The continuous oxidation of HMF to FDCA and the immobilisation and stabilisation of periplasmic aldehyde oxidase (PaoABC)

Article abstract of DOI:10.1039/c7gc01696d

By manipulating the reaction conditions, furandicarboxylic acid (FDCA) was prepared by the biooxidation of hydroxymethyl furfural (HMF) in a continuous one-pot reaction using galactose oxidase M_3-5, periplasmic aldehyde oxidase (PaoABC), catalase and horseradish peroxidase. In addition, PaoABC was successfully entrapped in a SiO₂ hydrogel and recycled 14 times without loss of activity. The catalyst was able to tolerate up to 200 mM DFF concentration giving FDCA in full conversion with very promising TOF and TON values.

Full text of DOI:10.1039/c7gc01696d

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The continuous oxidation of HMF to FDCA and the immobilisation
and stabilisation of periplasmic aldehyde oxidase (PaoABC)
S. M. McKenna,a P. Mines, b P. Law, b K. Kovacs-Schreiner, b W. R. Birmingham, c N.J. Turnerc,
Received 00th January 20xx,
Accepted 00th January 20xx
S. Leimkühler,d A. J. Carnell*a
DOI: 10.1039/x0xx00000x
www.rsc.org/
By manipulating the reaction conditions, furandicarboxylic acid Bio-based
5 could replace terephthalate in polyethylene
(FDCA) was prepared by biooxidation of hydroxymethyl furfural furanoate (PEF) and related bioplastics which have improved
(HMF) in a continuous one-pot reaction using galactose oxidase performance compared to their terephthalate counterparts.
M3-5
, periplasmic aldehyde oxidase (PaoABC), catalase and Notably BASF and Avantium have recently developed a
horseradish peroxidase. In addition, PaoABC was successfully partnership ‘Synvina’ to manufacture biobased FDCA using a
entrapped in a SiO2 hydrogel and recycled 14 times without loss of chemocatalytic approach for PEF production.8
activity. The catalyst was able to tolerate up to 200mM DFF
5-hydroxylmethylfurfural (HMF) 1 which is obtained from
concentration giving FDCA in full conversion with very promising the dehydration of lignocellulose9-11 can be converted to FDCA
TOF and TON values.
5
via intermediates DFF
2, HMFCA
3
and FFCA
4
(Figure 1).
O
O
OH
With the rapid growth of the world population and
increasing consumption of fossil fuels, there is an urgent need
to produce chemicals from renewable sources.1 Biomass is
cheap and abundant and can provide a potential substitute for
chemicals production.2 75% of biomass is composed of
carbohydrates, so developing efficient approaches to
transform biomass derived carbohydrates into value-added
chemicals is a high priority.3
HO
HMFCA 3
HO
O
O
OH
HO
O
H
H
O
O
O
O
O
HO
HO
OH
O
H
O
H
DFF 2 hydrate
FDCA 5
HMF 1
FFCA 4
disfavoured
equilibrium
H
O
O
HO
O
HO
OH
H
O
H
O
2,5-Furandicarboxylic acid (FDCA)
5 is one of 12 identified
DFF 2
important platform chemicals derived from biomass and a
highly efficient, sustainable and cost effective integrated
process to achieve the conversion is in demand.4 FDCA can
serve as a polymer building block for the production of
biopolymers such as polyamides and polyesters.5,6 The
polyethylene terephthalate (PET) packaging market was worth
$48.1 billion in 2014 and this is expected to increase to $60
billion in 2019.7
FFCA 4 hydrate
Figure 1 Intermediates in the oxidation of HMF 1 to FDCA 5
Chemocatalytic approaches12 used for HMF oxidation include
Au–C modified with Pd,13 Au–hydrotalicite,14 Pt–C,15
Co/Mn/Br16 and MnOx-CeO2.17 However, these reactions
require high pressure/temperature and additives and due to
the instability of
1, side products are often produced.
a Department of Chemistry, University of Liverpool
Biocatalysts have emerged as a potential solution to this
problem since they operate under mild reaction conditions
such as ambient temperature/pressure and physiological pH
and often exhibit excellent regio/chemo selectivity, decreasing
the formation of side products.18 The first biocatalytic
approach for HMF oxidation used chloroperoxidase which gave
mixtures of oxidation products.19 A whole-cell fed batch
process using recombinant P. putida showed promise,
although product isolation was challenging.20 More recently
biocatalysts such as hydroxymethyl furfural oxidase (HMFO)21
and fungal aryl alcohol oxidase (AAO) plus unspecific
peroxygenase (UPO)22 have been employed to carry out the
Crown Street, Liverpool (UK). Fax: +44 1517943500; Tel: +44 151
7943534; E-mail: acarnell@liv.ac.uk
b Biome Bioplastics, North Road, Marchwood, Southampton, SO40 4BL
(UK).
c School of Chemistry, Manchester Institute of Biotechnology, University of
Manchester, 131 Princess Street, Manchester, M1 7DN, (UK). Fax: +44 161
2751311; Tel: +44 161 3065173; E-mail: nicholas.turner@manchester.ac.uk
d Institute of Biochemistry and Biology, University of Potsdam
D-14476 Potsdam, Germany. Fax: +44 331/977-5128; Tel: +49 331/977-
5603; E-mail: sleim@uni-potsdam.de
†
Electronic Supplementary Information (ESI) available:
[Supporting information for this article including HPLC data
and NMR spectra is available XX]. See DOI: 10.1039/b000000x/
This journal is © The Royal Society of Chemistry 20xx
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oxidation of to 5 under mild reaction conditions in good
Journal Name
1
Continuous oxidation of HMF to FDCA
conversion, albeit at low substrate concentration (4mM).
Although inherently green, a limitation of the HMFO is that it
has been shown to require the hydrated form of 2,5-
Under continuous conditions using galactose oxidase
GOaseM3-5 and aldehyde oxidase PaoABC, two oxidation
products can be formed initially. The alcohol oxidation
diformylfuran (DFF)
(FFCA) . Fraaije and coworkers have shown that although
well hydrated, the degree of hydration of at pH 7.0 is low
2
and of 5-formylfuran-2-carboxylic acid
product, diformylfuran (DFF)
product, 5-hydroxymethylfuran-2-carboxylic acid (HMFCA)
However, we have previously shown that is a poor substrate
2 and aldehyde oxidation
4
2
is
3.
4
3
(1.8%) and that this correlates with the low activity of the
biocatalyst. A HMFO double mutant showed a promising
increase in activity, although overall the activity remained
modest.21b Laccase-mediator systems also appear to have the
same requirement, giving predominantly FFCA from HMF.23
for GOaseM3-5 at high substrate concentrations in cascade
conditions (>20mM).24 Therefore, stepwise addition of the
biocatalysts was required to circumvent the unwanted
formation of
3.
To allow the continuous oxidation of
1
to 5, further
FDCA
5 was obtained by an initial oxidation of HMF by
directed evolution of GOaseM3-5 may well improve the activity
of GOase variants for HMFCA at higher substrate
concentrations. Although directed evolution has delivered
excellent biocatalysts, these strategies are often labour
intensive. Therefore, we chose to look at the reaction kinetics
of PaoABC to determine if we could manipulate reaction
galactose oxidase (GOase) followed by CAL-B/peroxide
oxidation of the intermediate 2,5-diformylfuran (DFF).23
However, the second step required a change in solvent and the
slow addition of hydrogen peroxide. In addition, residual HMF
from the first step was converted to 5-hydroxymethyl-2-furan
carboxylic acid (HMFCA, 3), which is not converted to FDCA by
the lipase/peroxide system.
conditions to furnish
the formation of HMFCA. The apparent Km and Vmax values
for PaoABC with HMF as substrate are 0.13mM and 0.92
mol/min/mg respectively (ESI Section 2.5). In addition,
uncompetitive inhibition was observed at higher HMF
concentration. For DFF , the apparent Vmax is similar (0.93
mol/min/mg) and the apparent Km is slightly higher 0.73mM.
5 in a continuous manner and minimise
We have previously reported the step-wise bio-oxidation of
1
1
using two isolated oxidases, galactose oxidase M3-5 variant
µ
(GOaseM3-5) and periplasmic aldehyde oxidase ABC (PaoABC)
with the addition of catalase for both enzymes to destroy the
hydrogen peroxide produced (Figure 2a).24 The advantage of
this methodology is that PaoABC catalyses the full double
oxidation of DFF to FDCA and does not require the hydrated
2
µ
However, DFF readily forms a hydrate when compared with
HMF.21 This suggests that DFF has a more electrophilic
carbonyl which would undergo attack by the nucleophilic
molybdenum-OH centre at the active site of PaoABC. Thus it
may be possible to preferentially oxidise DFF in the presence
of HMF. To achieve this, the rate at which GOaseM3-5 oxidises
HMF to DFF needs to be fast enough to provide a sufficiently
high concentration of DFF for PaoABC.
form of FFCA
4. In addition, PaoABC uses oxygen from air
rather than hydrogen peroxide used by the peroxygenase.22
This resulted in a much higher rate of conversion and in turn
higher substrate concentration being tolerated (>100 mM).
However, stepwise addition of the GOaseM3,5 and PaoABC was
required because the rate of HMF oxidation by GOase M3-5
could not compete with fast oxidation of
HMFCA (see Figure 1), itself a poor substrate for GOaseM3-5
thus creating
continuous oxidation of
1 by PaoABC to give
Although the precise mechanism of activation is unclear, horse
radish peroxidase (HRP) has been long known to activate
GOase,23,25,26 and addition of HRP to GOase catalysed oxidation
contributes to significantly increased activity and yield.26 The
inclusion of HRP did indeed increase the rate of oxidation of
3
,
a
bottleneck.
Herein, we describe the
5 using GOaseM3-5, PaoABC and
1
to
catalase with the addition of horse radish peroxidase (HRP)
(Figure 2b). In addition, immobilisation of PaoABC was
HMF
1 to give greater conversion to DFF 2 in a short, 1 hour
explored to facilitate future scale up of FDCA production.
(a) Previous - Stepwise (1. GOaseM3,5 + O2 (air)+ catalase; 2. PaoABC + O2 (air)+ catalase)
add PaoABC here
reaction (Table 1, cf. entries 1&2). Varying pH showed that pH
7.0 was the optimum for the oxidation of HMF (Table 1 entry
3). Recently, it was reported that WT GOase performs well in
unbuffered systems.23 This would decrease costs associated
with the scale up of this process, however, the M3-5 variant
performed poorly in unbuffered water (Table 1, entry 5). In
addition, KPi was previously shown to be a poor buffer for the
wild type enzyme. It was postulated that a precipitate of
Cu3(PO4)2 might be formed when the copper-dependent
GOase is incubated in phosphate buffer, thus leading to copper
deficiency and lower activities.23 However, the M3-5 variant
step 1
step 2
PaoABC
PaoABC
GOase M3,5
FFCA 4
FDCA 5
HMF 1
DFF 2
PaoABC
GOase M3,5
slow
HMFCA 3
unproductive
(b) Current - One Pot continuous (GOaseM3,5 + PaoABC + O2 (air) + catalase + HRP)
GOase M3,5
HRP
PaoABC
PaoABC
FFCA 4
DFF 2
FDCA 5
HMF 1
PaoABC
HMFCA 3
performed better in KPi, even at 100mM
Strangely, we identified that HMFCA is an excellent substrate
of GOaseM3-5 with and without HRP (Table S1). This
contradicts our previous cascade results in which we noted
that was not sufficiently oxidized under cascade conditions.
The likely reason for this is that produced in the dual enzyme
1 (Table 1, Entry 7).
GOase M3,5 + HRP fast
3
,
Figure 2 (a) Original stepwise and (b) new continuous one-pot conversion for HMF to
FDCA using HRP. For compound structures see Figure 1.
3
5
2 | J. Name., 2012, 00, 1-3
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system may partially inhibit GOaseM3-5 oxidation of HMFCA 3. This supports our initial observation that in the presence of
This suggests future work should include in-situ product HMF and DFF, PaoABC will oxidise DFF preferentially. Upon
removal of
With improved GOaseM3-5-HRP conditions in hand, we incomplete conversion to FDCA (55%) was observed and
turned our attention to PaoABC. By varying the concentration HMFCA was present as 45% of the reaction mixture (Table 2,
of PaoABC in the reaction we would be able to reduce the rate entry 4). Again, lowering the amount of PaoABC decreased the
of the off target oxidation of and limit HMFCA formation. We aldehyde group oxidation in , resulting in lower conversion to
were delighted to observe that at each concentration tested being obtained in 100% conversion (Table 2, entry
we were able to produce the desired
than stepwise manner (Table 2, Entries 1-3).
5
to allow optimal activity of GOaseM3-5
.
increasing the substrate HMF concentration to 100 mM,
3
1
1
3
and in 5
5
in a continuous rather 5).
100
90
80
70
60
50
40
30
20
10
HMF
DFF
HMFCA
FFCA
FDCA
Table 1 Optimisation of HMF
1 to DFF 2 conversion by GOase M3-5.
[HMF] 1
(mM)
% DFF
Entrya
pH
Buffer
2
1b
50
7.5
“
KPi
“
73
2
“
88
3
4
5
6
7
“
“
7.0
6.5
-
7.0
“
“
“
91
77
51
60
80
0
0
20
40
60
80
100
120
140
160
180
TIME (MIN)
“
“
100
Water
NaOAc
KPi
Figure 3 Time course analysis of the continuous oxidation of HMF to FDCA (Table 2,
Entry 1) Reaction conditions: Final volume 0.3 mL, 33 L catalase (3.3 mg/mL), 70
HRP (1.0 mg/mL), 103 L GOaseM3-5(3.0 mg/mL), 1 μL PaoABC (28.9 mg/mL) 0.2 M KPi
μ
μL
μ
[a]Reaction conditions: Final volume 0.3 mL, 33 μL catalase (3.3 mg/mL), 70 μL
HRP (1.0 mg/mL), 37° C, 103 μL GOaseM3-5, (3mg/mL), 1 hr. Conversion calculated
by RP-HPLC and adjusted with a 1:1:1 standard of HMF:DFF:FFCA. [b] No HRP
pH 7.0, 50 mM HMF, 37°C.
Time course analysis (Figure 3) indicated that at lower PaoABC
concentration (Table 2, entry 1) HMFCA remained low at all
times. At higher PaoABC concentrations (entries 2&3),
increasing amounts of HMFCA is formed as an intermediate (Cf
Figures S3-S5), although with HRP present, complete
conversion to FDCA is still observed over the same time period
(3h.).
Immobilisation and use of PaoABC
A substantial effort has been devoted to developing effective
immobilisation methods to increase the operational stability of
enzymes and to facilitate their recovery and recyclability.27
Combined-cross linked enzyme aggregates (combi-CLEAS) have
emerged as a multi purpose, novel and versatile carrier free
immobilised biocatalyst formulation for co-immobilisation of two or
more enzymes, and are capable of combining multi-step cascade
and non-cascade biotransformations into one catalytic pot.28,29
Combi-CLEAs have been used to co-immobilise glucose oxidase and
catalase with great success.30 We attempted the formation of a
combi-CLEA with PaoABC and catalase but were unsuccessful (ESI 4)
Although both enzymes retained activity after aggregation, cross-
linking appeared to remove all activity. PaoABC contains many
lysine residues around the active site31 and we believe that the bi-
functional glutaraldehyde may cross-link these residues, altering
the active site and leading to inactivity.
Table 2 Optimisation of PaoABC in the continuous oxidation of HMF to FDCA
GOase
(3 mg/ml)
(μL)
PaoABC
(28.9 mg/ml)
(μL)
1
Entry
% FDCA 5
(mM)
1
2
3
4
50
“
“
103
1
100
100
100
Classical immobilisation on solid supports is an alternative to CLEA
formation due to the many commercially available resins
available.30 Co-immobilisation of PaoABC with catalase on
Eupergit-CM showed good conversion after one cycle.
However, enzymatic deactivation was seen in subsequent
cycles (ESI, Table S8). We identified that catalase was
denaturing on the resin since the reaction could be rescued by
“
“
“
2.5
5
2.5
100
55 (6hr)
5
100
130
1
100 (6hr)
Reaction conditions: Final volume 0.3 mL, 70 μl HRP (1 mg/mL), 33 μL catalase
(3.3 mg/mL), 0.2 mM KPi pH 7.0, 37°C, 3 hr, pH adjusted with 2M NaOH.
This journal is © The Royal Society of Chemistry 20xx
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Journal Name
adding an additional portion of catalase in subsequent and presumably insufficient electrostatic interactions occur
reactions. We then created a CLEA of catalase (CAT-CLEA)30 with the enzyme.
which resulted in this biocatalyst being able to be recycled 20
We next looked at the formulation of PaoABC (Figure 5,
times without any loss in conversion in the test reaction S14-16) with catalase and to determine the recyclability of the
(Figure S9). With this modified procedure, the PaoABC immobilised enzyme. Enzyme immobilisation often leads to
immobilized on Eupergit-CM in combination with the CAT- increased stability of enzymes to denaturing conditions so we
CLEA could be recycled 5 times (Figure S8). However, the first tested to determine if PaoABC-gel could withstand
activity of Eupergit-CM immobilised PaoABC was low when reaction conditions (H2O2) without addition of catalase. This
compared with soluble PaoABC (Figure 4). This poor activity severely decreased the reaction rate (Figure 5, blue bars).
may result from reaction of surface epoxy groups on the resin When soluble catalase was co-entrapped with PaoABC (grey
with lysine residues near the enzyme active site or from bars), the formulation could be recycled three times,
interference with the enzyme’s quaternary structure.31
maintaining conversion above 70%. Interestingly the second
Physical entrapment of cells/isolated enzymes in polymer recycle (91%) gave better conversion than the first (78%). This
networks or organic/inorganic materials has been widely used could be due to swelling of the gel in the buffer which allows
in industrial applications.32,33 Proteins have been stabilised by better mass transfer of the substrate into the catalyst. We
sol-gel encapsulation in hydrated silicas, including flavoprotein have shown that soluble catalase is unstable under the
oxidases.34 These enzymes have been co-immobilised in sol- reaction conditions and so we opted to test the highly stable
gels with counter-charged polymers that form electrostatic CAT-CLEA encapsulated within the hydrogel formulation.
adducts with the enzyme (based on their isoelectric point) to However, this proved detrimental to the activity of the
aid stabilization during the sol process.
biocatalyst (Figure 5, yellow bars). However, when the CAT-
CLEA was combined with the already encapsulated PaoABC,
the biocatalysts could be recycled up to 14 times without loss
in yield (Figure 5, orange bars). Subsequent recycles resulted in
soluble PaoABC
Eupergit CM
Hydrogel
Hydrogel (PVI)
Hydrogel (PEI)
Hydrogel (PEG)
100
90
80
70
60
50
40
30
20
10
0
lower conversion to
5.
0
20
40
60
80
100
120
140
160
180
100
90
80
70
60
50
40
30
20
10
0
TIME (MIN)
Figure 4. Comparison of immobilised forms of PaoABC for oxidation of DFF 2 to FDCA 5.
Reactions include catalase-CLEA (ESI 4.2.4, 4.4.2). Reaction conditions: Final volume 0.3
mL, 5 mg catalase-CLEA, 0.065 mg soluble PaoABC or 50 mg PaoABC immobilised
hydrogel, 0.1 mM KPi pH 7.0, 0.2M DFF, 37°C, pH adjusted with saturated solution of
bicarbonate.
PaoABC was co-encapsulated in a tetramethyl orthosilicate
(TMOS) based sol-gel with stabilisers polyethylene imine (PEI),
polyvinyl imidazole (PVI) or polyethylene glycol and these
catalysts tested in conjunction with CAT-CLEA for oxidation of
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
CYCLE NUMBER
PaoABC hydrogel
Hydrogel + Cat CLEA
50 mM DFF
2 to determine the activity of the hydrogel-
Hydrogel encapsulated catalase Hydrogel encapsulated Cat-CLEA
encapsulated enzyme (Figure 4, Figures S10-13). Co-
encapsulating PaoABC with PVI made little difference to the
activity of the catalyst compared with the hydrogel
Figure 5. Recycling of different formulations of PaoABC-gel. Blue – PaoABC hydrogel
with no catalase; Orange – PaoABC hydrogel with separate catalase-CLEA; Grey –
PaoABC hydrogel co-encapsulated with catalase; Yellow – PaoABC hydrogel co-
encapsulated with pre-made catalase-CLEA; 300 µL of 0.3 M KPi pH 7.0, hydrogel (50
mg containing 0.065 mg PaoABC), 50 mM DFF, catalase (CLEA), 5 mg; reaction time 90
min.
formulation without
stabilisation of the protein. However, the PEI-stabilised
PaoABC solgel gave full conversion to after 90 minutes which
a
stabiliser. PEG showed some
5
is almost comparable to that of the soluble PaoABC (Figure 4).
The isoelectric points of PaoABC subunits are 5.5, 9.0 and 5.9
respectively35 and so more basic PEI (polycationic at neutral
pH) effectively binds to the polyanionic Pao A and C subunits.
This can be seen by a precipitation of the enzyme on addition
of PEI to soluble enzyme. PVI on the other hand, is less basic
PaoABC-Gel/CAT-CLEA was able to tolerate substrate
concentrations up to 200 mM (Figure S24). Soluble PaoABC
actually performed better at this higher substrate level (Figure
S23) which may be a result of insufficient mass transfer with
4 | J. Name., 2012, 00, 1-3
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the heterogeneous biocatalyst on a small scale. We also
identified the optimum temperature (ESI 4.4.6) for the
Acknowledgements
oxidation of DFF
2
as 35oC, although this and higher
The research leading to these results received support from
the Biotechnology and Biological Sciences Research Council
and (BBSRC BB/M028631/1 to SMM and AJC), Innovate UK
(Biome Bioplastics), The Royal Society Wolfson Merit Award
(to NJT).
temperatures resulted in lower TOF when using the hydrogel
(Table 3), which could be a result of hydrolysis of the hydrogel.
Nevertheless, the PaoABC hydrogel did show additional
thermostability at 45oC compared to the soluble enzyme
(Figures S29, S30)
. The turnover numbers (TON) and
frequencies (TOF) achieved with the biocatalyst are shown in
Table 3.
Notes and references
1
2
3
4
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Temperature
(oC)
TON
TOF
TOF
[s-1]
Catalyst
Soluble
[mol mol-1]
[hr-1]
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The ability to run cascades of enzyme bioconversions in a
single reaction process will be a major driving force in future
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(HMFO) or as combinations for HMF to FDCA conversion have
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to FDCA to give full conversion at a HMF concentration of
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active to speed up oxidation of HMF to DFF (and HMFCA
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lower amounts of PaoABC to limit the formation of HMFCA
3 to
3
from HMF. PaoABC could be entrapped as a solgel and re-used
14 times without loss of activity, in conjunction with catalase
CLEA for DFF
2 to FDCA 5 conversion at 200mM substrate
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with immobilised GOaseM3,5 and HRP for scale up of the HMF
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This journal is © The Royal Society of Chemistry 20xx
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6 | J. Name., 2012, 00, 1-3
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