Upgrading of Biomass Monosaccharides by Immobilized Glucose Dehydrogenase and Xylose Dehydrogenase

Article abstract of DOI:10.1002/cctc.201801335

Direct upgrading and separation of the monosaccharides from biomass liquors is an overlooked area. In this work we demonstrate enzymatic production of gluconic acid and xylonic acid from glucose and xylose present in pretreated birchwood liquor by glucose dehydrogenase (GDH, EC 1.1.1.47) and xylose dehydrogenase (XDH, EC 1.1.1.175), respectively. The biocatalytic conversions were compared using two different kinds of silica support materials (silica nanoparticles (nanoSiO₂) and porous silica particles with hexagonal pores (SBA 15 silica) for enzyme immobilization. Upon immobilization, both enzymes showed significant improvement in their thermal stability and robustness at alkaline pH and exhibited over 50 % activity even at pH 10 and 60 °C on both immobilization matrices. When compared to free enzymes at 45 °C, GDH immobilized on nanoSiO₂ and SBA silica displayed a 4.5 and 7.25 fold increase in half-life, respectively, whilst XDH immobilized on nanoSiO₂ and SBA showed a 4.7 and 9.5 fold improvement in half-life, respectively. Additionally, after five reaction cycles both nanoSiO₂GDH and nanoSiO₂XDH retained more than 40 % activity and GDH and XDH immobilized on SBA silica maintained around 50 % of their initial activity resulting in about 1.5–1.6 fold increase in biocatalytic productivity compared to the free enzymes.

Full text of DOI:10.1002/cctc.201801335

Accepted Article
Title: Upgrading of biomass monosaccharides by immobilized glucose
dehydrogenase and xylose dehydrogenase
Authors: Jakub Zdarta, Manuel Pinelo, Teofil Jesionowski, and Anne
Meyer
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Upgrading of biomass monosaccharides by immobilized
glucose dehydrogenase and xylose dehydrogenase
Jakub Zdarta*[a,b], Manuel Pinelo[b], Teofil Jesionowski[a], Anne S. Meyer[b]
Abstract: Direct upgrading and separation of the monosaccharides
from biomass liquors is an overlooked area. In this work we
demonstrate enzymatic production of gluconic acid and xylonic acid
from glucose and xylose present in pretreated birchwood liquor by
glucose dehydrogenase (GDH, EC 1.1.1.118) and xylose
dehydrogenase (XDH, EC 1.1.1.175), respectively. The biocatalytic
conversions were compared using two different kinds of silica
support materials (silica nanoparticles (nanoSiO2) and porous silica
particles with hexagonal pores (SBA silica) for enzyme
immobilization. Upon immobilization, both enzymes showed
significant improvement in their thermal stability and robustness at
alkaline pH and exhibited over 50% activity even at pH 10 and 60°C
on both immobilization matrices. When compared to free enzymes at
45°C, GDH immobilized on nanoSiO2 and SBA silica displayed a 4.5
and 7.25 fold increase in half-life, respectively, whilst XDH
immobilized on nanoSiO2 and SBA showed a 4.7 and 9.5 fold
improvement in half-life, respectively. Additionally, after five reaction
cycles both nanoSiO2GDH and nanoSiO2XDH retained more than
40% activity and GDH and XDH immobilized on SBA silica
maintained around 50% of their initial activity resulting in about
1.5-1.6 fold increase in biocatalytic productivity compared to the free
enzymes.
compounds from biomass[5]. Gluconic acid is a mild organic acid
that has multiple applications in the food industry but also in
pharmaceuticals synthesis[6]. Xylonic acid is used as a substrate
for synthesis of 1,2,4-butanetriol and 1,2,4-butanetriol trinitrate[7]
and has moreover been projected for various uses in the food,
pharmaceutical and agriculture industries[8]. However, the
practical biocatalytic conversion of biomass components using
GDH and XDH is limited due to their relatively low stability at
extreme pH and temperature conditions.
A possible approach to overcome these limitations and at the
same time increase the biocatalytic productivity via maximizing
enzyme “reuse” is enzyme immobilization[4,9]
.
Enzyme
immobilization may moreover reduce the complexity of enzyme
separation from the products after reaction[6,10]
Various
.
techniques and methods of immobilization such as adsorption,
covalent binding, entrapment or encapsulation have been
described previously[11-13]. Silica-based materials are frequently
used as support materials in enzyme immobilization due to their
thermal, chemical and mechanical resistance, good sorption
properties, and the presence of many hydroxyl groups that
facilitate enzyme binding[14-16]. Moreover, silica-based materials
are easy to obtain and relatively cheap, a feature of particular
significance in biomass valorization processes. Nonetheless,
data related to immobilization of glucose dehydrogenase and
xylose dehydrogenase on silica are limited.
Introduction
As data about immobilization of glucose dehydrogenase and
xylose dehydrogenase are limited, a simple protocol is required
to use efficient and stable support materials for immobilization of
GDH and XDH which additionally facilitating further separation of
products of enzymatic conversion. Thus, in the present study,
we examine immobilization, characterization and comparison of
GDH and XDH on two types of silica support materials – silica
nanoparticles and mesoporous silica with hexagonally ordered
pores. As part of the study, we also investigate the kinetics of
the enzymatic conversions of monosaccharides, and examine
the effect of various pH and temperature conditions on free and
immobilized GDH and XDH and evaluate the influence of these
parameters on the enzyme stability. The practical application of
the biocatalytic systems is validated by applying them for
conversion of glucose and xylose present in authentic pretreated
birch wood biomass liquors.
Bio-based conversion of biomass components by soluble or
immobilized enzymes has been presented as a promising and
efficient approach for sustainable production of valuable
chemical compounds under mild reaction conditions[1,2]. Use of
glucose dehydrogenase (GDH) (EC 1.1.1.118) and xylose
dehydrogenase (XDH) (EC 1.1.1.175) is of interest in this regard
because these NAD+ dependent enzymes catalyze conversion
of D-glucose into gluconic acid and D-xylose into xylonic acid,
respectively[3]. Besides transformation of monosaccharides, the
enzymatic conversion adds a charge to the products, which can
facilitate separation of acid products from a mixed product
stream, for instance using membrane technology[4]. Both
gluconic acid and xylonic acid are classified by the US
Department of Energy among the top 30 potential high-value
[a]
[b]
Dr Jakub Zdarta, Prof. Teofil Jesionowski
Institute of Chemical Technology and Engineering, Faculty of
Chemical Technology,
Poznan University of Technology
Berdychowo 4, PL-60965 Poznan, Poland
E-mail: jakub.zdarta@put.poznan.pl
Dr Jakub Zdarta, Prof. Manuel Pinelo, Prof. Anne S. Meyer
Center for BioProcess Engineering, Department of Chemical and
Biochemical Engineering
Technical University of Denmark,
SoltoftsPlads 229, DK-2800 Kgs. Lyngby, Denmark
Results and Discussion
GDH and XDH immobilization
In this study, silica nanopowder of particle size in the range of
10–20 nm and pore diameters of around 2 nm size (nanoSiO2)
and mesoporous SBA 15 silica (<150 μm particle size) with
hexagonal pore morphology and pore size up to 20 nm (SBA15)
were used. It should be emphasized that type of the support
material is known to affect place of enzyme binding. According
to the previously published articles, using silica nanoparticles,
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GDH and XDH are expected to be immobilized on the particle
surfaces, while in case of SBA silica, biomolecules are
presumably bound mainly inside the SBA 15 silica pores, as it is
unaltered after enzyme binding. Surface area of SBA 15 silica
was intact after immobilization, meanwhile significant changes
were noticed in pore size and pore volume indicating GDH and
XDH immobilization into the pores of the hexagonal silica.
schematically presented in Fig. 1[14,15]
.
(b)
(a)
Table 1. Porous structure parameters of nanoSiO2 silica and
hexagonal mesoporous silica SBA 15 before and after immobilization
of glucose dehydrogenase or xylose dehydrogenase.
BET surface
area (m2/g)
Pore volume
(cm3/g)
Pore size
(nm)
Sample name
219.6
118.3
121.6
579.6
567.2
570.2
0.098
0.087
0.091
0.868
0.476
0.513
2.048
2.046
nanoSiO2
Figure 1. Schematic presentation of immobilization of glucose dehydrogenase
and/or xylose dehydrogenase: (a) on silica nanoparticles and (b) in pores of
hexagonal mesoporous silica.
nanoSiO2GDH
nanoSiO2XDH
SBA15
2.047
19.211
14.754
15.634
These expectations were confirmed by the images from
transmission electron microscopy and changes of the porous
structure parameters of silica-based materials after GDH and
XDH immobilization (Fig. 2 and Table 1). After immobilization of
both enzymes surface area of the nanoSiO2 decreased around
two times and reached about 120 m2/g as its pore size was
SBA15GDH
SBA15XDH
100 nm
100 nm
100 nm
(a)
(b)
(c)
100 nm
100 nm
100 nm
(e)
(d)
(f)
Figure 2. TEM images of the: (a) SBA 15 silica and (b) nanoSiO2 silica before enzyme immobilization; (b) and (e) after immobilization of glucose
dehydrogenase; (c) and (f) after immobilization of xylose dehydrogenase.
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Table 2. Immobilization yield and amount of immobilized enzyme in different biocatalytic systems. Specific activity and activity retention of
free and immobilized GDH and XDH.
Analyzed parameter
freeGDH
nanoSiO2GDH
SBA15GDH
freeXDH
nanoSiO2XDH
SBA15XDH
Amount of immobilized
enzyme ’(mg/g);*(U/g)
2.8±0.1’
560±19*
2.5±0.1’
500±14*
-
-
534±16*
89±3.0
450±12*
75±2.7
Immobilization yield (%)
Specific activity (U/mg)
-
94±3.5
82±2.8
-
42.2+0.9
30.5±0.8
27.3±1.1
46.8±1.7 U/ml
29.0±0.7
24.6±0.8
The different ways of immobilization were thus anticipated to
produce different amounts of immobilized enzymes, highest on
the nanoparticles due to their high surface area: 2.8 mg (560 U)
megaterium glucose dehydrogenase and immobilized less than
0.5 mg of enzyme per 1 g of the support[20], whereas here we
report a loading capacity of silica nanoparticles of 2.8 mg/g,
of GDH and 534 U of XDH were immobilized on 1 g of nanoSiO2, probably due to higher density of hydroxyl groups on the surface
which was about 10–15% higher compared with SBA 15
(Table 2). Immobilization yield also followed this trend: when
nanoSiO2 was used, immobilization yield for GDH and XDH
reached 94 and 82%, whereas for SBA 15 it was 89 and 75%,
respectively, Hence, irrespective of the support material,
a higher quantity of the immobilized enzyme was noticed in case
of glucose dehydrogenase. Greater amounts of immobilized
GDH, as compared to XDH, are probably related to the three-
dimensional structure and amino acids composition of this
enzyme[17]. A possible explanation for this is that enzymes
molecules possessing more accessible amino acids in their
structure, like arginine, asparagine, glutamic acid or lysine are
more effectively immobilized, as it was previously reported for
of nanoSiO2.
Effect of temperature and pH on activity and stability of free
and immobilized GDH
Free GDH as well as both of the GDH-based, immobilized
systems exhibited the highest activity at 45°C (Fig. 3a). At
temperatures below optimum, free GDH was characterized by
slightly higher, but significantly different, activity than the
immobilized enzyme. But at higher temperatures (45–60°C),
immobilized GDH showed better activity than the free biocatalyst.
This is particularly noticeable in the case of the mesoporous
SBA 15 silica support and GDH immobilized on this material
showed higher residual activity (53%) than free enzyme (29%)
even at a temperature of 60°C. GDH is known as an enzyme
that exhibits catalytic activity only in multimeric form. In
a previous study, it has been shown that at temperatures above
50°C, multimers tend to dissociate, which leads to irreversible
glucose-6-phosphate dehydrogenase[18]
.
Nevertheless, both
enzymes are linked to the silica by adsorption immobilization, by
creation of electrostatic interactions and hydrogen bonds
between mainly amino (–NH2), hydroxyl (–OH) and carbonyl
(–COOH) groups of the enzymes and hydroxyl groups of the
silica-based support.
inactivation[21]
.
Since immobilization using silica provided
enzymes multipoint attachment and improved rigidity of the
enzyme, thermal dissociation of silica-bounded GDH multimers
could be prevented that lead to better activity retention at higher
temperatures.
The specific activity of GDH and XDH was 42.2 U/mg and
46.8 U/ml, respectively, indicating that both enzymes showed
similar catalytic activity in model reaction. The specific activity of
the immobilized enzymes was lower and reached 30.5 and
27.3 U/mg of the enzyme, respectively, for nanoSiO2GDH and
SBA15GDH, which corresponded to an activity retention of
72.3 and 64.7%, respectively. For nanoSiO2XDH and
SBA15XDH, these values were slightly lower and reached
29.0 and 24.6 U/mg, respectively. Also noticed was a lower
activity retention of 61.8% for nanoSiO2XDH and 52.6% for
SBA15XDH. The lower values of specific activity and activity
retention noticed for enzymes immobilized onto SBA 15
compared with nanoSiO2, might be explained by two factors:
(i) lower amount of immobilized biocatalysts and (ii) hindered
accessibility of the enzyme active sites for the substrates
molecules due to immobilization of enzymes mainly into the
pores of the support. Nevertheless, lower activity of immobilized
XDH compared with GDH is probably related to the larger
conformational changes of 3-D biomolecule structure that occur
Free and silica immobilized GDH showed similar pH profiles
which were, however, statistically significantly different over
whole analyzed pH range (6–10), as illustrated in Fig. 3b.
However, nanoSiO2GDH and SBA15GDH exhibited about
10 and 15% enhancement of catalytic activity, respectively,
particularly at basic conditions (8.5–10), compared to free
catalysts. A shift of the pH optima from 8 (free GDH) to 8.5
(immobilized GDH) was also observed. Changes in the pH and
temperature profiles could be explained by the fact that
immobilization, in general, leads to conformational changes of
the structure of the enzyme[22]. These changes occur mainly as a
result of ionization of side chains of active site amino acids.
However, type, nature and functional group of the matrix also
play a significant role. These factors lead to modifications of
microenvironment around the active site of the enzyme and in
consequence affect the pH and temperature profile of the
upon immobilization, as reported earlier by Li et al.[19]
.
immobilized biocatalysts[23]
.
There is no available literature about efficient immobilization of
XDH, and though there are some reports about immobilization of
GDH, results presented here go further than previously
published data. For example, Baron et al. used controlled pore
silica with average pore size of 500 Å, as a support for Bacillus
Evaluation of the thermal stability of the immobilized enzyme is
a crucial step in determining practical applications of the
produced biocatalytic systems. Binding of GDH to the silica
support significantly improved thermal stability of the enzyme:
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after 45 min of heating at 45°C, at pH 8, free enzyme maintained
42.5% of its activity, while nanoSiO2GDH and SBA15GDH
retained 47 and 53% of catalytic activity, respectively (Fig. 3c).
Significant improvement in the stability of the immobilized GDH
can also be observed in values of the inactivation constant (kD)
and enzyme half-life (t1/2) (Fig. 3d). kD and t1/2 for free glucose
Moreover, after 210 min of incubation under the same conditions, dehydrogenase was found to be 0.0174 1/min and 39.8 min,
no catalytic activity was observed for free GDH whereas
immobilized enzyme was more stable and retained about 30 and
35% of its activity when immobilized using nanoSiO2 and SBA
silica, respectively that might be explained by creation of a more
suitable microenvironment after immobilization as reported also
by Li et al.[24]. Since drop in the relative activity of free GDH
could be explained mainly by thermal and chemical inactivation
of the biocatalyst, decrease in the activity of the immobilized
enzyme could also partially reflect leakage of GDH from the
matrix. Nevertheless, the higher relative activity noticed for
SBA15GDH, as compared to nanoSiO2GDH, is related to
catalyst immobilization in the hexagonal pores of the support,
which ensures better protection of the enzyme molecules
against harsh reaction conditions.
respectively, whereas inactivation constants of nanoSiO2GDH
and SBA15GDH were, respectively, 4.5 fold and 7.2 fold
(0.0038 1/min and 0.0024 1/min) lower than of those of the free
enzyme. As a result, enzyme half-life was significantly improved
and reached 182.4 min for nanoSiO2GDH and 288.8 min for
SBA15GDH. These findings are in agreement with data reported
earlier by Twala et al. who immobilized GDH on functionalized
ReSynTM polymer microspheres. However, in their study,
immobilized enzyme half-life increased two fold after
immobilization after incubation at 45°C compared to the free
catalysts, while in the current study, enzyme half-life was
improved much more (for about 4.5 fold). That is probably
related to the more suitable chemical microenvironment created
by the silica nanoparticles compared to polymeric support[1].
(b)
(a)
100
100
90
90
80
70
60
80
70
60
freeGDH
nanoSiO₂GDH
SBA15GDH
freeGDH
nanoSiO₂GDH
SBA15GDH
50
50
40
40
30
30
5
6
7
8
9
10
11
25 30 35 40 45 50 55 60 65
pH
Temperature (°C)
(c)
(d)
5.0
4.0
3.0
2.0
1.0
0.0
freeGDH
nanoSiO₂GDH
SBA15GDH
100
80
60
40
20
0
freeGDH
nanoSiO₂GDH
SBA15GDH
0
30 60 90 120 150 180 210 240
time (min)
0
30 60 90 120 150 180 210 240
time (min)
Figure 3. (a) temperature profiles, (b) pH profiles and (c, d) thermal stability of free and silica immobilized glucose dehydrogenase (GDH). Thermal stability of free
and silica immobilized GDH was examined under optimal temperature (45°C) and pH (8) conditions. Inactivation constants (kD) were evaluated based on the
linear regression slope. All data are presented as means ± standard deviation.
Effect of temperature and pH on activity and stability of free
and immobilized GDH
temperature exceed 40°C, activity of free enzyme dropped
sharply while immobilized XDH retained significantly higher
activities. At 65°C nanoSiO2XDH and SBA15XDH retained
69 and 64%, respectively, of their residual activity while free
biocatalyst showed less than 50% residual activity. The higher
temperature optimum recorded for immobilized XDH and the
significant improvement of immobilized enzyme activity at higher
temperatures are probably a result of creation of enzyme-matrix
The optimum temperature of free XDH was 40°C, whereas for
nanoSiO2XDH and SBA15XDH, the optimum shifted slightly
upwards (45°C) (Fig. 4). Temperature profiles of free and
immobilized XDH were also statistically significantly different.
At temperatures below maximum, free enzyme showed higher
activity compared with immobilized enzymes. However, when
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interactions that form external enzyme backbones and increase
rigidity of the biocatalyst[25]. Such changes would have protected
the active sites of the XDH against conformational changes and
denaturation at higher temperatures, and thus help to maintain
high catalytic activity.
decreased (Table 2), stability of immobilized XDH was improved
compared to the free enzyme (Fig. 4c). After 180 min of
incubation at pH 8 and 40°C, free enzyme completely lost its
activity while nanoSiO2XDH and SBA15XDH retained more than
50% of their initial activity. Moreover, after 240 min of incubation
All tested XDH systems exhibited their optimum at pH 8 (Fig. 3b). at 45°C, XDH immobilized on silica nanoparticles or in
Moreover, their pH profiles were significantly different, however,
particularly at pH values close to neutral (6.5–8.5), they were
similar. Immobilization of XDH at basic pH (9–10) resulted in
nanoSiO2XDH and SBA15XDH showing about 10 and 20%
higher activity, respectively, compared to the free catalyst.
Under strongly acidic or basic conditions, ionic groups presented
in the enzyme structure might be protonated or deprotonated,
respectively. These changes result in formation of electrostatic
repulsion between these groups, and hence destruction and
degeneration of the enzyme active site and decrease in catalytic
properties of the enzyme[28]. Creation of the enzyme-matrix
interactions which limit dissociation of enzyme subunits and
stiffening the structure of the biomolecule after immobilization,
caused that susceptibility of the enzyme to conformational
changes in pH decreases, and leads to improvements of
enzyme activity under harsh pH conditions.
hexagonal silica retained over 25 and over 30% of activity,
respectively. As stability and catalytic activity of the enzyme after
immobilization were improved, inactivation constant (kD) and
enzyme half-life (t1/2) were also enhanced (Fig. 4d). kD and t1/2 of
free enzyme reached 0.0237 1/min and 29.2 min, respectively,
while after immobilization on silica nanoparticles the values were
0.005 1/min and 138.6 min, respectively. When hexagonal SBA
silica was used as support, an even higher enzyme half-life
(277.2 min) and lower inactivation constant (0.0025 1/min) were
obtained. It might be explained by the fact that due to
immobilization in the pores of the matrix, enzyme molecules are
better protected compared to attachment of biocatalysts onto the
surface of the matrix. Free enzyme on the other hand was
subject to total inactivation due mainly to thermal and chemical
and denaturation caused by heating and contact with base.
However, negative effect of harsh reactive conditions on GDH
and XDH was strongly hampered after enzyme immobilization
due mainly to stabilization of the entire biocatalyst structure.
Additionally, thermal stability of free and both silica-immobilized
XDH was studied. Although, after immobilization activity
(b)
(a)
100
100
90
80
70
60
90
80
70
60
freeXDH
nanoSiO₂XDH
SBA15XDH
freeXDH
nanoSiO₂XDH
SBA15XDH
50
50
40
40
30
30
5
6
7
8
9
10
11
25 30 35 40 45 50 55 60 65
Temperature (°C)
(d)
pH
(c)
5.0
4.0
3.0
2.0
1.0
0.0
100
80
60
40
20
0
freeXDH
nanoSiO₂XDH
SBA15XDH
freeXDH
nanoSiO₂XDH
SBA15XDH
0
30 60 90 120 150 180 210 240
time (min)
0
30 60 90 120 150 180 210 240
time (min)
Figure 4. (a) temperature profiles, (b) pH profiles and (c, d) thermal stability of free and silica immobilized xylose dehydrogenase (XDH). Thermal stability of free
and silica immobilized XDH was examined at optimal temperature (40°C for free enzyme and 45°C for immobilized enzymes) and pH (8) conditions. Inactivation
constants (kD) were evaluated based on the linear regression slope. All data are presented as means ± standard deviation.
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Table 3. Kinetic parameters for free and silica immobilized GDH and XDH.
Analyzed parameter
freeGDH
nanoSiO2GDH
SBA15GDH
freeXDH
nanoSiO2XDH
SBA15XDH
KM (mM)
Vmax
16.1
7.7 U/mg
79
21.8
5.1 U/mg
77
28.7
4.1 U/mg
75
0.31
0.81 U/mL
27
0.34
0.70 U/mg
21
0.45
0.64 U/mg
19
kcat (1/s)
kcat/Km (1/s*mM)
4.9
3.5
2.6
87.1
61.7
42.2
Kinetic parameters of free and immobilized enzymes
presented above (Table 2). It also should be emphasized that,
irrespectively of the used enzyme, higher values of KM were
noticed for enzymes immobilized using SBA silica, compared to
nanoSiO2, indicating lower substrate affinity to bounded GDH
and XDH. It is related to the fact that using mesoporous silica
(SBA 15) higher diffusional limitations occurred because
biomolecules are immobilized mainly into its pores, as in case of
nanoSiO2 enzyme is attached onto the surface of the support, as
presented in Fig. 1.
The KM Michaelis–Menten constant and the Vmax for free GDH
were found to be 16.1 mM and 7.7 U/mg, respectively. After
immobilization KM increased and reached 21.8 and 28.7 mM for
nanoSiO2GDH and SBA15GDH, respectively. Simultaneously,
Vmax dropped 35 and 45% for nanoSiO2GDH and SBA15GDH,
respectively, compared to free catalyst. Nevertheless, it should
be underlined that turnover number of immobilized biocatalysts
was maintained almost unaltered as kcat of nanoSiO2GDH and
SBA15GDH were 97 and 94% of that of free GDH, respectively
(Table 3). Since turnover numbers of free and immobilized
glucose dehydrogenase are comparable, increase in KM value
could be explained by the fact that after immobilization
diffusional limitations occurred. As a result, active sites of the
silica-bounded GDH are less accessible to the substrate and
cofactor molecules, however, additional effects of cofactor-
matrix and substrate-matrix interactions cannot be excluded
either[20]. Thus, as suggested earlier by Zhou et al., a higher
concentration of substrates is required to enhance their
interactions with immobilized catalysts[27]. Moreover, as a result
of enzyme attachment to the support, some of the active sites
could be blocked, which reduces reaction rate and leads to drop
in the maximum reaction velocity (Vmax) [30]. Baron et al., who
immobilized GDH from Bacillus onto DEAE-Sephadex modified
by glutaraldehyde, also made similar observations. However, in
their study KM was 4-fold greater after immobilization and Vmax
was simultaneously almost 4-fold lower compared to the free
Conversion of biomass liquors
Investigation of stability of free and immobilized enzymes was
carried out based on model solutions of xylose and glucose.
Thus, to evaluate practical applications of the biocatalytic
systems produced, for conversion of glucose xylose two different
real biomass solutions, (i)
a stream of monosaccharides
(glucose, xylose, arabinose) obtained after nanofiltration of PTL
and (ii) pretreated liquor (PTL), were used.
(a)
100
Monosaccharides
PTL
80
60
40
20
enzyme[20]
.
0
The KM and Vmax calculated for free XDH were 0.31 mM and
0.81 U/mL, respectively, and were comparable to those reported
in our previous study where Vmax has been found to be
0.56 U/mL [31]. The KM of nanoSiO2XDH and SBA15XDH were
about 10 and 50% higher, respectively, compared with KM of free
XDH. However, the increase of KM after immobilization observed
for XDH was less significant than for GDH immobilized in this
study. Simultaneously, a less significant drop of Vmax was also
noticed for immobilized XDH. These results could indicate that
diffusional limitations that occurred after XDH immobilization
were minimalized compared to GDH immobilization. This effect
freeGDH nanoSiO₂GDH SBA15GDH
(b)
100
Monosaccharides
PTL
80
60
40
20
0
is probably related to the smaller molecular weight of XDH[17]
.
freeXDH nanoSiO₂XDH SBA15XDH
Nevertheless, the turnover number of nanoSiO2XDH and
SBA15XDH were about 80 and 70%, respectively, of that of the
free enzyme, which suggested that some conformational
changes in the structure of active site amino acids occurred
upon immobilization[30]. These results support the data related to
the retention of catalytic properties by immobilized XDH
Figure 5. Conversion of (a) glucose to gluconic acid and (b) xylose to
xylonic acid catalyzed by free and silica immobilized GDH and XHD,
respectively, from monosaccharides solution and pretreated liquor (PTL).
Dosage of enzyme: 0.5 mg of free or immobilized GDH or 200 U of free
or immobilized XDH. All data are presented as means ± standard
deviation.
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10.1002/cctc.201801335
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Conversion of glucose to gluconic acid in the stream of
monosaccharides by free GDH, nanoSiO2GDH and SBA15GDH
reached 76, 64 and 56%, respectively (Fig. 5a), corresponding
to biocatalytic productivity values of 17.08, 14.41 and 12.56 mg
of gluconic acid per 1 mg of free, nanoSiO2 or SBA15
immobilized GDH, respectively (Table 4). By contrast,
transformation of xylose from the same feed solution was lower
and achieved 67, 57 and 52% for free, nanoSiO2XDH and
SBA15XDH, respectively (Fig 5b), corresponding to
a biocatalytic productivity of 0.180, 0.155 and 0.141 mg of
xylonic acid per 1 U of free, nanoSiO2 or SBA 15 immobilized
XDH, respectively as shown in Table 4. Higher conversion of
glucose to gluconic acid was mainly due to two factors. First of
all, a greater amount of GDH was immobilized on silica carriers,
moreover, attached GDH exhibited higher specific activity and
activity retention compared to immobilized XDH. Second,
concentration of glucose was about five times lower than xylose.
The drop in conversion of both monosaccharides which was
observed for immobilized enzymes compared to the free ones is
related to decrease in enzyme activity upon immobilization, and
of activity was higher than retention obtained when silica
nanoparticles were used as carrier, as after five reaction cycles
nanoSiO2GDH and nanoSiO2XDH maintained, respectively, only
43 and 39% of their catalytic properties with monosaccharides
solution, and 38 and 33%, respectively, of their catalytic
properties with PTL as feed solution.
The above results are in agreement with the data of biocatalytic
productivity. After five consecutive reaction cycles, productivity
of gluconic acid with the monosaccharides solution as the feed
by free GDH reached 32.11 mg of gluconic acid per mg of
enzyme. Productivity of nanoSiO2GDH was about 1.6 fold higher
(52.44 mg/mg), while productivity of SBA15GDH was about
1.4 fold higher (45.61 mg/mg) compared to the free enzyme
(Table 4). Even higher increase in biocatalytic productivity of
xylose was noticed using silica immobilized XDH and
monosaccharides solution. After five reaction steps free XDH
exhibited a productivity of 0.307 mg of xylonic acid per 1 unit of
XDH, while productivity of nanoSiO2XDH and SBA15XDH was
0.502 mg/U (1.7 fold) and 0.461 mg/U (1.6 fold), respectively.
It should also be mentioned that lower values of biocatalytic
productivity of gluconic and xylonic acid were observed using
PTL as a feed solution; this result is directly related to higher
concentration of glucose and xylose in PTL compared to in the
stream of monosaccharides. These results confirm that
reusability of the immobilized GDH and XDH was significantly
improved. Nevertheless, it should be emphasised that
irrespective of the feed solution used, during initial reaction
cycles (up to third cycle), enzymes immobilized onto silica
nanoparticles exhibited higher catalytic properties, but in further
conversion steps, SBA 15 based systems showed higher activity.
This phenomenon could be explained by the fact that enzyme
immobilized in pores of hexagonal silica is better protected
before inactivation and elution from the matrix, and thus its
activity is less affected during the sequential conversion process.
Higher retention of catalytic activity by enzymes used for
conversion of both glucose and xylose from monosaccharides
solution is probably related to the fact that the amount of
enzyme inhibitors such as metal ions, inorganic acids and furans
was significantly reduced compared to their presence in the PTL
solution. Nevertheless, gradual decrease in enzyme activity after
just a few catalytic cycles could be explained by several factors,
such as enzyme inhibition by product, structural modification of
the enzymes as well as by catalyst inactivation caused by their
thermal and pH denaturation[34]. It should be also mentioned that
due mainly to adsorption interactions, some enzyme leakage
from the matrix (up to 30% after last cycle) also occurred as
a contributory factor. Earlier studies reported that glucose-6-
phosphate dehydrogenase encapsulated in silica-based
hydrogels retained less than 40% of its initial activity just after
one reaction cycle[35], as in another study, 40% of activity
retention was observed after seven reaction cycles when
has also been observed in other studies[31,32]
.
Conversion of glucose and xylose presented in crude PTL
(containing inhibitors) followed the same trend (Fig. 5a,b).
However, compared to the stream of monosaccharides, in case
of the PTL feed solution, about 15 and 20% lower values of
conversion were achieved for glucose and xylose, respectively.
This is directly related to the presence of inhibitors, such as
formic and acetic acids or furans, in the feed solution, which
could inhibit enzyme catalytic properties. The presence of trace
amounts of phenols and monovalent (K+ or Na+) or divalent
(Mg2+ or Ca2+) ions could also affect activity of both enzymes as
reported earlier[33]. Nevertheless, the results presented clearly
show that investigated biocatalytic systems demonstrated
efficient conversion of monosaccharides and therefore could find
practical applications in the food industry as well as in
biorefinery.
Reusability of the free and immobilized GDH and XDH
One of the great advantages of immobilization is enhancement
of enzyme reusability in sequential conversion processes, which
leads to decrease in process costs. As can be seen, free GDH
and XDH gradually lost their catalytic properties: GDH was
inactivated after four catalytic cycles using a monosaccharides
stream and after four cycles using PTL as feed solution, while
XDH was inactivated after three catalytic cycles irrespective of
the feed solution used (Fig. 6). On the other hand, immobilized
GDH retained around 70% of its initial activity and immobilized
XDH maintained around 60% of its catalytic properties after
three reaction cycles irrespective of the support material and
feed solution. Moreover, after five reaction cycles, GDH and
XDH immobilized onto SBA 15 surface retained over 50% of
their initial activity during conversion of glucose and xylose from
monosaccharides solution, and over 40% of initial catalytic
properties when PTL was used as feed solution. This retention
glucose-6-phosphate
in alginate[23]
dehydrogenase
was
immobilized
.
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Table 4. Biocatalytic productivity of free and immobilized GDH (mg of gluconic acid per mg of enzyme) and free and immobilized XDH
(mg of xylonic acid per U of enzyme) after five consecutive catalytic cycles.
Biocatalytic productivity (GDH in mg/mg) and (XDH in mg/U)
Analyzed parameter
freeGDH
nanoSiO2GDH
SBA15GDH
freeXDH
nanoSiO2XDH
SBA15XDH
Monosaccharides
PTL
32.11
31.12
52.44
51.10
45.61
39.08
0.307
0.204
0.502
0.336
0.461
0.279
(b)
(a)
100
freeGDH
nanoSiO₂GDH
SBA15GDH
freeXDH
100
80
60
40
20
0
nanoSiO₂XDH
SBA15XDH
80
60
40
20
0
1
2
3
4
5
1
2
3
4
5
No of cycle
No of cycle
(d)
(c)
100
80
60
40
20
0
freeGDH
nanoSiO₂GDH
SBA15GDH
freeXDH
nanoSiO₂XDH
SBA15XDH
100
80
60
40
20
0
1
2
3
4
5
1
2
3
4
5
No of cycle
No of cycle
Figure 6. Reusability of the free and silica immobilized GDH and XHD evaluated based on consecutive conversion of glucose and xylose,
respectively, using monosaccharides solution after nanofiltration (a) and (b) and PTL (c) and (d) as feed solution. All data are presented as means ±
standard deviation.
Nevertheless, on both types of silica supports, the immobilized
enzymes attained improved thermal stability both silica-
immobilized enzymes are characterized by improved thermal
stability, with half-life of SBA15GDH and SBA15XDH being
Conclusions
increased 7.25 and 9.5 fold, respectively as compared to free
biocatalysts, after incubation for four hours at 45°C. However,
immobilization did not affect the pH optima of any of the two
enzymes, regardless of the type of silica immobilization material,
and the immobilized enzymes showed the same high activity
over a broad range of pH (over 50% activity at pH range 8–10).
Robustness at higher pH, above the pKa of gluconic and xylonic
acid, is of particular interest, because this feature facilitates
simple separation of the gluconic and xylonic acid from reaction
mixture even by a simple membrane filtration. Additionally, the
immobilized enzymes exhibited improved reusability; not only
was their catalytic activity retained after five consecutive reaction
cycles but the biocatalytic productivity was improved about 1.6
fold by nanoSiO2GDH and nanoSiO2XDH compared to the free
Our study provides proof-of-concept for the immobilization of
glucose dehydrogenase (GDH) and xylose dehydrogenase
(XDH) on silica nanoparticles and hexagonal mesoporous
SBA15 silica, and application of the obtained biocatalytic
systems for effective conversion of glucose and xylose from
biomass liquors. Specific activity of free GDH and XDH
(assessed as NADH consumption during reduction of glucose
and xylose, respectively) was comparable and reached 42.2
U/mg and 46.8 U/mL, respectively. After immobilization, both
enzymes exhibited high catalytic activity retention, but better
loadings and activity retention were obtained for GDH and XDH
immobilized on the surface of silica nanoparticles than on the
mesoporous silica particles, which confirms that type of support
material affects properties of the biocatalytic systems obtained.
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10.1002/cctc.201801335
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(mL) before and after immobilization, and msupport is the mass of the
support material (g).
enzymes. The data thus clearly shown that immobilization of
GDH and XDH is an attractive approach for efficient
transformation of glucose and xylose into valuable products.
Hopefully, the data may stimulate further development of the use
of monosaccharide dehydrogenases in biorefining and
separation of monosaccharides in genuine biomass liquors.
GDH and XDH activity
Activity assays for GDH and XDH were performed spectrophotometrically
during kinetic reduction of NAD+ to NADH, as presented below, using
a standard calibration curve of NADH. One unit of free or immobilized
enzyme activity was defined as the quantity of enzyme required to
produce 1 µM of NADH per minute under the assay conditions. Specific
activity values (U/mg) of free and immobilized enzymes are presented as
initial enzyme activity retained per unit mass of enzyme, and per unit
mass of enzyme and solid support, respectively. The activity
retention (%) of immobilized enzyme was defined as the percentage
activity of the immobilized GDH and XDH compared to the catalytic
activity of free enzymes. All measurements were done in triplicate and
error bars are presented as means ± standard deviation.
Experimental Section
Chemicals and reagents
Commercially available silica nanopowder of 10–20 nm particle size
(nanoSiO2) and mesoporous SBA 15 silica (<150 μm particle size) with
hexagonal pore morphology and pore size up to 20 nm (SBA15) used in
this study were provided by Sigma-Aldrich (Steinheim, Germany). Tris-
HCl buffer, phosphate buffer, β-nicotinamide adenine dinucleotide
hydrate (NAD+), β-nicotinamide adenine dinucleotide, reduced disodium
salt hydrate (NADH), D-glucose, D-xylose and glucose dehydrogenase
from Pseudomonas sp. (GDH) (EC 1.1.1.118) were purchased from
Sigma-Aldrich (Steinheim, Germany). Xylose dehydrogenase (XDH)
(EC 1.1.1.175) was provided by Megazyme (Bray, Wicklow, Ireland). All
chemicals were of analytical grade and were used as received from the
suppliers without further purification.
GDH activity assay
Catalytic activity of free and immobilized GDH was evaluated
spectrophotometrically by measuring the absorbance at λ=340 nm
(Shimadzu UV1280, Shimadzu, Japan) during kinetic reduction of NAD+
to NADH. The reaction was carried out for 2 min in a styrene cuvette with
a volume of 1 mL of reaction mixture containing 20U of free or
immobilized GDH, 3 mM NAD+ and 50 mM D-glucose in Tris-HCl buffer
at pH 8, 45C.
XDH activity assay
Real liquors
Activity
of
free
and
immobilized
XDH
was
determined
The pretreated liquor (PTL) used in this study was obtained after
combined acid and hydrothermal pretreatment of the biomass. For
pretreatment, diluted sulfuric acid and high temperature steam (180°C)
were injected in the reactor containing soaked wood for a 10 min. After
pretreatment, the liquid fraction was separated from the solids by
pressing in reactor. Prior to use, the PTL was filtered using the
microfiltration membrane GR40PP (MWCO 100 kDa) at 4 bar in an
Amicon 8050 (Millipore, Burlington, MA, USA) stirred cell to clarify the
solution. The concentration of monosaccharides, glucose, xylose and
arabinose was 13.3 g/L, 55.2 g/L, and 2.1 g/L, respectively. The stream
of monosaccharides without inhibitors (inorganic acids and furans)
containing glucose (10.3 g/L), xylose (48.8 g/L) and arabinose (1.9 g/L)
was obtained after nanofiltration of PTL at 40 bar using a NF90
nanofiltration membrane (MWCO 200–400 Da) in a stainless steel stirred
cell (HP4750, Sterlitech Corporation, Kent, WA, USA).
spectrophotometrically by following changes in the absorbance at λ=340
nm during kinetic reduction of NAD+ to NADH[37]. The reaction was
carried out in a styrene cuvette with a volume of 1 mL of reaction mixture
containing 30 U of free or immobilized XDH, 3 mM NAD+ and 70 mM
D-xylose in Tris-HCl buffer at pH 8 for 2 min at 45C.
pH profiles of free and immobilized enzymes
The pH profiles of free and immobilized GDH and XDH were examined
using the methodology described above, at pH values ranging from 6 to
10 at 45°C. The pH of the solution was adjusted using 0.1 M HCl and
0.1 M NaOH. All measurements were made in triplicate and error bars
are presented as means ± standard deviation.
Temperature profiles of free and immobilized enzymes
The temperature profiles of free and immobilized glucose dehydrogenase
and xylose dehydrogenase were evaluated based on the above-
mentioned methodology at temperatures varying from 30°C to 60°C in
steps of 5°C, at pH 8. Prior to spectrophotometric measurements, free
and immobilized enzymes were incubated at the desired temperature for
30 min. All measurements were made in triplicate and error bars are
presented as means ± standard deviation.
Immobilization of GDH and XDH
For the immobilization of GDH and XDH, 50 mg of silica nanoparticles or
silica SBA 15 was immersed in 2 mL of enzyme solution in phosphate
buffer at pH 7 containing 0.15 mg (30U) of GDH or 30U of XDH. The
immobilization was carried out by incubation of the silica support
(nanoSiO2 or SBA 15) with each enzyme solution for 3 h at 4°C in an IKA
KS 4000i control incubator (IKA WerkeGmbH, Germany) with mixing at
200 rpm. The immobilized enzymes (nanoSiO2GDH, SBA15GDH,
nanoSiO2XDH, SBA15XDH) were recovered from the solution by
centrifugation at 4000 rpm for 15 min (Sigma 4K15, Sigma
Laborzentrifugen GmbH). The protein content in the samples after
immobilization (amount of immobilized enzyme (mg/g)) was determined
by the Bradford method (eq. 1) as the difference in the initial enzyme
dosage protein and the concentration of protein present in the
supernatant after immobilization[36] and considering mass of the support
material, according to eq. 1. From the results of the Bradford method,
immobilization yield (%) was calculated (eq. 2).
Stability of free and immobilized enzymes
Stability of both free and immobilized enzyme over time was evaluated
after incubating the native and immobilized enzymes for 240 min under
optimum pH and temperature conditions. For free and silica immobilized
GDH, the optima were 45°C and pH 8, while for free and silica
immobilized XDH, the optima were pH 8 and 40°C and pH 8 and 45°C,
respectively. The relative activity of free and immobilized enzyme was
further determined after a specified period of time. The initial activity of
free GDH and XDH was defined as 100% activity. The inactivation
constant (kD),and half-life (t1/2) were evaluated based on the linear
regression slope. All measurements were made in triplicate and error
bars are presented as means ± standard deviation.
푐 푉 –푐
푉
푠
푠
푖
푖
Amount of immobilized enzyme =
(eq. 1)
(eq. 2)
푚
푠푢푝푝표푟푡
Immobilization yield (%) = 푐 푉 –푐 푠 ⋅ 100%
푉
푠
푖
푖
Kinetic parameters of free and immobilized enzymes
푐 푉
푖
푖
Examination of the kinetic parameters - the Michaelis–Menten constant
(KM), maximum reaction rate (Vmax), turnover number (kcat) and specificity
where ci and cs denote concentration of enzyme before and after
immobilization (mg/mL or U/mL), Vi and Vs denote volume of the solution
constant (kcat/KM)
- of free and immobilized GDH and XDH was
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10.1002/cctc.201801335
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performed based on the model enzymatic reactions (see section GDH
and XDH activity), using various concentrations of NAD+. The kinetic
parameters were calculated based on Hanes-Wolf plots under optimum
assay conditions.
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enzyme immobilization, silica, biomass conversion
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Immobilization of glucose and xylose
dehydrogenase using silica-based
support materials improved enzymes
stability and reusability as well as
enhanced efficient conversion of
biomass components in bioreactors,
under mild conditions.
Jakub Zdarta*, Manuel Pinelo,
Teofil Jesionowski, Anne S. Meyer
Page No. – Page No.
Upgrading of biomass
monosaccharides by immobilized
glucose dehydrogenase and xylose
dehydrogenase
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