Bioconversion of d-glucose into d-glucosone by immobilized glucose 2-oxidase from Coriolus versicolor at moderate pressures

Article abstract of DOI:10.1016/j.procbio.2010.08.002

The immobilized glucose 2-oxidase (pyranose oxidase, pyranose:oxygen-2- oxidoreductase, EC 1.1.3.10) from Coriolus versicolor was used to convert d-glucose into d-glucosone at moderate pressures, up to 150 bar, with compressed air in a modified commercial batch reactor. Several parameters affecting biocatalysis at moderate pressures were investigated as follows: pressure, different forms of immobilized biocatalysts, glucose concentration, pH, temperature and the presence of catalase. Glucose 2-oxidase (GOX2) was purified by immobilized metal affinity chromatography on epoxy-activated Sepharose 6B-IDA-Cu(II) column at pH 6.0. Purified enzyme and catalase were immobilized into a polyethersulfone (PES) membrane in the presence of glutaraldehyde and gelatin. Enhancement of the bioconversion of d-glucose was done by the pressure since an increase in the pressure with compressed air increases the conversion rates. The optimum temperature and pH for bioconversion of d-glucose were found to be 62 °C and pH 6.0, respectively and the activation energy (E _a) was 28.01 kJ mol^-1. The apparent kinetic constants (V′max, K′m, K′cat and Kcat/K′m) for this bioconversion were 2.27 U mg^-1 protein, 11.15 mM, 8.33 s^-1 and 747.38 s^-1 M^-1, respectively. The immobilized biomass of C. versicolor as well as crude extract containing GOX2 activity were also useful for bioconversion of d-glucose at 65 bar with a yield of 69.9 ± 3.8% and 91.3 ± 1.2%, respectively. The immobilized enzyme was apparently stable for several months without any significant loss of enzyme activity. On the other hand, this immobilized enzyme was also stable at moderate pressures, since such pressures did not affect significantly the enzyme activity.

Full text of DOI:10.1016/j.procbio.2010.08.002

Process Biochemistry 46 (2011) 168–173
Contents lists available at ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
Bioconversion of d-glucose into d-glucosone by immobilized glucose 2-oxidase
from Coriolus versicolor at moderate pressures
∗
Amin Karmali , José Coelho
Chemical Engineering and Biotechnology Research Center, Department of Chemical Engineering, Instituto Superior de Engenharia de Lisboa, Rua Conselheiro Emídio Navarro,
1959-007 Lisboa, Portugal
a r t i c l e i n f o
a b s t r a c t
Article history:
The immobilized glucose 2-oxidase (pyranose oxidase, pyranose:oxygen-2-oxidoreductase, EC 1.1.3.10)
from Coriolus versicolor was used to convert d-glucose into d-glucosone at moderate pressures, up to
Received 19 January 2010
Received in revised form 29 June 2010
Accepted 2 August 2010
1
50 bar, with compressed air in a modified commercial batch reactor. Several parameters affecting bio-
catalysis at moderate pressures were investigated as follows: pressure, different forms of immobilized
biocatalysts, glucose concentration, pH, temperature and the presence of catalase. Glucose 2-oxidase
Keywords:
(
GOX2) was purified by immobilized metal affinity chromatography on epoxy-activated Sepharose 6B-
IDA-Cu(II) column at pH 6.0. Purified enzyme and catalase were immobilized into a polyethersulfone
PES) membrane in the presence of glutaraldehyde and gelatin. Enhancement of the bioconversion of
Biocatalysis at moderate pressures
Immobilized glucose 2-oxidase from
Coriolus versicolor
Immobilization in polyethersulfone
membranes
Catalase
Bioconversion of d-glucose into
d-glucosone
(
d-glucose was done by the pressure since an increase in the pressure with compressed air increases
the conversion rates. The optimum temperature and pH for bioconversion of d-glucose were found to
◦
−1
be 62 C and pH 6.0, respectively and the activation energy (Ea) was 28.01 kJ mol . The apparent kinetic
ꢀ
ꢀ
ꢀ
ꢀ
−1
−1
constants (V
and 747.38 s
, K , K and Kcat/K ) for this bioconversion were 2.27 U mg protein, 11.15 mM, 8.33 s
max
−1
m
−1
cat
m
M
, respectively. The immobilized biomass of C. versicolor as well as crude extract con-
Apparent kinetic constants
taining GOX2 activity were also useful for bioconversion of d-glucose at 65 bar with a yield of 69.9 ± 3.8%
and 91.3 ± 1.2%, respectively. The immobilized enzyme was apparently stable for several months without
any significant loss of enzyme activity. On the other hand, this immobilized enzyme was also stable at
moderate pressures, since such pressures did not affect significantly the enzyme activity.
©
2010 Elsevier Ltd. All rights reserved.
1
. Introduction
is very slow and time-consuming at atmospheric pressure [4,5].
Moreover, GOX2 activity from basidiomycete strains (i.e. Coriolus
versicolor and Phanerochaete chrysosporium) is highly unstable [6,7]
and there is a great need to stabilize the enzyme activity by several
ways such as by protein engineering and by immobilization in a
suitable support [8].
Immobilization of enzymes to solid carriers is the most widely
used strategy to improve stability of biocatalysts such as storage
and operational stabilities. Moreover, this strategy can increase
selectivity towards other substrates compared with the corre-
sponding free enzymes. The four main methods of immobilization
are as follows: (a) adsorption, (b) covalent binding, (c) entrapment
and (d) membrane confinement. In general, thermal stability of bio-
catalyst is due to the molecular rigidity introduced by binding to
a rigid support and creation of a protected microenvironment for
the biocatalyst [9,10].
Enzymes are ideal biocatalysts for stereo- and regioselective
reactions which take place in aqueous medium under mild con-
ditions [1]. Fungal glucose 2-oxidase (GOX2, pyranose oxidase,
pyranose:oxygen-2-oxidoreductase, EC 1.1.3.10) has been used
to catalyse the oxidation of d-glucose at C-2 position producing
2
-keto-d-glucose (d-glucosone, d-arabino-hexos-2-ulose) which
is an important precursor for biosynthesis of the antibiotic cor-
talcerone [2]. Moreover, this enzyme also exhibits activity over
other carbohydrates such as d-galactose, l-sorbose and 1-deoxy-
d-glucose producing the corresponding keto-sugars [3]. Therefore,
dicarbonyl sugars are widely used for synthesis of rare sugars such
as antibiotics, pyrrolidine and piperidine aminosugars. However,
the bioconversion of d-glucose and other carbohydrates in the pres-
ence of GOX2 and catalase into their corresponding keto-sugars
Enzyme reactions can be carried out under high pressure (i.e.
over 2 kbar) or in supercritical fluids which is a new and promis-
ing field of enzyme engineering [11]. High pressure is responsible
for direct conformational changes in enzymes which affect their
biological activities [12,13]. However, such high pressures may
inactivate some enzymes since undesirable changes may occur in
∗ Corresponding author at: Chemical Engineering and Biotechnology Research
Center, Instituto Superior de Engenharia de Lisboa, Rua Conselheiro Emídio Navarro,
1
, 1959-007 Lisboa, Portugal. Tel.: +351 218317052; fax: +351 218317267.
E-mail address: akarmali@deq.isel.ipl.pt (A. Karmali).
1
359-5113/$ – see front matter © 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.procbio.2010.08.002

A. Karmali, J. Coelho / Process Biochemistry 46 (2011) 168–173
2.5. Protein assay
169
enzyme conformation which results in enzyme inactivation [13].
Moreover, pressure also influences the reaction rate constant which
changes according to transition state theory and standard thermo-
dynamics [14]. There are many reports in the literature on the effect
of high pressure (i.e. over 2 kbar) on enzyme structure and activ-
ity [12,15]. However, few published work have been found in the
literature regarding the use of moderate pressures, in the range of
Protein concentration was determined by Coomassie blue dye binding method
19].
[
2
.6. Immobilization of GOX2 and catalase, crude extract and biomass of C.
versicolor
Either crude extract containing GOX2 activity or purified enzyme and catalase
1
–150 bar, on enzyme structure and activity. Therefore, the present
were immobilized by covalent bonding in PES membranes (discs of 3.0 cm diameter)
by using the following reaction mixture: 80 U of purified GOX2, 10 mg of catalase
work is concerned with the use of moderate pressure up to 150 bar
by compressed air in a pressure batch reactor in order to increase
the rate of bioconversion of d-glucose since oxygen is one of the
substrates of the reaction catalysed by immobilized GOX2. Several
parameters affecting this bioconversion will be investigated such as
pressure, different forms of biocatalysts, d-glucose concentration,
pH, temperature and the presence of catalase.
−
1
(1000 U mg protein), 2 ␮l of glutaraldehyde (5%, v/v) and 20 ␮l gelatin (25%) in
50 mM phosphate buffer pH 6.0 in a final volume of 200 ␮l. The mixture was imme-
diately transferred to membrane discs which were dried and washed several times
with 50 mM phosphate buffer pH 6.0 [20]. In some experiments, GOX2 was immo-
bilized on PES membranes without catalase by using the methodology described
−1
above. Alternatively, crude extract (0.21 U mg protein) was immobilized in PES
membranes by using the following reaction mixture: crude enzyme having 80 U
−
1
of activity, 10 mg catalase (1000 U mg protein), 10 ␮l of glutaraldehyde (5%, v/v)
and 50 ␮l gelatin (25%) as described above. Regarding biomass immobilization, 1.0 g
2. Materials and methods
(
wet weight) of biomass of C. versicolor having 80 U of enzyme activity, was mixed
with 1.0 ml of glutaraldehyde (5%, v/v) and 1.0 ml gelatin (25%) in 50 mM phosphate
buffer pH 6.0. The biomass was allowed to dry, washed with 50 mM phosphate
buffer pH 6.0 and subsequently it was wrapped in a dialysis membrane (P10) for
biocatalysis in a batch reactor. Alternatively, biomass of C. versicolor was wrapped
in a dialysis membrane (P10) and used for biocatalysis. All immobilized forms of
2
.1. Materials
C. versicolor was isolated from old growth forest of Olympic Peninsula (Port
Townsend, Washington, USA). Corn-steep liquor was kindly donated by COPAM,
Portugal. O-dianisidine, iminodiacetic acid, d-glucose, d-glucosone, 1,4-butanediol
diglycydil ether, glutaraldehyde, catalase and peroxidase were purchased from
Sigma Chemical Company (USA). Sepharose 6B was obtained from GE Health Care,
Sweden.
Sugar Pak column was obtained from Waters (USA) and discs of 3.0 cm diameter
of modified polyethersulfone (PES) membranes (Ultrabind, US450 0.45 ␮m) were
purchased from Pall Gelmam Laboratories (USA).
◦
biocatalysts were stored at 4 C in 50 mM phosphate buffer pH 6.0.
2
2
.7. Biocatalysis at moderate pressure
.7.1. Apparatus set-up
The experimental set-up consists basically of one batch reactor (Micro Reactor
Parr Instruments CO, 4843) with 25 ml of capacity equipped with agitation, temper-
ature and relative pressure reading devices. Two principal valves inlet and outlet
connections and a rupture disk set a 250 bar were used to perform the enzyme
reactions. In each experiment samples were withdrawn at appropriate intervals to
determine product concentration by using an HPLC system (Jasco Instruments).
Biocatalysis at moderate pressure was carried out by using either immobilized
2
.2. Enzyme production and purification
GOX2 from C. versicolor was produced by submerged fermentation by using a
basal culture medium containing agro-industrial wastes as carbon sources such as
whey powder (2.5%) and other components as follows: corn-steep liquor (0.7%),
MgSO4 (0.15%) CaCl2 (0.0125%) and KH2PO4 (0.1%). They were all dissolved in tap
water which also contained trace amounts of salts as follows: 1.0 ␮M CuSO4, 1.0 mM
CaCO3, 0.06 mM MgCl2, 0.9 ␮M AlCl3, 1.3 ␮M MnCl2, 0.01 mM NaNO3, 0.85 ␮M NaF,
−
1
crude extract and catalase (0.05 U mg protein) in PES membranes, immobilized
−
1
purified enzyme and catalase in membranes (1.5 U mg protein), biomass wrapped
in a dialysis membrane or immobilized biomass from C. versicolor (1.0 g) wrapped
in a dialysis membrane. The reaction mixture contained appropriate concentration
of d-glucose in 50 mM phosphate buffer pH 6.5 (19.0 ml) and appropriate form of
1
.7 ␮M FeCl3, 1.1 ␮M ZnCl and 0.1 mM NaCl. The pH of the medium was adjusted
◦
to 5.5 and sterilized at 121 C for 20 min in an autoclave. Alternatively, other agro-
industrial wastes could be used such as tomato pomace and rice bran.
◦
enzyme which was stirred at 110 rpm at 23 C. The reaction was performed at several
◦
relative pressures at 23 C by using compressed air. Aliquots (100 ␮l) were removed
Erleynmeyer flasks (500 ml) containing 100 ml of sterile medium were inoc-
ulated with two pieces of 0.5 cm × 0.5 cm plugs of the appropriate culture grown
from the batch reactor at suitable time intervals and the substrate and product of
the reaction were analysed by HPLC as described below.
◦
in petri dishes and the culture was grown at 200 rpm, 25 C for several days. By
this time it was possible to detect GOX2 activity in the biomass. This inoculum
2
.7.2. Calibration curve for d-glucose and d-glucosone
Several concentrations of d-glucose and its corresponding keto-sugar were pre-
was used to inoculate batch fermenter (2.5 l) containing the same culture medium
◦
(
2.0 l) and the culture was grown at 200 rpm, pH 5.5 and 25 C for several days and
pared in 50 mM phosphate buffer pH 6.5. These samples were analysed in an HPLC
system (Jasco) by using a Sugar Pak column at 75 psi, 90 C, 0.1 mM Ca(II) EDTA in
GOX2 production was followed as a function of time. Aliquots (10 ml) were removed
daily from the fermenter under aseptic conditions; the biomass was recovered by
filtration and washed with saline. The biomass was resuspended in 2 volumes of
◦
−
1
millipore water as the solvent and at a flow rate of 0.5 ml min
.8. Assay of GOX2 by HPLC analysis
Samples (25 ␮l) from reaction mixtures were injected into the HPLC system and
.
5
0 mM phosphate buffer pH 6.5 and sonicated at 100 W for 2 min in an ice bath. The
2
suspension was centrifuged at 10,000 × g for 5 min and the cell-free supernatant
was the source of GOX2 activity. The enzyme was purified by immobilized metal
affinity chromatography on epoxy-activated Sepharose 6B-IDA-Cu (II) as described
previously [16].
the chromatograms were obtained in 12 min time since the retention time of d-
glucose and d-glucosone were 9.3 and 10.3 min, respectively. The peak area was
determined as a function of either d-glucose or d-glucosone concentration.
2.3. Electrophoretic analysis
2
.9. Kinetic characterization of purified preparation of immobilized GOX2 and
Crude extract and purified enzyme were analysed for purity by SDS-PAGE and
native PAGE as reported previously [17,18].
catalase at moderate pressures
Immobilized GOX2 activity containing co-immobilized catalase in PES mem-
branes was used throughout these kinetic studies. It was assayed by using d-glucose
as the substrate and one unit of GOX2 activity was defined as the amount of
enzyme required to oxidize 1 (mol substrate per min. under the same experimental
condition. Assays in pressure batch reactor were performed at suitable moderate
pressures, up to 120 bar, in 50 mM of the appropriate buffer and pH, and suitable
aliquots were removed from the pressure reactor at appropriate time intervals and
analysed by HPLC as described above. The optimum pH of GOX2 activity was inves-
tigated at 110 bar by using purified immobilized enzyme preparation in a reaction
mixture containing 0.1 M d-glucose in several buffers as follows: 50 mM citrate pH
4.6 and 5.6, 50 mM phosphate buffer pH 6.0 and 7.0 and 50 mM Tris pH 7.0 and
8.0. The optimum temperature of GOX2 activity was investigated by using puri-
fied immobilized enzyme preparations at 110 bar in a reaction mixture containing
2
.4. Enzyme assays
For routine enzyme assays in fungal cultures and chromatographic fractions,
GOX2 activity at atmospheric pressure was determined by using o-dianisidine,
d-glucose (0.1 M) and peroxidase (1 U) in 50 mM phosphate buffer pH 6.5. The
reaction mixture contained 0.79 ml of o-dianisidine (6 mg/100 ml), 0.01 ml peroxi-
−1
dase (1 mg ml ), 0.1 ml glucose (1 M) and 0.1 ml of crude extract. After 10 min, the
◦
3
−1
−1
absorbance was recorded at 450 nm at 25 C (ε = 8.3 × 10 cm
M ). Alternatively,
enzyme activity was assayed at atmospheric pressure by using 0.1 ml of appropriate
concentration of d-glucose in 50 mM phosphate buffer pH 6.0 and suitable amount
of either crude extract or purified enzyme in a final volume of 1 ml by bubbling
with air. Aliquots were removed from the reaction mixture at suitable time inter-
vals which were analysed by HPLC in terms of substrate and product as described
below. Catalase activity was assayed in the presence of H2O2 as reported previously
100 mM d-glucose in 50 mM phosphate buffer pH 6.0 which was incubated at dif-
ferent temperatures (i.e. 25, 35, 45, 55, 62, 66, 70 and 80 ◦C) for 5 min. The activation
[16].
energy for immobilized GOX2 reaction was determined by measuring the enzyme

1
70
A. Karmali, J. Coelho / Process Biochemistry 46 (2011) 168–173
activity at different temperatures and transforming them according to the Arrhenius
equation:
Ea
1
Ln(v) = A − R × T
where v is reaction rate; T is the absolute temperature (K), Ea is the activation energy
and R is gas constant.
ꢀ
ꢀ
ꢀ
ꢀ
The apparent kinetic constants (Vmax, K , K and Kcat/K ) for d-glucose were
m
cat
m
determined by Michaelis–Menten plot by using the software from Sigma Plot 2004
version 9.01).
(
2.10. Biocatalysis at atmospheric pressure
For comparative purposes, biocatalysis at atmospheric pressure, was also car-
ried out by using either immobilized cell-free extract and catalase in PES membranes
−
1
(
0.05 U mg protein), immobilized biomass of C. versicolor (1.0 g wet weight) and
−
1
purified immobilized enzyme and catalase in PES membranes (1.5 U mg protein).
The reaction mixture contained appropriate concentration of d-glucose in 50 mM
phosphate buffer pH 6.5 (19.0 ml) and appropriate form of enzyme/biocatalyst
which was stirred at 110 rpm in a magnetic stirrer. The reaction was performed
by using the reaction mixture bubbled with air ([O2] = 8.89 mg l ). The solubility of
O2 in the solution is assumed as in fresh water, using the toolbox and results from
Fig. 1. Progress curve of consumption of d-glucose by free and immobilized GOX2.
Atmospheric pressure: immobilized GOX2 and catalase (ꢀ) and free GOX2 (9.0 U)
and catalase (10 mg) (ꢁ); 72 bar: immobilized form (ꢂ) and free form (᭹).
−
1
[21] and the respective corrections when O2 is pure against the concentration in the
air.
several reports in the literature have described this effect on sev-
eral enzyme reactions [12,23]. The oxidation of d-glucose in the
presence of immobilized GOX2 and catalase was also carried out
at several working pressures at room temperature as shown in
Fig. 2A. These data suggest that by increasing the pressure there
is an increase in the conversion of d-glucose compared with atmo-
2
.11. Stability of immobilized GOX2 and catalase at moderate pressures
Membrane preparations containing immobilized GOX2 and catalase were stored
◦
◦
in 50 mM phosphate buffer pH 6.0 at 4 C and assayed for activity in a pressure
batch reactor at 100 bar, 110 rpm at 23 C in a reaction mixture containing 0.1 M
d-glucose in 50 mM phosphate buffer pH 6.0 as described above. Such membranes
were assayed at appropriate time intervals for several months in order to investi-
gate its stability. Alternatively, PES membrane preparations containing immobilized
spheric pressure because [O ] is much higher in batch reactor than
2
at atmospheric pressure. Several experiments were carried out by
increasing the pressure up to 160 bar in the batch reactor which
exhibited an increase in rate of reaction catalysed by immobilized
GOX2 and catalase (data not shown). Furthermore, the conversion
of d-glucose into d-glucosone was investigated by using differ-
ent enzyme preparations such as immobilized crude extract and
immobilized biomass from C. versicolor, either in a batch reactor
or at atmospheric pressure (Fig. 2B). The data presented in Fig. 2B
revealed that crude extract exhibited a higher degree of conversion
compared with purified enzyme preparation by using the same
amount of enzyme activity. Surprisingly, the biomass of C. versi-
color containing GOX2 activity was also useful for conversion of
d-glucose into d-glucosone at moderate pressure. This rather low
degree of bioconversion for the biomass compared with the crude
extract may be due to mass transfer limitations (Fig. 2B) which
has been reported by several researchers [24,25]. The aim of this
experiment was to show that it was possible to immobilize several
forms of biocatalysts and use them in batch reactor for biocatalysis
either at moderate or atmospheric pressures. However, the com-
parative analysis of these biocatalysts is difficult because: 1. the
amount of glutaraldehyde used for each biocatalyst was different
since the total amount of protein in each case was also different;
2. the microenvironment as well as steric hindrance of each immo-
bilized biocatalyst in PES membrane is different; 3. mass transfer
limitations for each immobilized biocatalyst are also different and
4. the amount of immobilized enzyme may be different for each bio-
catalyst. Biocatalysis at atmospheric pressure revealed a low degree
of d-glucose bioconversion with different immobilized biocatalysts
compared with the batch reactor (Fig. 2B). Immobilized biomass
of C. versicolor could be used several times for bioconversion of
d-glucose and about 2/3 of GOX2 activity was lost after the 10th
re-utilization of immobilized biomass in batch reactor (Fig. 2C). In
fact, the data in Fig. 2C revealed that the initial velocity in the 1st
GOX2 were incubated 50 mM phosphate buffer pH 6.0 (20 ml) in batch reactor at
◦
7
0 bar, 100 rpm at 23 C for several days. Membrane preparations were removed
at suitable time intervals and immobilized GOX2 activity was measured at atmo-
spheric pressure by HPLC. For comparative purposes, the stability of the immobilized
enzymes (i.e. GOX2 and catalase) in PES membranes was also investigated at atmo-
◦
spheric pressure for several days at 100 rpm in a magnetic stirrer at 23 C. Membrane
preparations were removed at suitable time intervals and immobilized GOX2 activ-
ity was measured by HPLC.
3
. Results and discussion
3.1. Biocatalysis at moderate and atmospheric pressures
The calibration curves for d-glucose and d-glucosone were car-
ried out by HPLC [22] which exhibited linear relationship between
the peak area (mV) and either d-glucose or d-glucosone concentra-
tion (data not shown).
The immobilization of GOX2 and catalase on PES membranes
revealed that the recovery of immobilized enzyme activity was
1
4.1 ± 0.92% and 11.5 ± 1.01%, respectively (data not shown). The
amount of GOX2 immobilized on PES membranes was found to
be 5.1 mg which was determined by the indirect method (i.e.
Coomassie blue dye binding method) because extensive washing
of PES membrane was not required. This protein value is five-
fold higher than the manufacturer data for this membrane in
the absence of glutaraldehyde. However, in the present work the
enzyme was immobilized in the presence of glutaraldehyde which
may increase the membrane capacity.
The formation of d-glucosone and the consumption of d-glucose
were followed by HPLC when the reaction was carried out at
atmospheric pressure and batch reactor by using either purified
immobilized enzyme and catalase or purified soluble enzyme and
catalase (Fig. 1). These data revealed that the enzyme reaction is
very slow at atmospheric pressure (Fig. 1), compared with the reac-
−
1
−1
tion in a batch reactor (i.e. 70 bar, [O ] = 2960.0 mg l ) (Fig. 1). This
use of biomass was 0.507 mmol d-glucosone h whereas the initial
2
−
1
result may be explained on the basis that molecular O is one of the
velocity in the 10th re-use of biomass was 0.156 mmol h . There-
fore, after 10th re-utilization of biomass, the initial velocity was
30.77% of the 1st use of biomass. Moreover, the yield of d-glucosone
from d-glucose by using either the immobilized biomass of C. versi-
color or immobilized crude extract was found to be 69.9 ± 3.8% and
2
substrates of this enzyme reaction and its concentration is much
higher in batch reactor at 70 bar compared with atmospheric pres-
−1
sure ([O ] = 8.89 mg l ). However, the activating effect of moderate
2
pressures on immobilized GOX2 activity cannot be ruled out since

A. Karmali, J. Coelho / Process Biochemistry 46 (2011) 168–173
171
Fig. 3. The effect of temperature and pH on the initial velocity of the reaction
catalysed by immobilized GOX2 and catalase at 110 bar. (A) Temperature and (B)
pH.
Fig. 2. (A) The effect of pressure on bioconversion of d-glucose by immobilized
◦
GOX2. The reaction at atmospheric pressure was carried out at 23 C by bubbling
the reaction mixture with air. (B) Bioconversion of d-glucose by different forms of
biocatalysts by applying 70 bar of pressure (white bar) and without pressure (dark
bar). (C) The reusability of immobilized biomass from C. versicolor on the rate of
bioconversion of d-glucose at 65 bar at 1st use of biomass (᭹) and 10th re-utilization
of biomass (ꢀ).
9
1.3 ± 1.2%, respectively (data not shown). In addition, the presence
of catalase in PES membranes increased significantly the degree of
conversion of d-glucose (data not shown) suggesting that H O acts
2
2
as a powerful inhibitor for this enzyme which is in agreement with
published reports [6].
3.2. Kinetic characterization of immobilized GOX2 from C.
versicolor at moderate pressures
The bioconversion of d-glucose in the presence of immobilized
GOX2 and catalase was carried out at several working temperatures
at moderate pressures as shown in Fig. 3A. These data suggest that
optimum temperature for maximum conversion of d-glucose into
◦
d-glucosone under these experimental conditions is 62 C which
◦
is higher than the value of 50 C reported for the free enzyme at
atmospheric pressure [26]. From the linear range of the Arrhenius
−
1
plot, the activation energy (Ea) was determined to be 28.01 kJ mol
−1
which is lower than the value of 34.6 kJ mol reported for this sol-
uble enzyme at atmospheric pressure [4]. There are few reports on
immobilization of GOX2 for biocatalysis in the literature [10,27–29]
Fig. 4. The effect of d-glucose concentration on the initial velocity of the reac-
tion catalysed by immobilized GOX2 and catalase at pH 6.0 and 110 bar.
Michaelis–Menten (A) and Lineweaver–Burk (B) plots.

1
72
A. Karmali, J. Coelho / Process Biochemistry 46 (2011) 168–173
ditions for this enzyme since linked enzyme assays with peroxidase
were used in such kinetic studies [24–26,29].
3.3. Storage stability of immobilized GOX2 at moderate pressures
The effect of moderate pressures on the stability of immobi-
lized GOX2 activity was studied by incubating several preparations
of immobilized enzyme in batch reactor for several days. The data
presented in Fig. 5 revealed that it did not affect significantly the
enzyme activity at 70 bar compared with the enzyme stored at
atmospheric pressure. In addition, the storage stability of immo-
bilized GOX2 on PES membranes containing catalase was also
investigated which revealed that this enzyme preparation was
apparently stable for several months without any significant loss
of enzyme activity (Fig. 6). The operational stability of this immo-
bilized enzyme was studied at moderate pressures which revealed
that it was stable for at least 30 days without any substantial loss
of GOX2 activity (data not shown). On the other hand, this is also
the first report on the use of either immobilized crude extract or
biomass from C. versicolor for bioconversion of d-glucose at mod-
erate pressures. Such membrane supports are highly suitable for
immobilization of GOX2 which exhibit high stability for several
months.
Fig. 5. Stability of immobilized GOX2 at moderate pressures. Several PES mem-
branes containing GOX2 and catalase (1.2 U) were incubated in 50 mM phosphate
buffer pH 6.0 either in batch reactor at 70 bar (᭹) or at atmospheric pressure (ꢀ).
which have described that there are no differences in optimum
temperature of activity of the free and immobilized forms of this
enzyme [29]. The effect of pH on the bioconversion of d-glucose
into d-glucosone at moderate pressure was also investigated which
revealed that the optimum pH is 6.0 for maximum conversion of
d-glucose into d-glucosone by using immobilized GOX2 and cata-
lase (Fig. 3B). These data are in agreement with the pH value of 6.2
reported for this soluble enzyme at atmospheric pressure [3,26].
The effect of d-glucose on initial velocity of immobilized GOX2-
catalysed reaction at moderate pressures of 110 bar was carried
out by using PES membrane containing GOX2 and catalase (Fig. 4).
4. Conclusions
The data presented in this work have revealed that at moder-
ate pressure, there are several parameters that affect the degree
of conversion of d-glucose into d-glucosone namely, temperature,
pH, different forms of immobilized biocatalysts and the presence
of catalase. The optimum pH and temperature for this reaction at
moderate pressures were slightly different compared with the data
published for this enzyme at atmospheric pressure. It is important
to stress that this is the first report on biocatalysis at moderate pres-
sures regarding immobilized GOX2 for bioconversion of d-glucose
into d-glucosone by using compressed air.
ꢀ
ꢀ
ꢀ
ꢀ
The apparent kinetic constants (i.e. V
, K , K
and Kcat/K
)
max
m
cat
m
were determined by using Sigma Plot which were found to
−1
−1
−1
−1
be 2.27 U mg
protein, 11.15 mM, 8.33 s
and 747.38 s
M ,
respectively (Fig. 4). These results are lower than the data reported
in the literature for soluble enzyme preparations which may be due
to mass transfer limitations as well as the use of different assay con-
Acknowledgements
The authors would like to thank Instituto Politécnico de Lisboa
(
(
Project No. 25/2003) and Funda c¸ ão para a Ciência e a Tecnologia
POCTI/EQU/48812/2002) for research grants.
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