Hydrogenation of crude and purified d-glucosone generated by enzymatic oxidation of d-glucose

Article abstract of DOI:10.1039/d0ra05512c

D-Fructose is an important starting material for producing furfurals and other industrially important chemicals. While the base-catalyzed and enzymatic conversion of d-glucose to d-fructose is well known, the employed methods typically provide limited conversion. d-Glucosone can be obtained from d-glucose by enzymatic oxidation at the C2 position and, subsequently, selectively hydrogenated at C1 to form d-fructose. This work describes an investigation on the hydrogenation of d-glucosone, using both chromatographically purified and crude material obtained directly from the enzymatic oxidation, subjected to filtration and lyophilization only. High selectivities towards d-fructose were observed for both starting materials over a Ru/C catalyst. Hydrogenation of the crude d-glucosone was, however, inhibited by the impurities resulting from the enzymatic oxidation process. Catalyst deactivation was observed in the case of both starting materials.

Full text of DOI:10.1039/d0ra05512c

RSC Advances
View Article Online
View Journal | View Issue
PAPER
Hydrogenation of crude and purified D-glucosone
generated by enzymatic oxidation of D-glucose
Cite this: RSC Adv., 2020, 10, 30476
a
b
a
Atte Aho,b Dmitry Yu. Murzin
and Reko Leino
*
Robert Lassfolk,
D-Fructose is an important starting material for producing furfurals and other industrially important
chemicals. While the base-catalyzed and enzymatic conversion of D-glucose to D-fructose is well known,
the employed methods typically provide limited conversion. D-Glucosone can be obtained from D-
glucose by enzymatic oxidation at the C2 position and, subsequently, selectively hydrogenated at C1 to
form D-fructose. This work describes an investigation on the hydrogenation of D-glucosone, using both
chromatographically purified and crude material obtained directly from the enzymatic oxidation,
subjected to filtration and lyophilization only. High selectivities towards D-fructose were observed for
both starting materials over a Ru/C catalyst. Hydrogenation of the crude D-glucosone was, however,
inhibited by the impurities resulting from the enzymatic oxidation process. Catalyst deactivation was
observed in the case of both starting materials.
Received 23rd June 2020
Accepted 12th August 2020
DOI: 10.1039/d0ra05512c
rsc.li/rsc-advances
synthesis of D-fructose involves glucose isomerase enzyme.17
The main challenges with this method are related to thermo-
Introduction
dynamic limitations, typically yielding a conversion of around
50%.18 Separation of the two sugars (i.e., D-fructose and D-
glucose) requires an expensive ion-exchange resin in the Ca2+
form, operating at 60–70 ꢀC and resulting in up to 90% purity.19
An alternative path toward D-fructose is based on selective
hydrogenation of D-glucosone.20,21 For example, Sun et al. have
investigated the one-pot enzymatic oxidation of D-glucose to
D-glucosone, followed by catalytic hydrogenation of D-glucosone
to D-fructose.21 To ensure efficiency of the enzymatic reaction,
Development of new synthetic methods for conversion of
abundant carbohydrates, such as D-glucose, to other rare sugars
and further to platform chemicals remains as a topical research
eld. D-Glucosone can be obtained from D-glucose by enzymatic
oxidation at the C2 position (Scheme 1).1–6 In 6 h, 80% yield of D-
glucosone can be achieved and in 10 h >95% is obtained.6
Similar to other carbohydrates, D-glucosone exhibits congu-
rational equilibrium in aqueous solutions, where only hydrated
forms of D-glucosone have been observed.7 Several hours are
required to achieve equilibrium, the composition of which
varies depending on temperature, pH, and concentration.8
While until now the use of D-glucosone has been limited due to
its high price and limited availability, it has been shown
recently, for example, that certain ne chemicals such as kojic
acid could potentially be produced from this source.6 New
enzymes, more suitable for industrial scale, are currently being
developed and are likely to open the door for broader utilization
of D-glucosone.6
Established methods exist already for producing various
value-added chemicals, including furfurals, from D-fructose.9,10
The well-known base-catalyzed isomerization of D-glucose to D-
fructose is performed in aqueous solutions using both homo-
and heterogeneous catalysts.11–16 Such methods typically
provide high selectivities, albeit at the expense of low yields due
to unfavorable thermodynamics. The conventional process for
a
˚
Laboratory of Molecular Science and Technology, Abo Akademi University, 20500
Turku, Finland. E-mail: reko.leino@abo.
bLaboratory of Industrial Chemistry and Reaction Engineering, Abo Akademi
Scheme 1 Formation of D-fructose (3) from D-glucose (1) via D-glu-
cosone (2), showing the composition of 2 in aqueous solution.
˚
University, 20500 Turku, Finland
30476 | RSC Adv., 2020, 10, 30476–30480
This journal is © The Royal Society of Chemistry 2020

View Article Online
Paper
RSC Advances
Fig. 3 The conversion of D-glucosone in the continuous reactor.
Conditions: 0.1 mol lÀ1 purified D-glucosone in H2O (0.5 ml minÀ1),
400 mg 0.7 wt% Ru/C catalyst, 20 bar H2 (25 ml minÀ1), 110 ꢀC.
Fig. 1 Conversion of purified D-glucosone in the batch reactor.
Conditions: 100 ml 0.1 mol lÀ1 purified D-glucosone in H2O, 100 mg
4.6 wt% Ru/C catalyst, 20 bar H2, 110 ꢀC.
impurities leover from the enzymatic process on the catalyst
performance in D-glucosone hydrogenation is, therefore,
addressed.
mild conditions were required. Thus, long reaction times were
necessary with the total time for converting D-glucose to
D-fructose of 36 h. The mass balance and analysis of insoluble
products were not addressed in their study. In addition
substantial amounts of non-fructose carbohydrates were
present meaning that purication is needed.
Results and discussion
Efficient methods for conversion of D-glucose to D-fructose
are essential for production of ne chemicals and biofuels from
biomass with D-fructose being one of the key starting materials
for further processing.10,22–24 A continuous method for D-gluco-
sone production, allowing large quantities of D-glucosone to be
accessed, combined with subsequent hydrogenation to produce
pure D-fructose with minimal purication steps, would be
a viable alternative to the existing methods.
Prior to a detailed technological and economical analysis of
the two-step pathway involving enzymatic oxidation of D-glucose
and the subsequent hydrogenation of D-glucosone, feasibility of
the hydrogenation step using a real feedstock should be
established. Purity of the feedstock and its inuence on the
catalytic performance has been seldom addressed in connec-
tion with bioreneries, as oen in previous studies only model
compounds have been used. However, it is well known that the
presence of impurities can signicantly deteriorate catalytic
activity.25 In the present study, an evaluation of the effect of
Different catalysts were rst screened in a batch reactor using
puried D-glucosone as the starting material, before testing
crude D-glucosone. Theꢀ conditions used in the batch reactor
were 20 bars H2 at 110 C. These were selected based on prior
investigations on carbohydrate hydrogenations.26,27 The crude
D-glucosone, obtained from MetGen Oy, was only subjected to
ltration through a 0.45 micron lter in order to remove enzyme
particles aer the enzymatic process where D-glucose is oxidized
to D-glucosone.6 A portion of the crude D-glucosone was then
further puried by column chromatography to obtain the
puried D-glucosone used in the hydrogenation test reactions.
The catalysts screened in the batch reactor were Ru/C, Ru/Al2O3,
Ru on nitrogen doped carbon nanotubes (NCNT), Cu/SiO2, and
RANEY® nickel. The Ru/Al2O3 catalyst showed traces of D-fruc-
tose aer 180 min and a D-glucosone conversion of 51%, while
the Cu/SiO2 catalyst exhibited traces of D-fructose, D-sorbitol,
and D-mannitol, and a D-glucosone conversion of 66% aer
180 min. The RANEY® nickel catalyst displayed 72% conversion
Fig. 4 Productivity of the catalyst in the continuous reactor over time-
Fig.
2
Conversion of crude D-glucosone in the batch reactor. on-stream. Conditions: 0.1 mol lÀ1 purified D-glucosone in H2O (0.5
Conditions: 100 ml 0.08 mol lÀ1 crude D-glucosone in H2O, 100 mg ml minÀ1), 400 mg 0.7 wt% Ru/C catalyst, 20 bar H2 (25 ml minÀ1),
4.6 wt% Ru/C catalyst, 20 bar H2, 110 ꢀC.
110 ꢀC.
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 30476–30480 | 30477

View Article Online
RSC Advances
Paper
Table 1 Measured values of the spent and fresh catalysts from the batch experiments using N2 adsorption and desorption
Surface area
Pore volume
Median pore
width (A)
(m2 gÀ1
)
(cm3 gÀ1
)
˚
Catalyst
Fresh catalyst
700
85
99
0.26
0.11
0.15
4.9
7.4
6.9
Spent catalyst with puried D-glucosonea
Spent catalyst with crude D-glucosoneb
a
b
Conditions: 100 ml 0.1 mol lÀ1 puried D-glucosone in H2O, 100 mg 4.6 wt% Ru/C catalyst, 20 bar H2, 110 ꢀC, 150 min. Conditions: 100 ml
0.08 mol lÀ1 crude D-glucosone in H2O, 100 mg 4.6 wt% Ru/C catalyst, 20 bar H2, 110 ꢀC, 120 min.
aer 120 min, resulting in 36% D-fructose yield without any
Experiments in the continuous reactor showed clear deacti-
D-sorbitol or D-mannitol detected. The Ru/NCNT catalyst gave vation of the catalyst when the commercial 0.7% Ru/C was used
66% conversion aer 180 min with 19% D-fructose yield and as the catalyst (Fig. 3). The change in loading is justied
neither D-sorbitol nor D-mannitol. The most active catalyst was considering potential industrial implementation of the catalyst.
Ru/C, which then was selected for further experiments. Aer Extrudates of the commercial catalyst were rst crushed to
1 h, 90% conversion of D-glucosone was observed (Fig. 1) with appropriate size prior to their use in the reactor operating in
a subsequent conversion of D-fructose to D-mannitol and continuous mode. Conversion of D-glucosone steadily decreased
D-sorbitol.
with time on stream to 90% aer 1 h, dropping to <50% aer
Aer initial screening with puried D-glucosone, the crude D- 140 min. Aer 60 min, neither D-mannitol nor D-sorbitol were
glucosone was tested to investigate whether the impurities from produced, with D-fructose being the only product. As seen from
the enzymatic oxidation of D-glucose to D-glucosone substan- Fig. 4, the catalyst productivity, based on D-glucosone conver-
tially inuence the hydrogenation reaction. During the enzy- sion, starts to decrease signicantly aer 60 min and aer
matic process, NH4OH is added to maintain the pH at 6.6. The 160 min the productivity is below 50% of the initially observed,
crude D-glucosone used contained ca. 18 wt% more impurities consistent with catalyst deactivation. Leaching is not the
compared to the puried D-glucosone, as veried by HPLC underlying reason for the observed deactivation: only 0.05% of
analysis. Most likely, these impurities consist of salts and the total ruthenium used in the reactor had leached over
enzyme leovers from the oxidation process. As seen from 160 min, as determined by measuring the ruthenium concen-
Fig. 2, conversion of crude D-glucosone remains incomplete tration in the effluent solution by ICP. This conrms that the
aer 120 min of the reaction and no further transformations of deactivation results from some other source. Similar to the
D-fructose to D-mannitol and D-sorbitol were observed. The batch reactor, a change in mass balance closure is observed
results are consistent with catalyst deactivation or inhibition during the continuous experiment from 47% to 62%, indicating
due to lower conversion of D-glucosone and a lower yield of that less side product is formed under catalyst deactivation
D-fructose compared to the hydrogenation of puried conditions.
D-glucosone.
Next, deactivation was investigated in more detail by ana-
The initial catalyst productivity, based on the conversion of lysing the spent catalysts from the batch reactions. As shown in
D-glucosone to D-fructose, was 2.6 mol gÀ1 hÀ1 for the puried
D-glucosone compared to 2.2 mol gÀ1 hÀ1 for crude D-glucosone.
It has been established that salts and impurities signicantly
inhibit the catalyst activity during hydrogenation of carbohy-
+
drates.25,27 It was concluded in earlier work that NH4 gave the
most dominant deactivation among the salts tested during
hydrogenation of carbohydrates on Ru/C catalyst.27 The added
NH4OH in the enzymatic process of D-glucose oxidation forms
+
NH4 , which most likely inhibits the catalyst in this work.
From both Fig. 1 and 2 it can be observed that the total
concentration of analysed carbohydrates is decreasing. Using
the puried D-glucosone, about 60% of the mass balance
closure for carbohydrates was achieved aer 60 min and for the
crude D-glucosone this value is 91%, meaning that the impuri-
ties slow down the hydrogenation, but also the formation of
side products. The formed side products could not be analysed
using HPLC, indicating that they are insoluble in water and
could be responsible for some type of deactivation, which was
further investigated in the continuous reactor.
Fig. 5 Batch reactor set-up for transformation of glucosone.
30478 | RSC Adv., 2020, 10, 30476–30480
This journal is © The Royal Society of Chemistry 2020

View Article Online
Paper
RSC Advances
pathway from D-glucose to D-fructose viable for larger scale
production.
Experimental
D-Glucosone was obtained from MetGen Oy, produced by an earlier
described method.6 The crude D-glucosone used in this work was
obtained by ltration (0.45 micron) and freeze drying of the initially
produced material obtained directly from the manufacturing
process. Further purication was then carried out by column
chromatography, using silica gel 60 (0.040–0.060 mm) as the
stationary phase and MeOH/CH2Cl2 as the eluent. The 4.6% Ru/C
catalyst had a Ru particle size of 2.5 nm and a catalyst size of
<100 mm.26 The 3.6% Ru/NCNT catalyst with nitrogen doped carbon
nanotubes as a support (Bayer Technical Services) had a Ru particle
size of 3.3 nm and was crushed to a catalyst size of 125–250 mm.26
The 5.0% Ru/Al2O3 catalyst (Fluka) was used as such. The 20% Cu/
SiO2 catalyst (BASF, catalyst H3-11) was crushed to <100 mm and
used as such. The RANEY® nickel catalyst (Grace, RANEY® 3110)
was likewise used as received. The 0.7% Ru/C catalyst (Engelhard)
had a Ru particle size of 2.9 nm and a catalyst size of 250–355 mm.26
The catalysts are pre-reduced in hydrogen prior to storage. In situ
pre-treatment of ruthenium catalysts prior to experiments at mild
conditions was done to remove the oxide layer. The hydrogenation
reactions were followed by high-performance liquid chromatog-
raphy (HPLC) (HITACHI Chromaster HPLC) equipped with
a refractive index (RI) detector. A Biorad HPX-87C carbohydrate
column was used with 1.2 mM CaSO4 (0.2 ml minÀ1 ow rate) as the
mobile phase. Temperature of the column and the detector were
80 ꢀC and 40 ꢀC, respectively. An accuracy within 3% was obtained
with the HPLC. Leaching was investigated by measuring the
concentration of ruthenium using a PerkinElmer ELAN 6100 DRC
Plus, with a detection limit of 0.003 mg lÀ1. The fresh and spent
catalysts were analyzed using Micrometrics MicroActive 3Flex 3500
instrument for surface area and pore size measurements, with an
accuracy within 2%.
Fig. 6 Continuous reactor set-up for transformation of glucosone.
Table 1, the surface area of the Ru/C catalyst decreases signi-
cantly when using both puried and crude D-glucosone, to 12%
and 14%, respectively, of the fresh catalyst surface area. Simi-
larly, substantial changes in the pore volumes were observed
when hydrogenating puried and crude D-glucosone, leading to
a decrease of 42% and 58% respectively of the fresh catalyst.
The smaller pores are blocked entirely due to fouling,
increasing the apparent average pore size measured by nitrogen
physisorption. Previously, it has been shown that D-glucosone
has a tendency to oligomerize or polymerize at temperatures
above 50 ꢀC.6 The difference in mass balance closures probably
results from the formation of polymers, which are deposited on
the walls and adsorbed on the catalyst due to low solubility in
water, resulting in fouling.
The mass balance is calculated as the ratio of the total
carbohydrate concentration C to the initial one C0, i.e. C/C0.
The productivity was calculated for the batch reactor as
DC/t  mRu
,
Conclusions
where DC is the difference in the D-glucosone concentration,
while for the continuous reactor the following expression was
used for productivity
As demonstrated in this study, selective hydrogenation of D-
glucosone to D-fructose is possible using both puried and
crude D-glucosone. Salts applied in the enzymatic oxidation of D-
glucose to D-glucosone, such as NH4+, are the most probable
catalyst inhibitors. Signicant deactivation took place in the
continuous reactor with the puried D-glucosone starting
material. The main reason for catalyst deactivation is fouling,
most likely due to oligomerization or polymerization of D-glu-
cosone. Such deactivation could be clearly observed in the
continuous reactor, where the productivity decreased signi-
˜
Dn/mRu
,
˜
where Dn is the difference in D-glucosone molar ow at the inlet
and outlet of the reactor.
Batch reactor
cantly with the time on stream. When using the crude D-glu- The catalyst (100 mg) was fed into the reactor (300 ml) and
cosone, 48% conversion to D-fructose was achieved aer 2 h, ushed with Ar and then with H2 (Fig. 5). The reactor was
ꢀ
while for the puried D-glucosone conversion of 42% was heated to 110 C and the H2 pressure was kept at 4 bars for 1–
reached already aer 1 h. Further investigations on minimiza- 1.5 h. D-Glucosone (2 g) was dissolved in 100 ml deionized water
tion of the polymerization and fouling are needed to make this and heated to 80 ꢀC. The D-glucosone solution was added to the
This journal is © The Royal Society of Chemistry 2020
RSC Adv., 2020, 10, 30476–30480 | 30479

View Article Online
RSC Advances
Paper
¨¨
reactor and the hydrogen pressure was raised to 20 bars. The
reaction started with stirring at 1200 rpm. The samples were
taken at desired intervals and analyzed by HPLC. Small catalyst
particles were used under vigorous stirring (1200 rpm) to avoid
internal and external mass transfer limitations.
6 R. Lassfolk, A. Suonpaa, K. Birikh and R. Leino, Chem.
Commun., 2019, 55, 14737–14740.
7 S. Freimund, L. Baldes, A. Huwig and F. Giorn, Carbohydr.
Res., 2002, 337, 1585–1587.
¨
8 S. Freimund and S. Kopper, Carbohydr. Res., 2004, 339, 217–
220.
Continuous reactor
9 L. Rigal, A. Gaset and J. P. Gorrichon, Ind. Eng. Chem. Prod.
Res. Dev., 1981, 20, 719–721.
The hydrogenation catalyst (400 mg) was mixed with glass beads
(9.6 g) in a column through which the reaction solution was
pumped at different rates. The reactor (diameter: 12.5 mm) was
packed by rst lling it with neat glass beads (425–600 mm) and
then with the catalyst mixture and nally neat glass beads
(Fig. 6). The substrate contact time to the catalyst was 2.4 min.
The reactor was ushed with Ar and then with H2. The reactor
was then heated to 110 ꢀC and H2 pressure was raised to 20 bars.
A 0.1 mol lÀ1 D-glucosone solution was prepared and pumped
into the reactor using an HPLC-pump (Agilent 1100 Quaternary
pump) at a rate of 0.5 ml minÀ1. The H2 ow was adjusted to 25
ml minÀ1. The samples were taken every 20 min and analyzed
by HPLC.
10 J. B. Binder and R. T. Raines, J. Am. Chem. Soc., 2009, 131,
1979–1985.
˜
11 S. Lima, A. S. Dias, Z. Lin, P. Brandao, P. Ferreira,
M. Pillinger, J. Rocha, V. Calvino-Casilda and A. A. Valente,
Appl. Catal., A, 2008, 339, 21–27.
12 M. Moliner, Y. Roman-Leshkov and M. E. Davis, Proc. Natl.
Acad. Sci., 2010, 107, 6164–6168.
13 C. Moreau, R. Durand, A. Roux and D. Tichit, Appl. Catal., A,
2000, 193, 257–264.
14 R. O. L. Souza, D. P. Fabiano, C. Feche, F. Rataboul,
D. Cardoso and N. Essayem, Catal. Today, 2012, 195, 114–
119.
15 S. S. Chen, D. C. W. Tsang and J. P. Tessonnier, Appl. Catal.,
B, 2020, 261, 118126.
16 H. S. El Khadem, S. Ennifar and H. S. Isbell, Carbohydr. Res.,
1987, 169, 13–21.
17 L. M. Hanover and J. S. White, Am. J. Clin. Nutr., 1993, 58,
724S–732S.
18 N. E. Lloyd and K. Khaleeluddin, Cereal Chem., 1976, 53,
270–281.
Conflicts of interest
There are no conicts to declare.
Acknowledgements
Robert Lassfolk thanks the Magnus Ehrnrooth Foundation and
Waldemar von Frenckell Foundation for funding. The authors
thank MetGen Oy for supplying the glucosone, Ekaterina
Kholkina for the kind help with the surface area analysis and
Sten Lindholm for assistance with the ruthenium concentration
measurements by ICP.
19 Y.-J. Wang, X.-W. Jiang, Z.-Q. Liu, L.-Q. Jin, C.-J. Liao,
X.-P. Cheng, B.-X. Mao and Y.-G. Zheng, Can. J. Chem. Eng.,
2016, 94, 537–546.
20 J. Geigert, S. L. Neidleman and D. S. Hirano, Carbohydr. Res.,
1983, 113, 159–162.
21 J. Sun, H. Li, H. Huang, B. Wang, L.-P. Xiao and G. Song,
ChemSusChem, 2018, 11, 1157–1162.
22 A. Voll and W. Marquardt, Biofuels, Bioprod. Bioren., 2012,
292–301.
23 H. Zhao, J. E. Holladay, H. Brown and Z. C. Zhang, Science,
2007, 316, 1597–1600.
Notes and references
1 A. Karmali and J. Coelho, Appl. Biochem. Biotechnol., 2011,
163, 906–917.
2 A. Huwig, H.-J. Danneel and F. Giorn, J. Biotechnol., 1994,
32, 309–315.
´
24 Y. Roman-Leshkov, C. J. Barrett, Z. Y. Liu and J. A. Dumesic,
Nature, 2007, 447, 982–985.
3 G. Treitz, G. Maria, F. Giorn and E. Heinzle, J. Biotechnol.,
2001, 85, 271–287.
25 D. Y. Murzin and T. Salmi, Catal. Lett., 2012, 142, 676–689.
26 A. Aho, S. Roggan, O. A. Simakova, T. Salmi and
D. Y. Murzin, Catal. Today, 2015, 241, 195–199.
¨
4 S. Freimund, A. Huwig, F. Giorn and S. Kopper, Chem.–
Eur. J., 1998, 4, 2442–2455.
¨
27 J. Hilgert, S. Lima, A. Aho, K. Eranen, D. Y. Murzin and
5 H. Seto, H. Kawakita and K. Ohto, Biochem. Eng. J., 2009, 48,
36–41.
R. Rinaldi, ChemCatChem, 2018, 10, 2409–2416.
30480 | RSC Adv., 2020, 10, 30476–30480
This journal is © The Royal Society of Chemistry 2020