2010, 68, 154–
161.
[17] J. Onken, R. G. Berger, Appl. Microbiol. Biotechnol. 1999, 51, 158–63.
[18] A. Wangler, R. Canales, C. Held, T. Q. Luong, R. Winter, D. H. Zaitsau,
S. P. Verevkin, G. Sadowski, Phys. Chem. Chem. Phys. 2018, 20,
11317-11326.
[19] C. Held, G. Sadowski, Annual Review of Chemical and Biomolecular
Engineering, 2016, 7, 395-414.
[20] P. G. Bowers, C. Hofstetter, C. R. Letter, R. T. Toomey, J. Phys. Chem.
1995, 99, 9632–9637.
[21] J. H. Northrop, J. Gen. Physiol. 1925, 7, 373–387.
[22] F. Escalettes, N. J. Turner, ChemBioChem 2008, 9, 857–860.
[23] P. J. Kersten, Proc. Natl. Acad. Sci. USA 1990, 87, 2936–2940.
[24] L. D. Kwiatkowski, D. J. Kosman, Biochem. Biophys. Res. Commun.
1973, 53, 715–721.
[25] M. R. Chapman, M. H. Kwan, G. King, K. E. Jolley, M. Hussain, S.
Hussain, I. E. Salama, C. González Nino, L. A. Thompson, M. E. Bayana,
A. Clayton, B. N. Nguyen, N. J. Turner, N. Kapur, A. J. Blacker, Org.
Process Res. Dev. 2017, 21, 1294–1301.
[26] B. Bechi, S. Herter, S. McKenna, C. Riley, S. Leimkühler, N. J. Turner,
A. J. Carnell, J. Clayden, F. Hollmann, N. J. Turner, Green Chem. 2014,
16, 4524–4529.
[27] S. M. McKenna, S. Leimkühler, S. Herter, N. J. Turner, A. J. Carnell, D.
P. Heuts, D. B. Janssen, M. W. Fraaije, A. T. Martinez, R. Ullrich, M.
Hofrichter and A. T. Martínez, Green Chem. 2015, 17, 3271–3275.
[28] V. Köhler, K. R. Bailey, A. Znabet, J. Raftery, M. Helliwell, N. J. Turner,
Angew. Chemie Int. Ed. 2010, 49, 2182–2184.
[29] J. H. Schrittwieser, B. Groenendaal, V. Resch, D. Ghislieri, S. Wallner,
E.-M. Fischereder, E. Fuchs, B. Grischek, J. H. Sattler, P. Macheroux, N.
J. Turner, W. Kroutil, Angew. Chem. Int. Ed. Engl. 2014, 53, 3731–3734.
[30] D. Ghislieri, A. P. Green, M. Pontini, S. C. Willies, I. Rowles, A. Frank, G.
Grogan, N. J. Turner, J. Am. Chem. Soc. 2013, 135, 10863–10869.
[31] J. M. Foulkes, K. J. Malone, V. S. Coker, N. J. Turner, J. R. Lloyd, ACS
Catal. 2011, 1, 1589–1594.
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COMMUNICATION
Layout 2:
COMMUNICATION
Dr Michael R. Chapman, Dr Sebastian
C. Cosgrove, Prof Nicholas J. Turner,*
Prof Nikil Kapur,* Prof A. John Blacker*
Page No. – Page No.
Highly Productive Oxidative
Biocatalysis in Continuous-Flow by
Surpassing the Aqueous Equilibrium
Solubility of Oxygen
Text for Table of Contents
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