Synthesis of 2,5-furandicarboxylic acid by a TEMPO/laccase system coupled withPseudomonas putidaKT2440

Article abstract of DOI:10.1039/d0ra03089a

As a useful and renewable chemical building block from biomass, 2,5-furandicarboxylic acid (FDCA) has become an increasingly desirable platform chemical as a terephthalic acid replacement for polymerization. In this work, an efficient and highly selective biocatalytic approach for the synthesis of FDCA from 5-hydroxymethylfurfural (HMF) was successfully developed using a TEMPO/laccase system coupled withPseudomonas putidaKT2440. TEMPO/laccase afforded the selective oxidation of the hydroxymethyl group of HMF to form 5-formyl-2-furancarboxylic acid as a major product, which was subsequently oxidized to FDCA byP. putidaKT2440. Manipulating the reaction conditions resulted in a good conversion of HMF (100percent) and an excellent selectivity of FDCA (100percent) at substrate concentrations up to 150 mM within 50 h. The cascade catalytic process established in this work offers a promising approach for the green production of FDCA.

Full text of DOI:10.1039/d0ra03089a

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PAPER
Synthesis of 2,5-furandicarboxylic acid by
a TEMPO/laccase system coupled with
Cite this: RSC Adv., 2020, 10, 21781
Pseudomonas putida KT2440†
a
abc
a
a
Lihua Zou,‡ Zhaojuan Zheng,‡
Huanghong Tan, Qianqian Xu
abc
and Jia Ouyang
*
As a useful and renewable chemical building block from biomass, 2,5-furandicarboxylic acid (FDCA) has
become an increasingly desirable platform chemical as terephthalic acid replacement for
a
polymerization. In this work, an efficient and highly selective biocatalytic approach for the synthesis of
FDCA from 5-hydroxymethylfurfural (HMF) was successfully developed using a TEMPO/laccase system
coupled with Pseudomonas putida KT2440. TEMPO/laccase afforded the selective oxidation of the
hydroxymethyl group of HMF to form 5-formyl-2-furancarboxylic acid as a major product, which was
subsequently oxidized to FDCA by P. putida KT2440. Manipulating the reaction conditions resulted in
good conversion of HMF (100%) and an excellent selectivity of FDCA (100%) at substrate
concentrations up to 150 mM within 50 h. The cascade catalytic process established in this work offers
a promising approach for the green production of FDCA.
Received 6th April 2020
Accepted 1st June 2020
a
DOI: 10.1039/d0ra03089a
rsc.li/rsc-advances
9
U.S. Department of Energy. It has been considered to have
a huge market potential because it can be used as a ‘green’
Introduction
The massive depletion of nonrenewable fossil resources and the substitute of terephthalic acid for the production of poly(-
1
0
emission of greenhouse gases have produced the widest range ethylene terephthalate).
of changes in the world. There has been growing interest in
1
In the past few years, intensive research efforts have been
developing renewable and sustainable fuels and platform conducted to synthesize FDCA by the oxidation of HMF through
2
–5
11
chemicals. Lignocellulosic biomass is an important renew- chemical pathways, such as the use of traditional stoichio-
12
13
able carbon resource, and its derived carbohydrates can be metric oxidants, homogeneous catalysts, and heterogeneous
converted into various biobased platform compounds for the catalysts (various noble metal catalysts, such as supported Au,
production of high-value products. Among these compounds, Pt, and Pd catalysts).
6,7
14
However, these chemocatalytic
5
-hydroxymethylfurfural (HMF) is such a compound that has approaches are characterized by problems associated with
a furan ring, a hydroxymethyl group and an aldehyde group, harsh reaction conditions, such as high temperature, high
which makes it an appealing starting material for catalytic pressure, and the presence of metal salts and organic solvents,
upgrades. It can be selectively oxidized to versatile building which render the process expensive and polluting. In addition,
blocks, including 5-hydroxymethyl-2-furancarboxylic acid the selectivity of some catalysts is poor in specic reactions,
1
5,16
(
HMFCA), 2,5-diformylfuran (DFF), 5-formyl-2-furancarboxylic resulting in the formation of byproducts.
acid (FFCA), and 2,5-furandicarboxylic acid (FDCA). Among
8
Biocatalysis is emerging as a valuable tool to address these
them, FDCA, obtained through the full oxidation of the problems in the context of green chemistry since it is typically
hydroxymethyl and aldehyde moieties in HMF, is only listed as performed under mild conditions, usually requires fewer and
one of the 12 key value-added chemicals from biomass by the less toxic reagents and solvent than chemocatalysis, and
17
exhibits excellent selectivity. Currently, biocatalytic produc-
tion of FDCA mainly involves microbial and enzymatic conver-
sion approaches. Since the FDCA concentration produced by
a
Jiangsu Co-Innovation Center of Efficient Processing and Utilization of Forest
Resources, College of Chemical Engineering, Nanjing Forestry University, Nanjing
18
2
b
10037, People's Republic of China. E-mail: hgouyj@njfu.edu.cn
wild-type strains is too low, gene modication or metabolic
Key Laboratory of Forestry Genetics & Biotechnology (Nanjing Forestry University), engineering becomes an indispensable step for the microbial
Ministry of Education, Nanjing 210037, People's Republic of China
conversion process. Two notable examples of microbial
c
Jiangsu Province Key Laboratory of Green Biomass-based Fuels and Chemicals,
bioconversion are the recombinant strains Pseudomonas putida
Nanjing 210037, People's Republic of China
20
S12 (ref. 19) and Raoultella ornithinolytica BF60. Although both
of them give relatively high FDCA titers, they still suffer from
several disadvantages: complex molecular operation and low
†
1
‡
Electronic supplementary information (ESI) available. See DOI:
0.1039/d0ra03089a
These authors contributed equally to this work.
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productivity or yield of FDCA. Enzymatic bioconversion is enzyme. Oxidation of ABTS was followed by absorbance
4
ꢁ1
ꢁ1
24
another promising alternative approach, but there are limited increase at 420 nm (3420 ¼ 3.6 ꢂ 10 M cm ) in 3 min. One
examples in the literature. An HMF oxidase was identied with unit of laccase activity was dened as follow: the amount of
a remarkable capability of oxidizing HMF to FDCA, which was laccase that can oxidized 1 mmol of ABTS per minute. The
21,22
conducted at very low concentrations (2–4 mM).
The full concentrations of proteins were determined by the Bradford
oxidation of HMF more oen needs a combination of two assay using bovine serum albumin as the standard. The specic
ꢁ1
enzymes, as the production of FDCA entails three consecutive activities of laccases from T. versicolor was 5.8 U mg
.
oxidation steps. For example, aryl-alcohol oxidase coupled with
23
an unspecic peroxygenase, galactose oxidase coupled with Oxidation of the hydroxymethyl group of HMF by TEMPO/
24
lipase, and galactose oxidase variant M coupled with alde- laccase
3
–5
25
hyde oxidase have been reported in the past ve years.
Compared with the corresponding bioconversion by microbes,
the bioconversion of HMF to FDCA proceeded by enzymes
needs extra expression and purication steps. Sometimes, the
reaction conditions of two enzymes do not match well, which
further complicates the whole process.
In a typical experiment, 4 mL buffer or deionized water con-
taining 30 mM HMF and TEMPO/laccase were incubated in
a 50 mL centrifuge tube capped with a septum at 150 rpm with
air bubbling for 3 min each 12 h. Parameters affecting the above
typical bio-oxidation experiment were investigated, including
temperature, buffer pH, and laccase concentration. The
The shortcomings of the existing methods and the potential
importance of FDCA production motivate us to further explore
efficient and robust oxidation method. In this study, we
designed a method of coupling reactions to produce FDCA from
HMF. The key feature of this approach is the oxidation of the
hydroxymethyl group via a TEMPO/laccase system and the
oxidation of the aldehyde moiety by P. putida KT2440 cells.
Combining the advantages of two catalysts, a satisfactory HMF
conversion and FDCA selectivity were realized even at high HMF
concentrations (150–200 mM). This study provides a highly
green and efficient biocatalytic alternative for the production of
FDCA from HMF.
parameters varied as follows. Temperature ranged from 20 to
ꢀ
30 C, and pH was varied from 4.5 to 7.0 using different buffers
3 3 2 4
(50 mM) (CH COOH–CH COONa for pH 4.5–6.0 and NaH PO –
Na
2
HPO
4
for pH 6.0–7.0, and distilled water as the control). The
ꢁ1
enzyme dosage was set from 0 to 7.5 mg mL . Aliquots were
withdrawn from the reaction mixtures at specied times and
diluted with the corresponding mobile phase prior to HPLC
analysis. All experiments were conducted in triplicate, and all
the data are expressed as the mean ꢃ standard deviation.
Representative procedure for the synthesis of FDCA
Experiments for the biotransformation of HMF to FDCA by the
TEMPO/laccase system coupled with P. putida KT2440 were
performed as follows. Typically, HMF (30–200 mM), a certain
proportion of TEMPO (molar TEMPO/HMF) and laccase were
added to acetate buffer (50–200 mM, pH 6.0), and the reaction
Experimental section
Chemical materials
DFF (>98%), FFCA (>98%) and FDCA (>98%) were purchased from
ꢀ
was incubated at 25 C, 150 rpm in a shaking incubator capped
TCI (Japan), while the laccase from Trametes versicolor, HMF
0
with a septum. When the rst step oxidation was completed by
the TEMPO/laccase system, the pH was carefully monitored and
adjusted to 6.0 with NaOH, which was followed by an addition
of P. putida KT2440 cells and calcium carbonate (at same
molarity as the substrate). The reaction progressed for a few
(
>99%), TEMPO (>98%) and 2,2 -azinobis(3-ethylbenzylthiozoline-
6-sulfonate) (ABTS) were purchased from Sigma-Aldrich (USA).
Preparation of whole-cell biocatalysts
For preparation of seed cultures, P. putida KT2440 cells were
ꢀ
hours in the incubator at 30 C, 200 rpm. Aliquots were with-
ꢀ
precultivated at 30 C and 200 rpm for 12 h in Luria Broth (LB)
drawn at specied time intervals from the reaction mixture. The
medium containing 0.5% yeast extract, 1% tryptone, and 1%
NaCl. Then, 2% seed culture was incubated in 1 L shake asks
containing 200 mL fresh LB medium. Aer incubation for 12 h
under the same conditions, the cells were harvested by centri-
ꢀ
samples were treated at 100 C for 5 min to denature the cells
and then diluted prior to HPLC analysis.
Analytical methods
ꢀ
fugation (6000g, 8 min, 4 C) and washed twice with phosphate
The reaction mixtures were analyzed using an Agilent 1260
HPLC system using an external standard calibration curve
method. The sample was separated by an Aminex HPX-87H
buffer (0.2 M, pH 6.0), followed by dispersing in corresponding
buffer for subsequent experiments. The biomass concentra-
tions were measured by determining the optical density at
ꢀ
column (Bio-Rad, USA) at 55 C, with elution performed by
6
00 nm and converted to the dry cell weight (dcw) using the
ꢁ
1
ꢁ
1
the use of 5 mM H SO at 0.6 mL min and detection per-
2 4
following equation: dcw (g L ) ¼ 0.348 ꢂ optical density at
00 nm [OD600] ꢁ 0.76.
formed with a UV detector at a xed wavelength of 268 nm. The
conversion (%) of HMF and the selectivity (%) of oxidation
products were calculated as follows:
6
Laccase activity assay
converted HMF ðmMÞ
The laccase activity was determined spectrophotometrically in
a sodium acetate buffer (0.1 M, pH 5.0) containing 0.5 mM ABTS
as the substrate at room temperature and a suitable amount of
HMF conversion ð%Þ ¼
ꢂ 100 (1)
starting HMF ðmMÞ
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product formed ðmMÞ
total products ðmMÞ
addition of the commercially available fungal laccase. When
HMFCA was incubated with the TEMPO/laccase system in
acetate buffer, almost 64% FFCA and only 6% FDCA were
detected aer 24 h of incubation, and these percentages
changed slightly during the subsequent incubation (approxi-
mately 19% FDCA aer 60 h) (Fig. 1A). The TEMPO/laccase
oxidation of HMFCA resulted in mixtures in which FFCA was
dominant. Qin and coworkers ever reported that TEMPO/
laccase could convert HMF into DFF as initial dominate inter-
mediate and further transform into FFCA, but its oxidation
Selectivity of productð%Þ ¼
ꢂ 100 (2)
Statistical analysis
Statistical analyses were performed using SPSS statistics 16.0.
The differences of the corresponding values between exposed
groups were tested by one-way analysis of variance (ANOVA). p <
0
.05 was considered to be a signicant difference.
24
efficiency of FFCA into FDCA was poor. Our results further
conrmed the low activity of the TEMPO/laccase system on
FFCA. In general, the hydrated form (gem-diol) of aldehydes act
as the key intermediates play a central role in the aldehyde
oxidation catalyzed by TEMPO/laccase system. Whereas, only
Results and discussion
Design and selection of routes for FDCA production
The synthesis of FDCA from HMF requires a catalyst to act on
both hydroxymethyl and aldehyde groups. Interestingly,
a previous study in our group demonstrated that wild-type P.
putida KT2440 could effectively convert HMF into HMFCA, and
no DFF, FFCA or FDCA formation was observed, which shows
perfect selective oxidation toward the aldehyde group of HMF.
For the oxidation of the hydroxymethyl group, TEMPO/laccase
22
a small fraction of FFCA may exist in hydrated form. Thus,
route 1 will not be sufficiently active to produce FDCA (Fig. 1C).
Next, the second route was evaluated. HMF was mixed with
TEMPO/laccase to test the ability of this system to oxidize it.
Most of the HMF (80% conversion) was converted into FFCA in
26
48 h, and very little DFF and FDCA were formed. The proportion
24,27
system has been employed in the literature toward HMF.
of FFCA slowly decreased over time because of the formation of
low amounts of FDCA (4.5% selectivity aer 48 h) (Fig. 1B).
Then, the ability of P. putida KT2440 to oxidize the intermedi-
ates of DFF and FFCA was tested (Fig. S1†). The results revealed
that, given enough time, P. putida KT2440 can fully oxidize the
aldehyde groups of both DFF and FFCA to yield FDCA. This
means that the oxidation of HMF to FDCA via route 2 is indeed
feasible (Fig. 1D). For this route, a critical task is determining
how to improve the conversion of HMF to 100%, as the surplus
HMF in the TEMPO/laccase system would be completely con-
verted to HMFCA by P. putida KT2440, increasing the difficulty
and cost of the separation and purication of FDCA.
Herein, we attempted to combine the abilities of the TEMPO/
laccase system and P. putida KT2440 to oxidize the hydrox-
ymethyl group and the aldehyde group, respectively, hoping
that they match perfectly and efficiently achieve FDCA produc-
tion under mild reaction conditions.
Based on the above assumptions, there are two possible
pathways for the oxidation of HMF to FDCA (Scheme 1). In the

rst pathway, the aldehyde group of HMF is rst oxidized by P.
putida KT2440 to yield HMFCA. Then, TEMPO/laccase is added
to the reaction mixtures to further oxidize the hydroxymethyl
group of HMFCA and thus complete full oxidation of HMF. In
this pathway, it is uncertain whether TEMPO/laccase can
oxidize the hydroxymethyl of HMFCA. In the second pathway,
the TEMPO/laccase catalysis operates prior to P. putida KT2440.
In this way, the conversion of the hydroxymethyl group of HMF
is rst catalyzed by the TEMPO/laccase system to generate furan
mixtures, which are then converted to FDCA by P. putida
KT2440. Regarding this pathway, we have not detailed and
comprehensively investigated the biocatalytic ability of P. putida
KT2440 toward other furanics except HMF.
To verify the feasibility of the rst route, considering the
oxidation of HMF to HMFCA by P. putida KT2440 had been
proved, we set up a test reaction using HMFCA with the TEMPO/
laccase system to identify whether the hydroxymethyl group of
HMFCA could be oxidized. The reaction was started with
Fig. 1 Time course of oxidation of HMFCA (A) and HMF (B) by TEMPO/
laccase system and them coupled with P. putida KT2440 via route 1 (C)
and route 2 (D) respectively. Reaction conditions: 30 mM HMFCA or
HMF, 4 mL acetate buffer (50 mM, pH 4.5), 20 mol% TEMPO, 2.6 mg
ꢁ1
ꢀ
mL laccase, air bubbling for 3 min each 12 h, 25 C, 150 rpm.
Scheme 1 Two pathways for oxidation of HMF to FDCA.
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Effects of key reaction conditions on HMF oxidation
To obtain higher FDCA selectivity and concomitantly eliminate
HMFCA generation in the second step, a series of experiments
were performed to improve the conversion of HMF. The inu-
ence of temperature on the oxidation of HMF in the TEMPO/
ꢀ
laccase system was tested in the range of 20–30 C for 48 h
(
Fig. 2A). The highest conversion of HMF (nearly 90%) was ob-
ꢀ
ꢀ
tained at 20 C. But considering that 25 C is closer to room
temperature, we selected 25 C for the next experiment. It has
ꢀ
been reported that the type of buffer and pH used can inuence
28
the conversion of substrates in reaction systems. For this
reason, the effects of buffer and pH on the oxidation of HMF
ꢀ
were explored at 25 C for 24 h. It was found that buffer type and
pH exerted a signicant effect on the catalytic performance.
Compared to unbuffered aqueous solutions, all the buffered
solutions tested were capable of considerably enhancing HMF
conversion apart from phosphate buffer at pH 7.0. The use of
sodium acetate buffer at pH 6.0 led to an almost perfect HMF
conversion (100%). At the same pH of 6.0, the conversion of
HMF in phosphate buffer decreased to 67.6%. For phosphate
buffer, the best results were observed at pH 6.5 (HMF conver-
sion of 83.1%) (Fig. 2B). In general, the results suggested that
a buffered solution was necessary for the conversion of HMF
because of the production of acidic intermediates during the
reaction course. Thus, we selected sodium acetate buffer of pH
6.0 for subsequent work. During the aerobic oxidation of
laccase/TEMPO, laccase can easily oxidise the stable oxyl-radical
form of TEMPO to the oxoammonium ion. The oxoammonium
ion of TEMPO as the actual oxidant could selectively oxidize the
27
hydroxymethyl group of HMF. Therefore, the dose of laccase
was also crucial to HMF conversion (Fig. 2C). A control experi-
ment conrmed that no substrate transformation occurred in
the absence of laccase under the investigated conditions, which
was in good agreement with previous reported results in the
29
literature. A maximum conversion of 95.6% was achieved at
ꢁ
1
2
.6 mg mL laccase. When the amount of enzyme was more
ꢁ1
than 2.6 mg mL , there was no signicant improvement in the
conversion of HMF. Therefore, considering the cost of enzyme, Fig. 2 Effects of reaction temperature (A), buffer pH (B) and laccase
ꢁ1
concentration (C) on HMF conversion in TEMPO/laccase system.
a laccase dose of 2.6 mg mL was used for the subsequent
studies.
Reaction conditions: (A) 4 mL acetate buffer (50 mM, pH 4.5), 30 mM
ꢁ
1
HMF, 20 mol% TEMPO, 2.6 mg mL laccase, 150 rpm, the designated
temperature, air bubbling for 3 min each 12 h. (B) 4 mL buffer (50 mM,
CH
3
COOH–CH
3
COONa for pH 4.5–6.0, NaH
2
PO
4
–Na
2
HPO
4
for pH
The oxidation of HMF in the TEMPO/laccase system at high
substrate concentrations
ꢀ
6.0–7.0, and distilled water), 25 C, others were the same as (A). (C)
mL acetate buffer (50 mM, pH 6.0), laccase of the designated
4
The excellent conversion at high concentrations of HMF ob- concentration, others were the same as (B). Different letters represent
significant differences between treatments (p < 0.05).
tained in the rst step is conducive to the production of FDCA in
the second step. Thus, the transformation of 10–200 mM HMF
by the TEMPO/laccase system was rstly assessed under the
30
optimum conditions obtained above. As presented in Fig. 3, TEMPO decomposition and pH drop with time. Based on the
aer 24 h of reaction, 100% conversion was achieved when the above speculation, we next investigated the inuence of these
HMF concentration was #30 mM. For 50 mM and 70 mM HMF, two factors on HMF conversion. First, the inuence of TEMPO
the conversion was slightly decreased to 96% and 89%, concentration on the oxidation of HMF by the TEMPO/laccase
respectively. However, the conversion was reduced markedly to system was evaluated. As shown in Fig. 4A, when the laccase
7
9% and 34% at HMF concentrations of 100 and 200 mM. The amount was constant, the conversion of HMF was promptly
major reason for the unsatisfactory conversion at high increased with increasing TEMPO concentration. When
concentrations might be the inappropriate amount of catalyst a TEMPO concentration of 80 mol% was used a high HMF
and poor buffer capacity in the reaction system because of conversion of nearly 100% was obtained aer 12 h in the
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Fig. 3 Effects of the initial HMF concentration on HMF conversion in
TEMPO/laccase system. Reaction conditions: 4 mL acetate buffer
(
50 mM, pH 6.0), HMF of the designated concentration, 20 mol%
ꢁ1
ꢀ
TEMPO, 2.6 mg mL laccase, air bubbling for 3 min each 12 h, 25 C,
50 rpm. Different letters represent significant differences between
1
treatments (p < 0.05).
presence of 70 mM HMF, and further increasing the TEMPO
concentration resulted in no signicant changes in the
conversion of HMF. This result means that a suitable TEMPO
amount is crucial for the efficient conversion of HMF. A higher
mediator/laccase ratio could greatly promote the conversion of
HMF at high concentrations. Therefore, 80 mol% TEMPO was
used for further research.
Upon examining the second possibility for unsatisfactory
conversion, we found that the continuous accumulation of
intermediates (mainly FFCA) resulted in an appreciably
decreased pH in the reaction system, which affected the prog-
ress of the reaction. For this reason, we investigated the effect of 6.0), 100 mM HMF, 80 mol% TEMPO, 2.6 mg mL laccase, air bubbling
buffer concentrations (50, 100, 200 mM) on the catalytic reac- for 3 min each 12 h, 25 C, 150 rpm. Different letters represent
Fig. 4 Optimization of the TEMPO dosage (A) and buffer concentra-
tions (B) for HMF conversion. Reaction conditions: (A) 4 mL acetate
buffer (50 mM, pH 6.0) containing 70 mM HMF, different concentra-
tion of TEMPO (0–150 mol%); (B) 4 mL acetate buffer (50–200 mM, pH
ꢁ1
ꢀ
significant differences between treatments (p < 0.05).
tion in the case of a higher HMF concentration. Aer a reaction
period of 36 h, 100 and 200 mM sodium acetate buffer gave the
best performance, and HMF conversion was close to 100%
KT2440 cells exhibited good catalytic performances, and
a conversion of 100% was achieved aer the short reaction time
of 0.5 h. In addition, all FFCA was oxidized to FDCA with an
excellent selectivity of 100%. Better yet, P. putida KT2440
maintained its outstanding capability under all tested FFCA and
acetate buffer concentrations (Table S1†), which further
demonstrated its great potential for oxidizing various furan
aldehydes.
Based on the above experimental results, the TEMPO/laccase
system and P. putida KT2440 cells were coupled to test the
conversion of HMF to FDCA in a sequential manner at different
concentrations of HMF. When HMF was completely converted
by the TEMPO/laccase system, the pH of the reaction mixtures
was adjusted to 6.0 with NaOH, and P. putida KT2440 cells were
added for further oxidation. The results of the two-step cascade
oxidation of HMF to FDCA are summarized in Table 1. In most
cases, almost 100% conversion of HMF was acquired in the
TEMPO/laccase system. Increasing the initial concentration of
TEMPO and buffer was benecial for HMF conversion at high
concentrations. The complete oxidation of HMF by TEMPO/
laccase resulted in the predominant formation of FFCA and
(Fig. 4B).
Given these facts, we further increased the HMF concentra-
tion to 150 mM and 200 mM, and this TEMPO/laccase system
was still capable of oxidizing HMF with conversion of 100% and
98.2%, respectively (Table 1). In the literature, Qin et al. re-
ported a conversion of 79% aer 48 h starting with 30 mM
24
HMF. Apparently, the results reported in this work were better
than the previous results. To our knowledge, this is the best
HMF conversion at such a high substrate concentration in the
TEMPO/laccase system ever reported.
Synthesis of FDCA via the TEMPO/laccase system and P.
putida KT2440 in tandem
With improved HMF conversion, we turned our attention to the
second step. Considering that the intermediate FFCA and the
acetate buffer in the rst step might affect the biocatalytic
performance of P. putida KT2440 in the last step, the effects of
these factors on P. putida KT2440 were tested by employing the
commercially available FFCA as a substrate. As shown in
Fig. S2,† when the pH values varied from 4.5 to 6.0, P. putida
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Table 1 Two-step cascade oxidation of HMF to FDCA
HMF conversion
(%)
FDCA selectivity
(%)
Cell dosage (g
a
ꢁ1
b
Entry
HMF (mM)
Buffer (mM)
TEMPO (mol%)
t (h)
L
)
t (h)
1
2
3
4
5
30
70
100
150
200
50
50
100
200
200
20
80
80
80
80
100
100
100
100
98.2
24
12
36
48
60
100
100
100
100
82.4
6
1
1
1
2
5
10
10
16
24
a
b
The reaction time of TEMPO/laccase oxidation. The reaction time of P. putida KT2440 biotransformation.
minor amounts of FDCA. HMF to FDCA conversion was faltered putida KT2440 in this study can completely convert FFCA to
at the aldehyde acid stage (FFCA) because of the low activity of FDCA within few hours in the second step. These results sug-
the TEMPO/laccase system on FFCA. FFCA was not efficiently gested that P. putida KT2440 is an excellent biocatalyst for the
oxidized by TEMPO/laccase system, which correlates with the oxidation of FFCA. Besides, P. putida KT2440 has good
low degree of hydration. Aer P. putida KT2440 cells, which can compatibility with TEMPO/laccase system, which would likely
directly oxidize the aldehyde group of FFCA, were added, FDCA be a potential and promising workhorse in biological conver-
was synthesized quickly, and FFCA decreased sharply in a short sion. Meanwhile, it was observed that improvement of the
reaction time. A full conversion of intermediates from the biocatalyst concentration was pivotal to obtaining a full
previous step was realized even at HMF concentrations up to conversion of FFCA with good product selectivity. When the
150 mM. Moreover, excellent selectivity (100%) was retained concentration of starting HMF was increased to 200 mM, only
during the reaction. Yang and coworkers exploited Comamonas 82.4% selectivity of FDCA was observed. Unsatisfactory FDCA
testosteroni SC1588 cells for synthesis of FDCA from FFCA in production may be attributed to insufficient amount of bio-
31
TEMPO/laccase system. However, it took about 15 h in the last catalyst at higher substrate loadings.
step and achieved around 90% of conversion. Because a rela-
The results obtained in this work were compared with those
tively high concentration of TEMPO in the catalytic system of previous studies. As shown in Table 2, the electrochemical
signicantly inhibited the activity of the cells. However, P. oxidation of HMF to FDCA was achieved using a catalytic anode
Table 2 HMF oxidation catalyzed by various catalysts
a
b
Catalysts
HMF (mM) Reaction conditions
t [h] HMF C [%] FDCA S/Y [%] Ref.
ꢀ
c
Manganese oxide (MnO
x
) anode
20
pH 1H
¼
2
SO
4
solution, 60 C, 1400 rpm, E n.a. >99.9
53.8 (Y)
90 (Y)
32
13
2.0 V vs. RHE, 250C charge passed
ꢀ
Co/Mn/Br
ꢄ377
Co/Mn/Br ¼ 1/0.015/0.5, T ¼ 180 C, P ¼ 0.5 n.a.
3
7
0 bar (molar CO
/93 (v/v), n ¼ 1200 rpm
/O
2 2
¼ 1), H
2
O/HOAc ¼
ꢀ
Pt/C
150
100
690 kPa O
2
, 2 equiv. of NaOH, 22 C
6
100
79 (S)
89 (Y)
14
20
ꢀ
Recombinant Raoultella ornithinolytica
BF60
HMF oxidase (HMFO)
45 g per L cells, 30 C, 50 mM phosphate 170 n.a.
buffer (pH 8)
ꢀ
4
3
20 mM HMFO, 20 mM FAD, 25 C,
potassium phosphate buffer of pH 7
AAO (5 mM), UPO (0.65 mM), sodium
phosphate (pH 7)
20 mol% TEMPO, 8.1 mg mL laccase, 36
40 g per L cells, phosphate buffer
200 mM, pH 7)
24
100
95 (Y)
91 (Y)
87 (Y)
22
23
31
AAO + UPO successively
120 100
ꢁ
1
TEMPO/laccase + C. testosterone SC1588 100
100
100
1
(
ꢁ
1
TEMPO/laccase + P. putida KT2440 in
150
80 mol% TEMPO, 2.6 mg mL laccase, 50
100 (S)
This work
tandem
16 g per L cells, acetate buffer of pH 6
a
b
c
C: conversion. Y: yield, S: selectivity. n.a.: not available.
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2
under strongly acidic conditions. Although the method
appeared to be clean, the initial HMF concentration and FDCA
yield reported in the literature were relatively low and accom-
panied by the formation of byproducts. Homogeneous catalysts
have some problems in the synthesis of FDCA, such as high
FDCA yield usually being obtained at the expense of harsh
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Conflicts of interest
There are no conicts to declare.
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This study was supported by the National Key Research and 24 Y.-Z. Qin, Y.-M. Li, M.-H. Zong, H. Wu and N. Li, Green
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