Natural heterogeneous catalysis with immobilised oxidase biocatalysts

Article abstract of DOI:10.1039/d0ra03618h

The generation of immobilised oxidase biocatalysts allowing multifunctional oxidation of valuable chemicals using molecular oxygen is described. Engineered galactose oxidase (GOase) variants M₁and M_3-5, an engineered choline oxidase (AcCO6) and monoamine oxidase (MAO-N D9) displayed long-term stability and reusability over several weeks when covalently attached on a solid support, outperforming their free counterparts in terms of stability (more than 20 fold), resistance to heat at 60 °C, and tolerance to neat organic solvents such as hexane and toluene. These robust heterogenous oxidation catalysts can be recovered after each reaction and be reused multiple times for the oxidation of different substrates.

Full text of DOI:10.1039/d0ra03618h

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Natural heterogeneous catalysis with immobilised
oxidase biocatalysts†
Cite this: RSC Adv., 2020, 10, 19501
a
Ashley P. Mattey, a Jack J. Sangster,a Jeremy I. Ramsden,a Christopher Baldwin,
a
a
William R. Birmingham,
Rachel S. Heath,a Antonio Angelastro,
ab
ab
a
*
*
Nicholas J. Turner,
Sebastian C. Cosgrove
and Sabine L. Flitsch
The generation of immobilised oxidase biocatalysts allowing multifunctional oxidation of valuable
chemicals using molecular oxygen is described. Engineered galactose oxidase (GOase) variants M1 and
M3–5, an engineered choline oxidase (AcCO6) and monoamine oxidase (MAO-N D9) displayed long-term
stability and reusability over several weeks when covalently attached on a solid support, outperforming
their free counterparts in terms of stability (more than 20 fold), resistance to heat at 60 ꢀC, and tolerance
to neat organic solvents such as hexane and toluene. These robust heterogenous oxidation catalysts can
be recovered after each reaction and be reused multiple times for the oxidation of different substrates.
Received 7th April 2020
Accepted 18th May 2020
DOI: 10.1039/d0ra03618h
rsc.li/rsc-advances
Oxidation comprises one of the most important trans- a number of variants capable of oxidising a myriad of alcohols
formations in bulk synthetic chemistry, with biocatalytic and have been demonstrated in both batch and ow reactor
oxidations offering a green alternative to oen energy intensive congurations,1,16–20 including the implementation of GOase in
or toxic chemical processes.1–4 Although biocatalysis has seen the gram scale biocatalytic manufacture of the investigational
signicant developments over the past 20 years, there are still anti-HIV drug Islatravir.21 Additionally, MAO-N versatility has
major limitations that prevent widespread use in organic been extensively shown in amine resolution and functionaliza-
chemistry, mainly owing to their poor stability under process tion.15,22 Here, we demonstrate a general immobilisation
conditions.5 Biocatalyst immobilisation offers a base for strategy that can be applied to a number of oxidase enzymes,
addressing these limitations, with benets including increased enabling multifunctional catalysis that could be readily imple-
stability, productivity and solvent tolerance compared with mented in industrial processes.
soluble enzymes.6–9 Use of immobilised enzymes also allows for
For initial immobilisation studies GOase M1 was chosen as
ease of scalability and alignment to downstream processes, due the model biocatalyst as it displays potential for large-scale
to the simplication of catalyst recovery; this includes the functionalization of complex carbohydrates via selective oxida-
potential for continuous biocatalytic ow reactors, with tion of the C6–OH of terminal galactose moieties.23,24 Pure
immobilized preparations readily applied in packed bed reac-
tors.10,11 Biocatalyst immobilisation offers the selectivity of an
enzyme combined with the properties of a carrier, opening the
potential for enzymes to be used as reusable heterogeneous
catalysts (Fig. 1).12 Oxidases, unlike dehydrogenases, are not
cofactor dependant and carry out oxidations using molecular
oxygen as the sole oxidant with coupled reduction to H2O2.
Particularly attractive oxidase biocatalysts for immobilisation
are galactose oxidase (GOase), choline oxidase and monoamine
oxidase from Aspergillus niger (MAO-N), as all have demon-
strated broad applications in biocatalysis.13–15 Previous engi-
neering efforts for GOase and choline oxidase have developed
aDepartment of Chemistry, Manchester Institute of Biotechnology, University of
Manchester, 131 Princess Street, Manchester, M1 7DN, UK. E-mail: Sebastian.
cosgrove@manchester.ac.uk; Sabine.itsch@manchester.ac.uk
bFuture Biomanufacturing Research Hub, Manchester Institute of Biotechnology,
University of Manchester, 131 Princess Street, Manchester, M1 7DN, UK
Fig. 1 Immobilised oxidases have been used to oxidise a wide range of
† Electronic supplementary information (ESI) available. See DOI:
substrates with an improved tolerance to harsh reaction conditions.
10.1039/d0ra03618h
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GOase M1 was immobilised on Purolite epoxy/butyl methacry-
late beads (ECR8285). Stability of the oxidase was tested
throughout the immobilisation process by measuring the
activity of the supernatant using the HRP-ABTS assay previously
described (ESI†).16,25 Protein immobilisation yield was also
calculated throughout by spectrophotometric monitoring of the
decrease in soluble protein concentration, with a maximum
biocatalyst loading of 10 wt% (see ESI† for detailed immobili-
sation protocol). To test long term stability of the immobilised
catalyst we performed multiple biotransformations on the same
support over a number of days (Fig. 2). As can be seen below, the
half-life (t1/2) for the soluble enzyme was <24 h, whereas the
immobilised preparation still had full activity aer 21 days,
showing the t1/2 to be >3 weeks. Furthermore, a sample of the
ꢀ
immobilised catalyst was stored at 4 C for three months and
still retained around 50% of the initial activity (see ESI†). It is
important to note that the immobilised biocatalyst retained
100% of the activity of its soluble equivalent, and this was the
case for the duration of the three week stability experiments
(Fig. 2).
To further demonstrate this general immobilisation strategy,
an engineered choline oxidase (AcCO6),19 GOase M3–5 and MAO-
N D9 were immobilized using the same procedure. Similarly to
GOase M1, AcCO6, MAO-N D9 and GOase M3–5 retained activity
for over a week (ESI†). To facilitate oxidation of amines,
a previously described blocking strategy was used to prevent
nucleophilic amine substrates attaching to the support.26
Aer demonstrating the improved long-term stability and
reusability of the immobilised catalysts, the GOase M1 prepa-
ration was tested for thermal stability and activity (Fig. 3).
Reaction temperature has a great impact on the kinetics of
immobilised enzymes, with them oen being more stable than
their soluble counterparts at higher temperatures.12 In carbo-
hydrate chemistry, GOase activity at higher temperatures would
Fig. 3 (A) Thermal activity of immobilised GOase M1 (100 mg, 1 wt%)
and soluble M1 (1 mg mLÀ1) was determined through oxidation of
100 mM lactose, conversions were determined by 1H NMR analysis and
plotted relative to activity at 25 ꢀC. (B) Thermal stability of GOase M1
was determined by heating the immobilised catalyst (100 mg, 1 wt%)
and soluble enzyme (1 mg mLÀ1) for 17 h. Reaction was then run at
25 ꢀC for 16 h and conversion was determined by 1H NMR analysis.
be a desirable trait in the oxidation and functionalization of
polysaccharides due to their poor water solubility at lower
temperatures.27 Pleasingly, the immobilised GOase out-
performed the enzyme in solution, displaying a 50% increase in
activity and maintained stability at 60 ꢀC. In comparison,
activity of the free enzyme dropped by 50% in addition to a 60%
ꢀ
decrease when GOase was preheated at 60 C and run at room
temperature (Fig. 3A).
With this increased stability in hand we explored the appli-
cability of the immobilised biocatalysts as reusable, ‘heteroge-
neous’ catalysts through isolation and reapplication of the same
supported biocatalyst preparation for the oxidation of multiple
substrates in sequential reactions. Although GOase M1 per-
formed well when immobilised, its general use in biocatalysis is
limited by its narrow substrate scope (primarily galactosides).
Therefore GOase M3–5, AcCO6, and MAO-N D9 were selected as
they all possess broad substrate scopes.16,17,19 Increased use of
alcohol oxidases is advantageous as the selective generation of
aldehyde intermediates from alcohols has wide applications in
the ne chemical industry and as part of biosynthetic
cascades.19,28 MAOs have been broadly exploited for the resolu-
tion and functionalization of amines but can be limited by
substrate and product inhibition.29,30 Immobilisation of these
enzymes could overcome these limitations.31,32 Initially GOase
Fig. 2 Long term stability of immobilised GOase M1 (50 mg, 10 wt%)
was tested by running three hour oxidations of 100 mM lactose each M3–5 was immobilised at 10 wt% loading and tested against
day with continuous reuse of the same beads. Catalase and horse-
radish peroxidase were also added to the reactions.
several substituted benzyl alcohols (Table 1). For this procedure
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Table 1 Immobilised oxidases are used to facilitate oxidation of
a number of substrates with the same supported catalysta
Day
1
GOase M3–5
AcCO6
MAO-N D9
2
3
Fig. 4 Solvent tolerance of immobilised GOase (Grey), AcCO6 (Blue)
and MAO-N D9 (Green) was assessed by drying the immobilised
oxidase under reduced pressure, running the biotransformation in neat
solvent and plotting relative to buffer (NaPi pH 7.4 GOase, KPi pH 8
AcCO6, KPi pH 8 MAO-N D9). All reactions contained catalase (0.1 mg
mLÀ1). Conversions were determined by GC analysis.
4
Next, solvent tolerance was assessed (Fig. 4). Using organic
solvents offers a number of advantages over aqueous systems
such as increased solubility of hydrophobic substrates and
reduction of side product formation.33 GOase M3–5 demon-
strated near identical activity in hexane (aqueous solubility:
14 mg per L H2O) and toluene (50 mg per L H2O)34 to that of the
aqueous system in the oxidation of 3-uorobenzyl alcohol 15.
Furthermore, no over oxidation to the corresponding acid was
observed.14 The use of more water-soluble solvents did have
a detrimental effect on activity. AcCO6 performed well in some
of the solvents, with a relative activity >40% for EtOAc (8.7 g
per L H2O), hexane and cyclohexane (5.5 mg per L H2O).34 MAO-
N D9 demonstrated a retained activity of around 30% for all
tested solvents. In comparison, the soluble oxidase enzymes
displayed no activity when tested in neat solvents.
a
The same samples of immobilised GOase M3–5, AcCO6 and MAO-N D9
were used to sequentially oxidise a number of substrates. Conversions
for GOase and AcCO6 were determined by NMR and GC respectively.
Conversion for MAO-N were determined for the oxidation of the (S)-
enantiomer of the racemic substrate (see ESI for reaction conditions).
the bio-oxidation of benzyl alcohol was carried out and the
product 3 removed, the support was then washed with the
reaction buffer (with 25% DMSO), then aer washing the next
substrate was added and the subsequent oxidation was carried
out. This process was repeated four times to facilitate oxidation
of four different substrates generating 3–6 using the same
sample of supported biocatalyst. Immobilised GOase M3–5 cat-
alysed extremely efficient oxidations, with >90% conversion
achieved within one hour for each substrate (25 mM). Immo-
bilised AcCO6 (10 wt%) was also tested as a recoverable and
reusable heterogeneous catalyst, exploiting the broad substrate
scope towards aliphatic primary alcohols. Again, the immobi-
lised oxidase was able to achieve good conversions of >78% for
all substrates (10 mM) to generate products 7–10 within four
hours (Table 1). Finally, MAO-N D9 (10 wt%) was screened
against several substrates under the same conditions. Robust
biotransformations were demonstrated, fully oxidising achiral
substrate 11 and resolving racemic samples 12–14 (20 mM).
There have been several studies of immobilisation of some of
ˇ
the biocatalysts discussed here previously. Rebros and co-
workers used LentiKats polyvinyl alcohol resin to immobilise
whole-cell preparations of MAO-N D5.35 They demonstrated
impressive stabilisation of the immobilised preparations, with
samples being stored for 15 months and only showing ꢁ10%
loss in activity. Although immobilisation of whole cells reduces
the number of processing steps, ampicillin was required in the
storage buffer to prevent bacterial growth, further increasing
costs. Additionally, problems may arise with oxygen require-
ments for cell metabolism limiting the activity of the bio-
catalyst. The same group showed that crude CFE could also be
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Table 2 Comparison of key properties between immobilised and free
GOase M1
M028836/1) and ERC (788231-ProgrES-ERC-2017-ADG). S. C. C.
acknowledges the EPSRC/BBSRC Future Biomanufacturing
Research Hub (EP/S01778X/1). R. S. H. & A. A. acknowledge the
EPSRC for funding a Prosperity Partnership (EP/S005226/1). W.
R. B. gratefully acknowledges the BBSRC for the award of
a Discovery Fellowship (BB/S010459/1)
Free
Immobilised
Half-lifea
<24 h
0.39
0.38
No
>3 weeks
1.52
1.25
Thermal activityb
Thermal stabilityc
Solvent toleranced
Yes
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This work was supported by IBioIC, Prozomix Ltd. and Bioshape
Ltd., RCUK (BB/L013762/1; BB/M027791/1; BB/M02903411; BB/
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