Biochemical characteristics of Trametes multicolor pyranose oxidase and Aspergillus niger glucose oxidase and implications for their functionality in wheat flour dough

Article abstract of DOI:10.1016/j.foodchem.2011.10.041

Similar to glucose oxidase (GO), pyranose oxidase (P₂O) may well have desired functionalities in some food applications in general, particularly breadmaking. As its name implies, P₂O oxidises a variety of monosaccharides. P₂O purified from a culture of Trametes multicolor (P₂O-Tm) had high affinity towards d-glucose (K_M = 3.1 mM) and lower affinity to other monosaccharides. GO from Aspergillus niger (GO-An) had a K_M value of 225 mM towards glucose, which points to a significant difference in glucose affinity between the two enzymes. Furthermore, P₂O-Tm had higher affinity towards O₂ (K_M = 0.46 mM) than GO-An (K_M = 2.9 mM). Dehydroascorbic acid did not accept electrons in the reactions catalysed by P₂O-Tm and GO-An. For the same activity towards glucose in saturating conditions, the rate of ferulic acid oxidation in a model system and of thiol oxidation in a wheat flour extract were higher with P₂O-Tm, than with GO-An. The demonstrated differences in properties and functional features between P₂O-Tm and GO-An allow prediction of differences in functional behaviour of the enzymes, in food applications.

Full text of DOI:10.1016/j.foodchem.2011.10.041

Food Chemistry 131 (2012) 1485–1492
Contents lists available at SciVerse ScienceDirect
Food Chemistry
journal homepage: www.elsevier.com/locate/foodchem
Biochemical characteristics of Trametes multicolor pyranose oxidase and
Aspergillus niger glucose oxidase and implications for their functionality
in wheat flour dough
a,
⇑
a
b
c
a
Karolien Decamps , Iris J. Joye , Dietmar Haltrich , Jacques Nicolas , Christophe M. Courtin ,
Jan A. Delcour
a
a
Laboratory of Food Chemistry and Biochemistry and Leuven Food Science and Nutrition Research Centre (LFoRCe), Katholieke Universiteit Leuven,
Kasteelpark Arenberg 20 – box 2463, B-3001 Heverlee, Belgium
b
Division of Food Biotechnology, Department of Food Sciences and Technology, University of Natural Resources and Applied Life Sciences Vienna (BOKU Wien), Muthgasse 18,
A-1180 Wien, Austria
c
Conservatoire National des Arts et Métiers, UMR Ingénierie Procédés Aliments 1145 (AgroParisTech-CNAM-INRA), 292 Rue Saint-Martin, F-75141 Paris, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 July 2011
Received in revised form 6 September 2011
Accepted 11 October 2011
Available online 18 October 2011
Similar to glucose oxidase (GO), pyranose oxidase (P O) may well have desired functionalities in some
2
food applications in general, particularly breadmaking. As its name implies, P
monosaccharides. P O purified from a culture of Trametes multicolor (P O-Tm) had high affinity towards
-glucose (K = 3.1 mM) and lower affinity to other monosaccharides. GO from Aspergillus niger (GO-An)
had a K value of 225 mM towards glucose, which points to a significant difference in glucose affinity
between the two enzymes. Furthermore, P O-Tm had higher affinity towards O (K = 0.46 mM) than
GO-An (K = 2.9 mM). Dehydroascorbic acid did not accept electrons in the reactions catalysed by P O-
Tm and GO-An. For the same activity towards glucose in saturating conditions, the rate of ferulic acid oxi-
dation in a model system and of thiol oxidation in a wheat flour extract were higher with P O-Tm, than
with GO-An. The demonstrated differences in properties and functional features between P O-Tm and
2
O oxidises a variety of
2
2
D
M
M
2
2
M
Keywords:
M
2
Pyranose oxidase
Glucose oxidase
Ferulic acid
Thiol
2
2
Wheat
GO-An allow prediction of differences in functional behaviour of the enzymes, in food applications.
Ó 2011 Elsevier Ltd. All rights reserved.
1
. Introduction
GO,
180 kDa], catalyses the oxidation of C
-glucono-1,5-lactone and H -Glucono-1,5-lactone, in turn,
a
homodimeric flavoprotein [molecular mass (MM)
ꢀ
1
of b- -glucose by O to
D
2
Oxidoreductase enzymes acting on CH–OH groups of substrates,
D
2 2
O . D
using O
2
as electron acceptor (E.C. 1.1.3.x), are widely distributed
spontaneously hydrolyses to form gluconic acid. The sugar oxida-
tion is coupled to reduction of flavin adenine dinucleotide (FAD)
among micro-organisms, plants and animals. The E.C. 1.1.3.x group
contains sugar-acting oxidases such as: glucose oxidase (GO, E.C.
to FADH
FAD, while O
tein (MM ꢀ270 to 300 kDa), catalyses the oxidation of C
numerous mono- and disaccharides, by O , to their corresponding
dicarbonyl derivatives and H . P O has a higher specificity to-
wards glucose than GO and oxidises both - and b- -glucose,
while GO only shows activity towards (the more predominant)
b- -glucose (Giffhorn, 2000). Both enzymes obey a Ping Pong Bi
2
. In the second half-reaction, FADH
is reduced to H . P O, a homotetrameric flavopro-
or C of
2
is re-oxidised to
1
1
.1.3.4), hexose oxidase (E.C. 1.1.3.5), galactose oxidase (E.C.
.1.3.9), pyranose oxidase (P O, E.C. 1.1.3.10), -sorbose oxidase
-mannitol oxidase (E.C. 1.1.3.40) and alditol oxi-
dase (E.C. 1.1.3.41). We here focus on GO and P O. Both GO (b-
glucose:oxygen 1-oxidoreductase, glucose 1-oxidase) and P
2
2
O
2
2
2
L
2
3
(
E.C. 1.1.3.11),
D
2
2
D
-
2
O
2
2
2
O
a-
D
D
(
pyranose:oxygen 2-oxidoreductase, glucose 2-oxidase, pyranose
2
-oxidase) are of fungal origin (Leitner, Volc, & Haltrich, 2001;
D
Swoboda & Massey, 1965).
Bi mechanism. The term ‘‘Bi Bi’’ refers to a reaction model with
two substrates and two products, where product is only formed
after formation of a complex between the enzyme and the two
substrates. ‘‘Ping Pong Bi Bi’’ is a specialised Bi Bi mechanism in
which substrate binding and release of products are ordered and
the enzyme shuttles between a free and a substrate-modified
intermediate conformation.
0
Abbreviations: ABTS, 2,2 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid);
AH
2
, ascorbic acid; AXOS, arabinoxylan oligosaccharides; DHA, dehydroascorbic
acid; FA, ferulic acid; FAD, flavin adenine dinucleotide; GO, glucose oxidase; GO-An,
GO from Aspergillus niger; AU, Absorbance Units; MES, 2-(N-morpholino)ethane-
2 2 2
sulphonic acid; P O, pyranose oxidase; P O-Tm, P O from Trametes multicolor; SDS–
PAGE, Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis; U, units.
⇑
Corresponding author. Tel.: +32 16321920; fax: +32 16321997.
E-mail address: Karolien.Decamps@biw.kuleuven.be (K. Decamps).
In Nature, P
2 2 2
O provides H O to peroxidases of the fungal
ligninolytic system, which comprises lignin and manganese
0
308-8146/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodchem.2011.10.041

1
486
K. Decamps et al. / Food Chemistry 131 (2012) 1485–1492
peroxidases (Daniel, Volc, & Kubatova, 1994; Leitner et al., 2001).
One of its current applications is the production of 2-keto- -galact-
ose. The latter can be (bio)catalytically reduced on C with forma-
tion of -tagatose, a low caloric sweetener with prebiotic effects
Levin, 2002). 2-Keto- -glucose, another product of P O, is an inter-
mediary product in the ‘‘Cetus process’’, for conversion of -glucose
into -fructose. Furthermore, some white-rot fungi use P O to
convert -glucose, via 2-keto- -glucose, into the antibiotic cortalc-
dam, The Netherlands) [moisture content 13.1%, protein content
(N ꢁ 5.7) 11.5% (dry matter basis)]. Wheat bran arabinoxylan oligo-
saccharides(AXOS) with an arabinoseto xyloseratio of 0.37, an aver-
age degree of polymerisation of 6.6 and containing 9.1% ferulic acid
(FA, by mass), as a result of fractionation of an AXOS preparation iso-
lated from wheat bran, essentially as described by Swennen, Cour-
tin, Lindemans, and Delcour (2006), were kindly donated by Jeroen
Snelders (Laboratory of Food Chemistry and Biochemistry). A puri-
fied peroxidase from wheat germ, as described in Billaud, Louarme,
and Nicolas (1999), was used in the FA oxidation experiments.
D
1
D
(
D
2
D
D
2
D
D
eron (Giffhorn, 2000; Haltrich et al., 1998; Volc & Eriksson, 1988).
Wagner, Holte, Si, and Laufen (2000) and Arnaut, De Meyer, and
Van Haesendonck (2006) reported the use of P
2
O in breadmaking.
O (Bankar,
GO, which in Nature, has a role similar to that of P
2
2
2.2. Production and purification of P O from T. multicolor
Bule, Singhal, & Ananthanarayan, 2009), is used in pharmaceutical,
food, chemical, beverage and other applications. When used in food
and beverage applications, its function is to remove either glucose or
P
2
O [sequence accession number AY753305 (GenBank Nucleo-
tide Sequence Database, http://www.ncbi.nlm.nih.gov/Genbank/
O
2
. GO is, for example, used in the manufacture of egg powder to re-
index.html)] from the fungus T. multicolor MB 49 (further referred
duce product browning. Elimination of glucose also reduces the risk
of microbial spoilage, hence, improving product storage stability (Si-
sak, Csanadi, Ronay, & Szajani, 2006). Furthermore, GO allows con-
trolling non-enzymic browning during fruit processing and fruit
puree storage (Parpinello, Chinnici, Versari, & Riponi, 2002). It can
also be used for producing white wine with reduced alcohol level
2
to as P O-Tm) was heterologously expressed in Escherichia coli
BL21(DE3) (Invitrogen, Carlsbad, CA, USA) and purified according
to Spadiut et al., 2008.
The E. coli cultures were cultivated in Erlenmeyer flasks, or a
fermentor. For enzyme production in baffled Erlenmeyer flasks,
the E. coli transformants were grown at 37 °C, 160 rpm, in Terrific
Broth media, [12.0 g/l peptone from casein, 24.0 g/l yeast extract,
4.0 ml/l glycerol, 100 mM potassium phosphate buffer (pH 7.5)]
with ampicillin (100 mg/l) to an optical density at 600 nm, of about
(Pickering, Heatherbell, & Barnes, 1998). Moreover, the produced
2 2
H O
can inhibit wine spoilage by acid and lactic acid bacteria, and
thus reduces the dosage of preservatives needed (Malherbe, du Toit,
Otero, van Rensburg, & Pretorius, 2003).
0.5. Protein expression was then induced by adding D-lactose to a
In breadmaking, potassium bromate, an oxidant, and ascorbic
final concentration of 0.5% (w/v) and cultures were further grown
in baffled Erlenmeyer flasks in an incubator at 25 °C, 110 rpm. After
20 h, E. coli cells were harvested by centrifugation at 4 °C, 3500g,
for 30 min, suspended in binding buffer [50 mM potassium phos-
phate buffer containing 20 mM imidazole, 0.5 M NaCl and 0.1%
phenylmethylsulfonylfluoride (pH 6.5)] and lysed twice using a
pressure homogenizer. The cells were separated from the crude ex-
tract by ultracentrifugation at 4 °C, 92,570g, for 30 min. Cultivation
in the fermentor was performed by a procedure, similar to that
above, but on a larger scale (52 l Terrific Broth medium). The crude
extracts were loaded onto an Immobilized Metal Ion Affinity Chro-
matography column (200 ml, Amersham Biosciences, Vienna, Aus-
tria), equilibrated on the above imidazole containing binding
acid (AH
quickly oxidised to dehydroascorbic acid (DHA), by the action of
AH oxidase. Addition of GO to dough recipes induces various chem-
ical changes, including formation of disulphide bonds (Bonet et al.,
006; Joye, Lagrain, & Delcour, 2009), or, less importantly, dityrosine
crosslinks (Joye et al., 2009; Tilley et al., 2001) between gluten pro-
teins. Furthermore, the H formed can promote the formation of
2 2
), an anti-oxidant are used. During breadmaking, AH is
2
2
2 2
O
diferulate bridges in arabinoxylans, catalysed by peroxidases (Fig-
ueroa-Espinoza & Rouau, 1998). GO supplemented doughs show im-
proved viscoelastic and rheological properties. The baked breads
have improved crumb characteristics and/or larger volume (Bonet
et al., 2006; Rasiah, Sutton, Low, Lin, & Gerrard, 2005).
Whereas GO is used in a number of food applications, such use
2
buffer. The recombinant His-tagged P O-Tm was then eluted using
of P
2
O is rare. Furthermore, the biochemical characteristics of both
a linear increasing gradient of elution buffer [50 mM potassium
phosphate buffer containing 1.0 M imidazole, 0.5 M NaCl and
0.1% phenylmethylsulfonylfluoride, pH 6.5], over a 100 min time
interval (0–100%, 8.0 ml/min). Impurities, imidazole and NaCl,
were removed from the resulting eluate by ultrafiltration with a
hollow fibre module, with a selectively permeable membrane
(Microza, Asahi Kasei Chemicals, Tokyo, Japan). The obtained en-
zyme solution was diluted four times, with a 50 mM potassium
phosphate buffer (pH 6.5, further referred to as buffer A), to im-
prove the removal of small components by minimising membrane
blockage and avoiding enzyme activity loss. The enzyme was
washed from the module with the same phosphate buffer.
enzymes have, to the best of our knowledge, not been compared.
Hence, the purpose of the present work was to investigate the dif-
ferences and similarities between a P
2
O and a GO enzyme. To that
end, Trametes multicolor P O was recombinantly produced and
2
purified and its biochemical characteristics, and those of Aspergillus
niger GO, were more closely studied and compared. Furthermore,
some features of the enzymic reactions catalysed by both enzymes
were examined in model systems, including a water extract from
wheat flour. Based on these data, predictions about the potential
use of P
forward.
2
O in dough and breadmaking applications will be put
2
2.3. P O-Tm and GO-An activity assays
2
. Materials and methods
Enzyme activity was spectrophotometrically measured with the
2,2 -azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) as-
0
2.1. Materials
say of H
et al., 2001). An aliquot (10
zyme solution was added to 990
1.14 mM ABTS, 142 U peroxidase, and 20 mM glucose, which had
been preincubated for 5 min, at 30 °C. The initial O level, mea-
sured by an O electrode (Inlab 605; Mettler Toledo, Zaventem, Bel-
gium), was 0.22 mM. The H , produced in a reaction catalysed by
O-Tm or GO-An, oxidised ABTS in the presence of peroxidase. An
increased level of oxidised ABTS causes a change in absorbance at
2
O
2
(Danneel, Rossner, Zeeck, & Giffhorn, 1993; Leitner
l) of an (appropriately diluted) en-
l of buffer A, containing
GO, from A. niger, further referred to as GO-An, catalase from bo-
l
vine liver and horseradish peroxidase were obtained from Sigma–
Aldrich (Bornem, Belgium). 2-Keto- -glucose and chemicals used
l
D
for enzyme production and purification were of the purest grade
available and from Sigma–Aldrich, unless stated otherwise. All
chemicals, solvents and reagents used in the other experiments
were of analytical grade and also from Sigma–Aldrich, unless speci-
fied otherwise. The wheat flour used was Kolibri (Meneba, Rotter-
2
2
2 2
O
P
2

K. Decamps et al. / Food Chemistry 131 (2012) 1485–1492
1487
4
20 nm, which was monitored for 3 min and used to calculate en-
zyme activity. One enzyme unit (U) is the amount of enzyme
needed to oxidise 2 mol of ABTS, per min, under the conditions
of the assay. The molar absorbance coefficient of ABTS was
continuously. The rate of its consumption was used to determine
the kinetic constants for the Ping Pong Bi Bi model of both en-
zymes, as described by Segel (1975). Apparent kinetic constants
[apparent Michaelis Menten constant (KMApp) and apparent maxi-
mum reaction velocity (VmApp)] towards reducing substrates in
air saturated conditions were calculated using the following equa-
tions (Segel, 1975):
l
ꢂ1
ꢂ1
4
3.2 mM cm
.
Enzyme activity was also determined by polarography using a
Clark electrode (OSD 23 Oxymeter, Heito, Paris, France). Hence,
one enzyme U, expressed in nkatal (nkat), is the amount of enzyme
V
m
K
M
needed to consume 1 nmol of O
.4. Peroxidase activity assay
Peroxidase activity was measured spectrophotometrically, as
2
in the assay conditions.
V
mApp ¼ K
and
K
MApp ¼ KM
½O
:
M
O
O
2
2
þ 1
þ 1
½
O
2
ꢃ
2
ꢃ
2
2
2.8. Product inhibition of P O-Tm
described by Garcia, Rakotozafy, Telef, Potus, and Nicolas (2002).
A purified peroxidase from wheat germ, as described in Billaud
et al. (1999), or a wheat extract (prepared as described in Sec-
tion 2.10), were used as a source of peroxidase. Peroxidase solu-
Product inhibition tests of P
2 2 2
O-Tm with 2-keto-D-glucose or H O . Apparent kinetic constants
in air saturated solutions, at 30 °C, were determined by activity mea-
surements in the presence of different concentrations of glucose
2
O-Tm were performed by incubating
P
tions were diluted to obtain an activity, which decreased the
extinction by 5.2 ꢁ 10ꢂ3 Absorbance U(AU)/s.
(0.1–100 mM) and 2-keto-
lis Menten kinetics (SigmaPlot, Systat Software, San Jose, CA, USA).
The potential inhibition of P O-Tm by H was investigated by
comparing the reaction catalysed by P O-Tm with, and without, cat-
alase. In case of product inhibition by 2-keto- -glucose or H , the
D-glucose (1.0 or 120 mM), using Michae-
2
2 2
O
2.5. Enzyme purity
2
D
2 2
O
Protein purity was verified, and subunit MM, determined by So-
apparent inhibition constant (KIApp) can be calculated using the
inhibitor concentration, the apparent Michaelis Menten constant to-
wards glucose (KMApp) and the apparent Michaelis Menten constant
in the presence of the inhibitor (KMApp(I)):
dium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS–
PAGE). Silver-stained SDS–PAGE gels were made using the Phast
System unit (GE Healthcare, Uppsala, Sweden), as described in
the GE Healthcare separation technique files 110 and 210. The
low MM marker mix (GE Healthcare) included
bean trypsin inhibitor, carbonic anhydrase, ovalbumin, albumin
and phosphorylase b with MM of 14.4, 20.1, 30, 45, 66 and
a
-lactalbumin, soy-
KMApp
For competitive inhibition : KIApp ¼ ½Iꢃ ꢁ K
:
ð1Þ
M
AppðIÞ
ꢂ KMApp
9
7 kDa, respectively.
Enzyme preparations of P
catalase and peroxidase activities. Catalase activity was measured
colorimetrically, as described by Sinha (1972). Briefly, the reaction
2
O-Tm and GO-An were screened for
2.9. DHA as possible electron accepting substitute for the enzymic
reaction of P O-Tm or GO-An
2
of dichromate with H
2
O
2
, remaining after possible catalase activity,
The reactions catalysed by 0.09 U/ml P
2
O-Tm, or 0.3 U/ml GO-An,
in the presence of acetic acid results in chromic acetate, which
shows absorption at 570 nm. Peroxidase activity was also deter-
mined by the above described ABTS procedure. In this case, the
in small reaction vessels containing 30 ml of buffer A, 0.7 mM glu-
cose and 0.5 mM DHA, at 30 °C, were monitored. To study the poten-
tial, of the latter, as an electron accepting substitute, O
excluded from the reaction medium by continuously sparging nitro-
gen gas through the medium. O concentrations were measured by
an O electrode. Reactions were started by addition of the enzyme
when the O concentration in the reaction solution was less than
0.05 mg/l, to eliminate interference of O as an electron accepting
2
was
2 2
reaction solution only contained H O and ABTS.
2
2.6. Determination of optimum temperature and pH activity ranges
2
2
Optimum temperature and pH activity ranges, for the reactions
catalysed by P O-Tm and GO-An, were determined by screening
activity at different temperatures (4–80 °C) and pH values (pH
.0–10.0), with the above described ABTS method. The latter
experiments were performed in 100 mM buffers of sodium citrate
2
substitute. Samples were periodically withdrawn and boiled for
5 min to inactivate the enzymes. After a convenient dilution, the glu-
cose concentration was quantified by High Performance Anion Ex-
change Chromatography with Pulsed Amperometric Detection
2
2
(
(
pH 2.0–5.0), potassium phosphate (pH 6.0–7.5) or sodium borate
pH 8.0–10.0), during a 180 s incubation time.
analysis (20–100 lM), with a ThermoFisher Scientific system (Wal-
tham, MA, USA). The system consisted of a gradient pump (P4000),
an autosampler and an electrochemical detector (ED40; Dionex,
Amsterdam, The Netherlands) with a gold working electrode. Sepa-
2
.7. Determination of kinetic constants
ration of 20
CarboPac PA100 column (Dionex, flow rate 1.0 ml/min). Three elu-
ents were used for the gradient mobile phase: H O of 18.2 M con-
ductivity, 100 mM NaOH (Baker, Deventer, The Netherlands) and a
mixture of 500 mM CH COONa and 100 mM NaOH (Table 2). All elu-
ll samples was executed at room temperature with a
D
-Glucose,
crose and -maltose, and
possible substrates for P
Michaelis Menten constant K
) for the Ping Pong Bi Bi mechanism of P
wards the above defined sugar substrates, were determined by
varying the concentrations of sugar and O . Different concentra-
tions (0.23–300 mM) of each of the sugar substrates were tested
in the presence of different concentrations of O (0.03–0.22 mM).
The reaction solution, containing the sugar substrate, in buffer A,
was preincubated at 30 °C. Solutions with different O levels were
prepared by sparging the reaction solutions with nitrogen. The ini-
tial O concentration was determined by a Clark electrode. After
addition of P O-Tm or GO-An, the O concentration was measured
D
-galactose, L-arabinose, D-fructose, D-su-
D
-xylose,
-glucono-1,5-lactone were screened as
O-Tm and GO-An. Kinetic constants
and maximum reaction velocity
O-Tm and GO-An, to-
D
D
2
X
2
(
V
M
3
ents were degassed and kept under helium overpressure to prevent
accumulation of atmospheric carbon dioxide.
m
2
2
2.10. Preparation of wheat extract
2
Wheat extract was prepared by suspending 10.0 g wheat flour
in 20.0 ml buffer A. The mixture was shaken for 60 min
(150 rpm, room temperature) and subsequently centrifuged at
15 °C, 9800g, for 20 min. The extraction was performed a second
time and supernatants were combined.
2
2
2
2

1
488
K. Decamps et al. / Food Chemistry 131 (2012) 1485–1492
For FA oxidation measurements, an alternative protocol for
preparation of a wheat extract, used as a source of peroxidase,
was followed. Wheat flour (4.00 g) was suspended in 10.0 ml 2-
1
2
3
4
kDa
97
(
N-morpholino)ethanesulphonic acid (MES) buffer (50 mM), ad-
6
4
6
5
justed to pH 5.6 with 0.1 M NaOH, further referred to as buffer B.
The suspension was vortexed during 10 s, homogenised with an
Ultra Turrax (IKA T-25, IKA, Staufen, Germany) and centrifuged at
3
0
4
°C, 28,000g, for 20 min. The supernatant obtained is further re-
ferred to as wheat extract.
2
1
0.1
4.4
2
.11. Oxidation of FA in model systems
FA oxidation was determined by measuring the decrease in
absorbance at 310 nm, for FA, and at 325 nm, for AXOS, using a
diode array spectrophotometer (Hewlett–Packard, Pickering, ON,
Canada). The reaction mixture contained either FA or AXOS (both
Fig. 1. Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (SDS–PAGE) of
purified pyranose oxidase from Trametes multicolor (P O-Tm) and glucose oxidase
from Aspergillus niger (GO-An). Lanes 1 and 4: low molecular mass marker, lanes 2
and 3: P O-Tm and GO-An, respectively.
2
1
00
2 2
lM FA or FA equivalent) and H O (500 lM) in buffer B. The
2
reaction was started by addition of 20
l
l peroxidase solution,
ꢂ3
which decreased the absorbance by 5.2 ꢁ 10 AU/s.
Experiments, with different concentrations of glucose, in the
presence of P
2
O-Tm and GO-An were performed and the rate of
Solutions of the pure enzymes displayed neither catalase, nor, per-
oxidase activity.
FA oxidation was studied. The reaction solution contained glucose
(
0.3 M, 2.0 mM or 0.47 mM), FA (100
l
M) and 20
l
l peroxidase
ꢂ3
solution, which decreased the absorbance by 5.2 ꢁ 10 AU/s.
2
3.2. Biochemical characterisation of P O-Tm and GO-An
Reactions were started by addition of 10
solution, appropriately diluted to obtain a V
oxidation of 4.8 M O /s towards 0.3 M glucose in buffer B. The
effect of AH on FA oxidation was also tested in the presence of
different AH concentrations (50, 100 or 150 M).
2
ll P O-Tm or GO-An
m
corresponding to
In contrast to P
-galactose, -arabinose,
-sucrose and
O-Tm nor for GO-An. Table 1 lists the kinetic constants of P
Tm and GO-An towards different substrates. The K value towards
-glucose for P O-Tm (3.1 mM) was more than 25-fold lower than
that towards other sugar substrates (P78.4 mM). Moreover, the
value of GO-An towards -glucose (225 mM) was approxi-
mately 73-fold higher than that of P O-Tm. In addition, the K
value of GO-An towards O (2.9 mM) was approximately 6-fold
O-Tm (0.46 mM).
2
O-Tm, GO-An does not catalyse the oxidation
-xylose, or -glucono-1,5-lactone.
-maltose are substrates neither for
O-
l
2
of
D
L
D
D
2
D-Fructose,
D
D
2
l
P
2
2
The activity of peroxidases, endogenously present in the wheat
flour extract, prepared as described above, was tested by monitoring
M
D
2
the oxidation of FA, either upon addition of P
The reaction solution contained the same reagents as above, except
for the peroxidase, which was replaced by 20 l of wheat extract.
2 2 2
O-Tm, GO-An or H O .
K
M
D
l
2
M
2
2
.12. Oxidation of free thiols
higher than that of P
2
To 40.0 ml of a wheat extract, either no enzyme, or 0.001 U P
2
O-
Tm (enzyme stock solution of 27.7 U/ml) or GO-An (enzyme stock
solution of 21.9 U/ml), were added. The enzyme dosage was cho-
sen based on preliminary experiments, in which the appropriate
dilution of the enzymes was determined to obtain a measurable
decrease in free thiol groups, in the time scale of the designed
Table 1
Kinetic constants of pyranose oxidase from Trametes multicolor (P
oxidase from Aspergillus niger (GO-An) towards different substrates.
2
O-Tm) and glucose
Enzyme
Substrate
V
m
(U/mg)
K
M
(mM)
V
m
/K
M
(
U/mM ꢁ mg)
experiment. Samples (900, 1000 ll for control samples) were with-
P
2
O-Tm
D
-Glucose
-Xylose
27.9
3.7
3.1
9.0
drawn, in duplicate, at certain time points and put on ice to inhibit
0
D
1
78.4
514
0.46
0.17
0.0009
60.7
enzyme activity. 100
l
l of 0.1% 5,5 -dithio-bis(2-nitrobenzoic acid),
L-Arabinose
0.47
27.9a
in 50 mM sodium phosphate buffer, containing 2.0% Sodium Dode-
cyl Sulphate, 3.0 M urea and 1.0 mM ethylenediaminetetraacetic
acid (pH 6.6) was added to the samples and the mixtures were vor-
texed vigorously. After shaking for 20 min in the dark, the samples
O
2
GO-An
D-Glucose
168.2
168.2a
225
2.9
0.75
58.0
O
2
a
were centrifuged at room temperature, 12,000g, for 10 min. Exactly
Determined in presence of
D
-glucose.
0
4
5 min after adding the above, 5,5 -dithio-bis(2-nitrobenzoic acid)
containing buffer, the absorbance of the samples was measured at
12 nm. A calibration curve was made using 0, 2, 4, 6, 8 and 10 g/
ml of glutathione.
4
l
Table 2
Gradient conditions of High Performance Anion Exchange Chromatography with
Pulsed Amperometric Detection used for the quantification of glucose levels during
the enzymatic reactions catalysed by pyranose oxidase and glucose oxidase.
3
. Results
Time
H
2
O (conductivity
100 mM
100 mM NaOH in 500 mM
(
min)
18.2 M
X
) (%)
NaOH (%)
3
CH COONa (%)
3
.1. Production and purification of P O-Tm
2
0
5
5.1
10
10
0
0
0
0
0
20
100
100
0
90
90
80
0
0
90
90
1
1
20
2
2
3
Heterologous expression of P
purification yielded 2.6 l of enzyme solution, with an activity to-
wards -glucose of 28.4 U/ml.
A silver-stained SDS–PAGE gel (Fig. 1) showed MMs of ca. 75
and 85 kDa for monomeric P O-Tm and GO-An, respectively.
2
O-Tm in E. coli and subsequent
5
5.1
0.0
D
10
10
0
2

K. Decamps et al. / Food Chemistry 131 (2012) 1485–1492
1489
m M 2
The enzyme efficiency, defined as V /K , of P O-Tm towards
A 1
.6
.4
3
2
00 mM glucose
.0 mM glucose
D
-glucose (9.0 U/mM ꢁ mg), was significantly higher than that of
1
GO-An (0.75 U/mM ꢁ mg).
Under our assay conditions, P
2
O-Tm displayed its highest activ-
1.2
1
0.47 mM glucose
ity at 30 °C, in a pH range from 5.5 to 6.5, whereas, GO-An had its
highest activity at 35 °C, in a pH range from 5.0 to 6.5.
Product inhibition was studied by altering the product concen-
tration and measuring the enzyme activity, as described above. The
0
0
0
0
.8
.6
.4
.2
0
presence of 1.0 mM 2-keto-
kinetic values for P O-Tm. However, incubation with excess levels
of 2-keto- -glucose (120 mM) slightly increased the apparent K
1.0 mM without 2-keto-
cose). The corresponding apparent inhibition constant K
D-glucose did not impact the apparent
2
D
M
(
D
-glucose and 2.8 mM with 2-keto-
D
-glu-
was
0
100
200
300
400
I
Time (s)
6
7 mM, using the formula, as described above, for competitive
inhibition. Addition of catalase to the reaction medium did not
change the glucose oxidation rate (results not shown).
B
1.6
.4
3
2
00 mM glucose
.0 mM glucose
1
DHA did not serve as an alternative electron accepting substrate
in the reactions catalysed by either P
crease in glucose level was observed upon incubation of these en-
zymes in presence of DHA and glucose in O free conditions
results not shown).
2
O-Tm or GO-An, as no de-
1.2
1
0.47 mM glucose
2
0
0
0
0
.8
.6
.4
.2
0
(
2
3.3. Functional characterisation of P O-Tm and GO-An
With the same enzyme dosage (0.15 U/ml) and, in non-limiting
conditions, the decrease in the level of natively present glucose,
O
2
0
100
200
300
400
in a water extract of wheat was more pronounced with P
2
O-Tm,
Time (s)
than with GO-An (Fig. 2).
Fig. 3. Effect of different glucose concentrations on the oxidation of ferulic acid
absorbance decrease at 310 nm [mAbsorbance Units (mAU)] in function of
incubation time(s)] by H and purified wheat peroxidase. H was produced
Oxidation of FA was monitored in a model system containing
and purified wheat peroxidase. The change in absorbance at
10 nm for FA and at 325 nm for AXOS, as a function of incubation
[
2 2
H O
2
O
2
2 2
O
3
by the enzymic reaction catalysed by pyranose oxidase from Trametes multicolor
time, was monitored and was higher with FA than with AXOS. Fur-
thermore, a decrease in absorbance with both components was
2
(P O-Tm) (A) and glucose oxidase from Aspergillus niger (GO-An) (B).
also observed upon addition of P
the highest glucose concentration (0.3 M), the rate of absorbance
decrease was the same for both P O-Tm and GO-An. Lowering the
glucose concentration (2.0 or 0.47 mM) reduced the FA oxidation
rate and, for the same glucose concentration, the rate of FA oxida-
2
O-Tm and GO-An (Fig. 3). For
Addition of different levels of AH
FA oxidation (Fig. 4). The higher the AH
pronounced the delay and, thus, the later the decrease in absor-
bance was observed, for both enzymes.
2
(50, 100 or 150
lM) delayed
2
concentration, the more
2
Oxidation of thiols upon addition of P
itored in wheat extract (Fig. 5). The thiol concentration decreased
faster with P O-Tm than with GO-An. The final thiol concentration
seemed to reach a plateau, the value of which was higher for GO-
An than for P O-Tm.
2
O-Tm or GO-An was mon-
2
tion was consistently higher upon addition of P O-Tm, than when
adding GO-An. When wheat extract was used as source of peroxi-
dase, the same trend in absorbance decrease could be observed (re-
sults not shown). Likewise, when FA was replaced by AXOS, a
decrease in absorbance could be observed (results not shown).
2
2
4
. Discussion
0
0
0
0
0
.45
.4
.35
.3
.25
.2
.15
.1
P2O-Tm
GO-An
D
-Glucose is the preferred substrate for both P
as the other substrates tested have significantly higher K
Furthermore, P O-Tm has a higher affinity towards both glucose
and O than does GO-An (Fig. 6). The observed differences in bio-
chemical characteristics may well point to differences in function-
ality of P O and GO in food applications. In some complex food
matrices, the O concentration may well be a very important and
critical factor for the activities of both enzymes. Hence, the differ-
ence in O affinity can be highly relevant. During breadmaking e.g.
after dough mixing, O is quickly consumed both by yeast and in
reactions catalysed by endogenous, or added O consuming en-
zymes, hence rendering dough anaerobic early in the fermentation
stage (Fig. 6). Furthermore, in view of the optimal pH and temper-
ature conditions for enzyme activity, we expect both enzymes to
be highly active during mixing and the early fermentation stages
of the breadmaking process.
2
O-Tm and GO-An
0
M
values.
2
2
0
2
2
0
2
2
0
2
.05
0
0
20
40
60
80
Time (min)
A
high concentration of 2-keto-
medium resulted in slightly increased apparent K
-glucose, while the apparent V value remained unaffected.
D
-glucose in the reaction
Fig. 2. Oxidation of glucose upon addition of pyranose oxidase from Trametes
multicolor (P O-Tm, 0.15 U/ml) and glucose oxidase from Aspergillus niger (GO-An,
.15 U/ml) to wheat extract.
M
value towards
2
0
D
m

1
490
K. Decamps et al. / Food Chemistry 131 (2012) 1485–1492
Furumoto, Yoshimoto, Fukunaga, and Nakao (2003) found GO to
be competitively inhibited by H when present in an appreciable
concentration. However, in dough, endogenous wheat peroxidases
and catalase presumably quickly use the H formed (Fig. 6).
Therefore, we assume that the concentration of both reaction prod-
ucts in dough does not reach the threshold level needed for them
A
1.6
2 2
O
1
1
.4
.2
1
50 µM AH2
100 µM AH2
150 µM AH2
2 2
O
2
to act as inhibitors. Hence, P O and GO are probably not inhibited
by the products of their reactions in wheat dough.
DHA was not a valuable electron accepting substitute for O in
2
2
the reaction catalysed by either P O-Tm or GO-An. However, if it
would have been, this would have been relevant in the context
of breadmaking. Indeed, wheat flour is commonly enriched with
0
0
0
0
.8
.6
.4
.2
0
AH
ent AH
substitute for P
tition between O
hence, would have resulted in a lower level of produced H
Fig. 6). The potential of DHA to function as a substrate will also
have caused competition for DHA between e.g. glutathione dehy-
drogenase (Kaid, Rakotozafy, Potus, & Nicolas, 1997) and P O or
GO, consecutively affecting the redox processes in dough (Fig. 6).
In the presence of AH , oxidation of FA upon addition of P O-Tm
or GO-An by peroxidase, was delayed by the scavenging of FA rad-
icals by AH (Garcia et al., 2002) (Fig. 6). When no AH is left, the
2
, which is quickly converted to DHA by e.g. endogenously pres-
oxidase. If DHA would have acted as an electron accepting
O or GO, there would probably have been compe-
and DHA in AH supplemented doughs which,
2
2
0
200
400
600
Time (s)
800
1000
2
2
2 2
O
(
B
1.6
50 µM AH2
100 µM AH2
150 µM AH2
2
1
1
.4
.2
1
2
2
2
2
concentration of FA steadily decreases by formation of FA dimers
and oligomers. Hence, it can be hypothesised that, in wheat flour
0
0
0
0
.8
.6
.4
.2
0
containing a high level of AH
occurring, is delayed.
2 2 2
, the FA crosslinking by H O , if
The oxidation rate of FA, irrespective of whether it occurred
free or bound in AXOS, was higher for P O-Tm than for GO-An.
FA was also oxidised when H was added to the reaction med-
ium. The substrate selectivity and specificity differences between
the two enzymes probably result in faster H production and
higher production level with P O-Tm. In that way, more substrate
2
2 2
O
2 2
O
0
200
400
600
Time (s)
800
1000
2
is available for wheat endogenous peroxidases to perform their
oxidising (crosslinking) reactions. The oxidation rate for AXOS
was lower than that of free FA. Probably, steric hindrance is rate
limiting.
In wheat extract, a system that more closely resembles dough
systems than a buffer system, the decrease in glucose level when
Fig. 4. Effect of different ascorbic acid (AH
ferulic acid [absorbance decrease at 310 nm [mAbsorbance Units (mAU)] in
function of incubation time(s)] by H and purified wheat peroxidase. H was
produced by the enzymic reaction catalysed by pyranose oxidase from Trametes
2
) concentrations on the oxidation of
2
O
2
2 2
O
2
multicolor (P O-Tm) (A) and glucose oxidase from Aspergillus niger (GO-An) (B).
adding P
This can be explained by the higher affinity of P
cose and O than that of the former enzyme and by the differences
in substrate selectivity between both enzymes (Fig. 6). Thiol con-
centrations decreased upon addition of P O-Tm or GO-An. Again,
this decrease was more pronounced for P O-Tm than for GO-An.
The observed differences in substrate selectivity and specificity be-
tween P O-Tm and GO-An are probably at the basis of the more
pronounced effects of P O-Tm on the level of free thiols. Further-
more, it can be hypothesised that the plateau thiol concentration
is lower for P O-Tm than for GO-An, because the higher level of
produced in the case of P O-Tm provides a higher substrate
concentration for thiol oxidation.
Fig. 6 shows the complexity of wheat flour dough, in that it con-
tains numerous enzymes that use O or H . In this manuscript,
experiments were carried out in buffer systems and wheat ex-
tracts. Although there is a difference in reaction conditions (e.g.
other substrates, pH) and mobility between compounds in wheat
extract or buffer and in dough itself, some results found here al-
ready point to a potentially different effect of the enzymes in
dough. More research on dough systems will be needed to formu-
late hypotheses and to obtain a full understanding of the applica-
2
O-Tm was more pronounced than when adding GO-An.
2
O-Tm towards glu-
8
7
6
5
4
3
2
1
Control
GO-An
P2O-Tm
2
2
2
2
2
2
2
H O
2
2
0
0
10
20
30
40
50
60
2 2
O
2
Time (min)
Fig. 5. Change in thiol levels in wheat extract upon addition of pyranose oxidase
from Trametes multicolor (P
An), or without enzyme.
2
O-Tm), or glucose oxidase from Aspergillus niger (GO-
Hence, under such conditions, 2-keto-
competitive inhibitor of P O-Tm (apparent K
at low 2-keto- -glucose concentration,
D-glucose is only a weak
2
I
= 43.2 mM). Indeed,
bility of P
2
O as a bread improving agent and the dynamics and
in dough. Furthermore, more research will also
D
2
P O-Tm activity was
use of O and H O
2
2 2
unaffected. Under the experimental conditions, H
significantly inhibit the reaction catalysed by
2
O
P
2
did also not
2
O-Tm. Bao,
shed light on the exact interplay between the enzymes, their
substrates and products.

K. Decamps et al. / Food Chemistry 131 (2012) 1485–1492
1491
Fig. 6. Overview of enzymic reactions, which make use of O
2
(upper part) or H
2
O
2
(lower part), possibly occurring in wheat flour extract or dough to which pyranose oxidase
-glucose by P O or to -glucono-1,5-lactone by GO.
(
P
2
O), or glucose oxidase (GO), have been added. O consuming reactions compete with the oxidation of glucose to 2-keto-
2
D
2
D
Billaud, C., Louarme, L., & Nicolas, J. (1999). Comparison of peroxidases from barley
kernel (Hordeum vulgare L.) and wheat germ (Triticum aestivum L.): Isolation
and preliminary characterization. Journal of Food Biochemistry, 23(2), 145–172.
Bonet, A., Rosell, C. M., Caballero, P. A., Gomez, M., Perez-Munuera, I., & Lluch, M. A.
(2006). Glucose oxidase effect on dough rheology and bread quality: A study
from macroscopic to molecular level. Food Chemistry, 99(2), 408–415.
5
. Conclusion
The present study covers a biochemical and functional charac-
terisation of P O-Tm and GO-An. The observed differences in glu-
cose and affinity suggest differences in functional
characteristics of both enzymes in food applications and, more spe-
cifically, in systems where the O or glucose concentration is lim-
iting. Furthermore, in line with what has been observed for GO-
An, P O-Tm can oxidise both thiol groups and FA in wheat flour ex-
tracts. The oxidation rates are higher upon addition of P O-Tm than
of GO-An. These results are relevant with regard to the potential
use of P O-Tm in dough and breadmaking processes, which will
2
O
2
2 2
Daniel, G., Volc, J., & Kubatova, E. (1994). Pyranose oxidase, a major source of H O
during wood degradation by Phanerochaete chrysosporium, Trametes versicolor, and
Oudemansiella mucida. Applied and Environmental Microbiology, 60(7), 2524–2532.
2
Danneel, H. J., Rossner, E., Zeeck, A.,
& Giffhorn, F. (1993). Purification and
characterization of a pyranose oxidase from the Basidiomycete Peniophora
gigantea and chemical analyses of its reaction products. European Journal of
Biochemistry, 214(3), 795–802.
2
2
Figueroa-Espinoza, M. C., & Rouau, X. (1998). Oxidative cross-linking of pentosans
by a fungal laccase and horseradish peroxidase: Mechanism of linkage between
feruloylated arabinoxylans. Cereal Chemistry, 75(2), 259–265.
2
be the topic of further investigation.
Garcia, R., Rakotozafy, L., Telef, N., Potus, J., & Nicolas, J. (2002). Oxidation of ferulic
acid or arabinose-esterified ferulic acid by wheat germ peroxidase. Journal of
Agricultural and Food Chemistry, 50, 3290–3298.
Acknowledgements
Giffhorn, F. (2000). Fungal pyranose oxidases: Occurence, properties and
biotechnical applications in carbohydrate chemistry. Applied Microbiology and
Biotechnology, 54, 727–740.
Karolien Decamps gratefully acknowledges financial support
from the ‘Fonds voor Wetenschappelijk Onderzoek – Vlaanderen’
Haltrich, D., Leitner, C., Neuhauser, W., Nidetzky, B., Kulbe, K. D., & Volc, J. (1998). A
convenient enzymatic procedure for the production of aldose-free D-tagatose.
Enzyme Engineering Xiv, 864, 295–299.
(
FWO, Brussels, Belgium). This research is also part of the Methu-
Joye, I. J., Lagrain, B., & Delcour, J. A. (2009). Endogenous redox agents and enzymes
that affect protein network formation during breadmaking – A review. Journal of
Cereal Science, 50(1), 1–10.
Kaid, N., Rakotozafy, L., Potus, J., & Nicolas, J. (1997). Studies on the glutathione-
dehydroascorbate oxidoreductase (EC 1.8.5.1) from wheat flour. Cereal
Chemistry, 74(5), 605–611.
Leitner, C., Volc, J., & Haltrich, D. (2001). Purification and characterization of
pyranose oxidase from the white rot fungus Trametes multicolor. Applied and
Environmental Microbiology, 67(8), 3636–3644.
Levin, G. V. (2002). Tagatose, the new GRAS sweetener and health product. Journal of
Medicinal Food, 5(1), 23–36.
salem programme ‘‘Food for the Future’’ at the Katholieke Univer-
siteit Leuven.
References
Arnaut, F., De Meyer, K.,
comprising carbohydrate oxidase and/or pyranose oxidase. US Patent US/2006/
292263 A1.
Bankar, S. B., Bule, M. V., Singhal, R. S., & Ananthanarayan, L. (2009). Glucose oxidase
An overview. Biotechnology Advances, 27(4), 489–501.
& Van Haesendonck, I. P. (2006). Bakery products
0
Malherbe, D. F., du Toit, M., Otero, R. R. C., van Rensburg, P., & Pretorius, I. S. (2003).
Expression of the Aspergillus niger glucose oxidase gene in Saccharomyces
cerevisiae and its potential applications in wine production. Applied Microbiology
and Biotechnology, 61(5–6), 502–511.
–
Bao, J., Furumoto, K., Yoshimoto, M., Fukunaga, K., & Nakao, K. (2003). Competitive
inhibition by hydrogen peroxide produced in glucose oxidation catalyzed by
glucose oxidase. Biochemical Engineering Journal, 13(1), 69–72.

1
492
K. Decamps et al. / Food Chemistry 131 (2012) 1485–1492
Parpinello, G. P., Chinnici, F., Versari, A., & Riponi, C. (2002). Preliminary study on
glucose oxidase-catalase enzyme system to control the browning of apple and
Spadiut, O., Leitner, C., Tan, T. C., Ludwig, R., Divne, C., & Haltrich, D. (2008).
Mutations of Thr169 affect substrate specificity of pyranose 2-oxidase from
Trametes multicolor. Biocatalysis and Biotransformation, 26(1–2), 120–127.
Swennen, K., Courtin, C. M., Lindemans, G. C. J. E., & Delcour, J. A. (2006). Large-scale
production and characterisation of wheat bran arabinoxylooligosaccharides.
Journal of the Science of Food and Agriculture, 86(11), 1722–1731.
Swoboda, B. E. P., & Massey, V. (1965). Purification and properties of glucose oxidase
from Aspergillus niger. Journal of Biological Chemistry, 240(5), 2209–2215.
Tilley, K. A., Benjamin, R. E., Bagorogoza, K. E., Okot-Kotber, B. M., Prakash, O., &
Kwen, H. (2001). Tyrosine cross-links: Molecular basis of gluten structure and
function. Journal of Agricultural and Food Chemistry, 49(5), 2627–2632.
pear purees. Food Science and Technology
Technologie, 35(3), 239–243.
– Lebensmittel-wissenschaft &
Pickering, G. J., Heatherbell, D. A., & Barnes, M. F. (1998). Optimising glucose
conversion in the production of reduced alcohol wine using glucose oxidase.
Food Research International, 31(10), 685–692.
Rasiah, I. A., Sutton, K. H., Low, F. L., Lin, H.-M., & Gerrard, J. A. (2005). Crosslinking of
wheat dough proteins by glucose oxidase and the resulting effects on bread and
croissants. Food Chemistry, 89(3), 325–332.
Segel, I. H. (1975). Enzyme kinetics: Behavior and analysis of rapid equilibrium and
steady-state enzyme systems. New York: John Wiley & Sons, Inc.
Volc, J.,
& Eriksson, K. E. (1988). Pyranose 2-oxidase from Phanerochaete
Sinha, A. K. (1972). Colorimetric assay of catalase. Analytical Biochemistry, 47(2),
chrysosporium. Methods in Enzymology, 161, 316–322.
Wagner, P., Holte, D. K., Si, J. Q., & Laufen, D. K. (2000). Use of a pyranose oxidase in
baking. US patent WO/1997/022257.
3
89–394.
Sisak, C., Csanadi, Z., Ronay, E., & Szajani, B. (2006). Elimination of glucose in egg
white using immobilized glucose oxidase. Enzyme and Microbial Technology,
39(5), 1002–1007.