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Tropical Journal of Natural Product Research Original Research Article Kinetics and Thermodynamic Properties of Glucose Oxidase Obtained from Aspergillus fumigatus ASF4

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  • Dennis Osadebay University
Research Proposal

Tropical Journal of Natural Product Research Original Research Article Kinetics and Thermodynamic Properties of Glucose Oxidase Obtained from Aspergillus fumigatus ASF4

Trop J Nat Prod Res, March 2022; 6(3):438-445 ISSN 2616-0684 (Print)
ISSN 2616-0692 (Electronic)
438
© 2022 the authors. This work is licensed under the Creative Commons Attribution 4.0 International License
Tropical Journal of Natural Product Research
Available online at https://www.tjnpr.org
Original Research Article
Kinetics and Thermodynamic Properties of Glucose Oxidase Obtained from
Aspergillus fumigatus ASF4
Onosakponome Iruoghene1, Ezugwu A. Linus1*, Eze S.O. Onyebuchi1, Chilaka F. Chiemeka1
Department of Biochemistry, University of Nigeria, Nsukka Enugu State, Nigeria
Introduction
Gluconic acid (GA) is a polyhydroxy carboxylic acid
possessing both reactive hydroxyl and carboxyl groups. Gluconic acid
is a non-volatile, easily biodegradable, non-toxic, and non-corrosive
chemical produced from biomass using different approaches including
microbial fermentation, enzymatic oxidation of glucose, and oxidation
by molecular oxygen using heterogeneous catalysis.1 GA is usually
produced by glucose oxidation catalyzed by Glucose oxidase (GOx)
using molecular oxygen as the electron acceptor.2
GOx catalyzes glucose oxidation, by a ping-pong mechanism,2
involving glucose and molecular oxygen as the electron acceptor and
yielding hydrogen peroxide and gluconic acid.3-9 This enzyme is a
member of a vast family of oxidoreductases acting on glucose,
methanol and choline. GOx is a dimeric glycoprotein with two
identical subunits (80kDa), linked together by disulfide bonds.10,11
Structurally, each of the subunits contains a tightly but not covalently
bound FAD moiety as a co-factor.12 This cofactor acts as a redox
carrier during catalysis.10-14. The ability of GOx to oxidize glucose
with liberation of hydrogen peroxide underlines its use in glucose
biosensors for evaluation of antidiabetic properties of some medicinal
plants.
*Corresponding author. E mail: linus.ezugwu@unn.edu.ng;
Tel: +2348063743436
Citation: Onosakponome I, Ezugwu AL, Eze SOO, Chilaka FC.
Kinetics and Thermodynamic Properties of Glucose Oxidase Obtained
from Aspergillus fumigatus ASF4. Trop J Nat Prod Res. 2022; 6(3):438-445.
doi.org/10.26538/tjnpr/v6i3.22
Official Journal of Natural Product Research Group, Faculty of Pharmacy,
University of Benin, Benin City, Nigeria.
Glucose oxidase is known to possess antimicrobial properties arising
from the production of hydrogen peroxide and can be used to
supplement diets and enhance pathogen defense response.15,16 Also,
the accumulation of gluconic acid due to degradation of
gluconolactone lowers the pH of a solution thereby contributing to the
antimicrobial activity of GOx.6
GOx is obtained from different sources, including citrus fruits, red
algae, bacteria, plants, animals, insects, and fungi. Among these
sources, fungal sources are preferred and are extensively employed in
various sectors of food industries as in the production of dry egg
powder, beverages, gluconic acid, and baking products.14
Gluconic acid and its derivatives (such as sodium gluconate or
calcium gluconate) as well as hydrogen peroxide are of great
importance as essential intermediates in the food, pharmaceutical,
building, and textile industries.4,17 Also, derivatives of GA are added
to soft drinks and dairy products to enhance and preserve their sensory
properties. Sodium gluconate can remove bitterness from food and
chelate metal.4 They are attractive feedstock for green chemistry
applications. The need for green and sustainable development has
prompted the search for eco-friendly and cost-effective means of
production of chemical feedstock such as gluconic acid. However, the
eco-friendly and cost-effective bioconversion of glucose to gluconic
acid and hydrogen peroxide by glucose oxidase remains a serious
challenge. This is due to the dependence of the catalytic efficiency of
GOx on its stability. Sufficient heat stability of enzymes is vital for
enhancing product efficiency.18 Thermal instability and little
knowledge on the optimal conditions for GA production are the major
setbacks preventing the use of glucose oxidase for the eco-friendly
production of GA. This can be achieved by exploring locally sourced
microbial heat-stable glucose oxidase and determine suitable
conditions for its catalysis. In this study, we isolated and molecularly
characterized GOx producing fungi, determined the effect of different
ARTICLE I NFO
ABSTRACT
Article history:
Received 25 February 2022
Revised 17 March 2022
Accepted 24 March 2022
Published online 05 April 2022
Heat instability is a major setback that prevents the broader use of glucose oxidase (GOx) in
industries. This research explored the kinetic and thermodynamic parameters of Aspergillus
fumigatus ASF4 GOx to determine its potential for biotechnological applications. Aspergillus
fumigatus ASF4 GOx was purified 2.18-fold with a 6.25% yield after ammonium sulfate
precipitation (60%), dialysis, ion-exchange chromatography, and gel filtration. The pH and
temperature optima for GOx activity were 5.5 and 40°C, respectively. Metal ions, Ag2+ and Hg2+
had a remarkable inhibitory effect on GOx activity whereas Ca2+, Mg2+, and Mn2+ enhanced
GOx activity. The maximum velocity (Vmax) and Michaelis constant (KM) were 2000 µmol/min,
and 24 mM, respectively. The enzyme retained 85% and 90% of its initial activity at 40C and
30C, respectively after 120 min of incubation. At 50C and 45C, the enzyme retained more than
50% of its initial activity after 120 min of incubation. The k values at 37°C were the lowest
(0.002) whereas that at 70°C was the highest (0.011). The Z-value was 0.3 and the activation
energy (Ea) was 70.64 KJ/mol/K suggesting great sensitivity of GOx to temperature change. The
D-value of Aspergillus fumigatus ASF4 GOx ranged between 115.5 to 208.4 min. The
thermodynamic studies showed that glucose oxidation by Aspergillus fumigatus ASF4 GOx was
reversible (ΔS<0), endothermic (ΔH>0), and non-spontaneous (ΔG>0) at all temperatures tested.
The results on the optimum conditions for GOx activity and stability have shown that
Aspergillus fumigatus ASF4 GOx can find application in the industrial production of gluconic
acid.
Keywords: Aspergillus fumigatus ASF4, characterization, glucose oxidase, thermos-stability,
18S-rDNA sequencing.
Copyright: © 2022 Iruoghene et al. This is an open-
access article distributed under the terms of the
Creative Commons Attribution License, which
permits unrestricted use, distribution, and
reproduction in any medium, provided the original
author and source are credited.
Trop J Nat Prod Res, March 2022; 6(3):438-445 ISSN 2616-0684 (Print)
ISSN 2616-0692 (Electronic)
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© 2022 the authors. This work is licensed under the Creative Commons Attribution 4.0 International License
metal ion concentrations, pH, glucose concentration, and temperature
on the purified GOx activity as well as the effect of heat treatment on
the stability of Aspergillus fumigatus ASF4 glucose oxidase.
Materials and Methods
Chemicals
All chemicals used in this study were of analytical grade and were
products of BDH chemical limited (England), USA, Merck
(Germany), May, and Baker limited (England), Sigma Aldrich.
Isolation of glucose oxidase producing fungi
Pure fungal strains were isolated from soil collected from a fruit
garden in the University of Nigeria, Nsukka, Enugu State. The
microorganism was identified as described by Martin et al.19 The
fungal strains were screened for glucose oxidase production capability
according to the method explained by Park et al.20 The fermentation
medium containing glucose (80g), peptone (3.0g), (NH4)2 HPO4
(0.388g), KH2PO4 (0.188g), MgSO4 7H2O (0.156g), agar (20g), in one
liter of sodium acetate buffer (0.05M, pH 5.5) was sterilized at 121oC
for 15min, dispensed into Petri dishes to gel. The Petri dishes were
inoculated with the fungal strain using a cork borer and incubated at
35ºC for 3 days. The plates were then spread with a solution
containing agar 1% (w/v) in sodium acetate buffer (0.05M, pH 5.5),
glucose 5% (w/v), O-dianisidine 0.1% (w/v), glycerol 2% (v/v),
Horseradish peroxidase 60IU/ml, and incubated at 35ºC for 1hr. A
reddish-brown coloration indicates the presence of GOx. The strain
with the highest GOx production suggested by the intensity of reddish-
brown coloration was identified, selected, and maintained on the PDA
slant at 4ºC for further studies.
Molecular identification and characterization of the fungal strain
The genomic DNA (gDNA) of the isolated fungal strain was extracted
using AccuPrep® DNA extraction kits following the manufacturer’s
instructions. The primer pairs ITS 1(5’-TCCGTAGGTGAACCT
GCGG-3’) and ITS 4 (5’-GCTGCGTTCTTCTTCATGATGC-3’)
were used to amplify the fungal internal transcribed spacer (ITS)
regions. The conditions for PCR were: initial denaturation at 94oC for
3min, denaturation at 94oC for 40 sec, annealing at 54oC for 40 sec,
extension at 72oC for 40 sec, and final extension at 72oC for 10 sec.
Agarose gel electrophoresis was carried out using 1.5% agarose gel
(1.5g of agarose in 100ml of Tris-acetate-EDTA (TAE) buffer).
Agarose gel powder was dissolved by microwaving in 1 × TAE buffer.
The mixture was cooled to 55oC and 12µl of ethidium bromide was
added and allowed to cool for 30min at 37oC. DNA ladder (6µl) and
the amplicon (10µl) were loaded into the wells of agarose gel followed
by electrophoresis at 100V for 1hr. The DNA bands on the gel were
visualized using a UV lightbox/gel imaging system. The DNA
sequences obtained were subjected to BLAST (Basic Local Alignment
Search Tools for Nucleotides) search algorithm and aligned using
Multiple Sequence Alignment based on Fast Fourier Transform
(MAFFT) version 5. The phylogenetic analysis of the ITS sequence
data was conducted using the molecular evolutionary genetic analysis
(MEGA) version 7.
GOx production from Aspergillus fumigatus ASF4
The fermentation broth was charged for GOx production with glucose
80% (w/v), peptone (0.3%), MgSO4.7H2O (0.0156%), KH2PO4
(0.0188%), (NH4)2HPO4 (0.04%), CaCO3 (3.5%) in 100ml of 50mM
sodium acetate buffer pH 6.0.21 The flasks were sterilized and
inoculated with four discs (10 mm) of pure strains of Aspergillus
fumigatus ASF4. The flasks were incubated at 30ºC on an orbital
shaker at 150rpm for seven days, filtered, and centrifuged at 15000
rpm for 15 min. The supernatant was assayed for GOx activity.
Glucose oxidase assay and protein determination
GOx activity was assayed using glucose as a substrate, and O-
dianisidine as a coupling reagent.22 Reagents A, B, C, D, and E were
prepared for the assay. Reagent A was 0.05M sodium acetate buffer of
pH 5.5; reagent B was an o-dianisidine (0.21mM) solution dissolved
in 100ml of reagent A; reagent C was β- D-glucose solution (10%);
solution D was a mixture of reagent B (24ml) and 5ml of reagent C;
reagent E was a freshly prepared solution of horseradish peroxidase
type II. The reaction mixture contained 2.9ml of solution D, 0.1ml of
reagent E, and 0.1ml of enzyme solution. The absorbance was
measured every 15 sec for 5 min using a UV-Visible
spectrophotometer at 500nm. One unit of glucose oxidase activity is
defined as the amount of enzyme that catalyzes the conversion of
1µmole of β-D-glucose to D-gluconolactone and H2O2 per minute at
35°C and pH 5.5. The concentration of protein was determined using
the Lowry method and Bovine serum albumin (BSA) as standard.23
Purification of Aspergillus fumigatus ASF4 GOx
The crude GOx was brought to 60% saturation using (NH4)2SO4 salt
as described by Chilaka et al.24 The precipitated enzyme (20ml) was
dialyzed for 12 hr against sodium phosphate buffer (0.05 M, pH 7.0).
The dialysate was loaded into a (2.0 x 14 cm) DEAE chromatographic
column equilibrated with 50 mM sodium acetate buffer (pH 5.5). The
unbound proteins were washed using the same buffer. A stepwise
elution with 50 mM sodium acetate buffer containing 0.05 to 1M NaCl
was carried out at a flow rate of 2.5 ml/min. The active fractions were
pooled together and introduced into a (2.0 x 80 cm) Sephadex- G-200
gel chromatographic column, pre-equilibrated with 0.05 M sodium
acetate buffer (pH 5.5). enzyme fractions were collected at a flow rate
of 5ml/15min and assayed for GOx activity. The active fractions were
combined and stored at -10°C for further studies.
Effect of pH and temperature on Aspergillus fumigatus ASF4 GOx
activity
The pH optimum for GOx activity was monitored using 0.05M
sodium acetate (pH 3.5-5.5), 0.05M sodium phosphate (pH 6.0-7.5)
and 0.05M Tris-HCl (8.0-10.0). The GOx activity was assayed using
each buffer as described above. The temperature optimum for GOx
activity was evaluated by incubating the reaction mixture excluding
the enzyme at a temperature range of 30-75oC (5oC interval) before
initiating the reaction. The reaction was initiated by adding GOx
(0.15Uml-1) as described by Singh and Verma.25 The GOx activity was
assayed and plotted against temperature.
Effect of glucose concentration and metal ions on GOx activity
The effect of different glucose concentrations (4.0-25%, w/v) on GOx
activity was determined at pH 5.5 and 40oC as described by Sandalli et
al.26 The maximum velocity (Vmax) and Michaelis constant (KM) were
obtained from the Lineweaver-Burk plot of initial velocity values at
varying glucose concentrations. The effect of different metal ions
(Mg2+, Ca2+, Co2+, Mn2+, Zn2+, Cu2+, Ag2+, and Hg2+) on GOx activity
was determined as described by Yanmis et al.27 The purified enzyme
(100µl) was incubated with 0.9ml of different concentrations (10, 20,
and 50 mM) of metal ions for 20 min. After incubation, GOx activity
was assayed. The GOx activity in each case was compared with the
activity obtained without metal ions.
Effect of temperature on Aspergillus fumigatus ASF4 GOx stability
The effect of temperature on Aspergillus fumigatus ASF4 GOx
stability was determined by incubating Aspergillus fumigatus ASF4
GOx at different temperatures (37 to 60 oC) without substrate for 2 h.28
Aliquots (0.1ml) of the enzyme were collected at different intervals,
quickly cooled in ice for 20 min, and assayed for GOx activity. The
initial activity was assumed 100 % and was used to calculate the
percentage residual activity after every incubation period. The first
order inactivation constant, k values were obtained from the gradient
of the first-order enzyme inactivation equation as follows:
  
Where t is the time of enzyme inactivation.
The activation energy of the purified GOx was obtained from the
Arrhenius plot of lnk against 1/T. Arrhenius law is usually used to
explain the temperature dependence of the rate of inactivation
constants (k) and is given by
  
Trop J Nat Prod Res, March 2022; 6(3):438-445 ISSN 2616-0684 (Print)
ISSN 2616-0692 (Electronic)
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Where:
Ea = activation energy of inactivation,
A=Arrhenius constant,
R= Universal gas constant (8.314 J/mol.K) and
T= Absolute temperature.
The activation energy (Ea) values and the Arrhenius rate constant (k)
were used to obtain the Gibbs free energy change (ΔG), the entropy
change (ΔS), and the enthalpy change (ΔH) using equations below:
   
    
   
Where t, Ea, R, T, KB, and h are the time of inactivation, activation
energy, universal gas constant (8.314J/mol.K), absolute temperature,
Boltzmann’s constant (1.3806 x 10-23 J/K), and Plank’s constant
(6.6260 x 10-34 J.s).
The half-life of inactivation (t1/2) was calculated from:
 
The D-value (the time required for 90% reduction of the initial
activity) was obtained using the equation:
   
The Z-value was obtained by plotting log D values at different
treatment times against the respective temperature. The slope of the
line is equal to the negative reciprocal of the Z-value.
Results and Discussion
The glucose oxidase-producing fungi were isolated and identified as
Aspergillus species based on morphological and microscopic
characteristics. The fungal strain was further confirmed as Aspergillus
fumigatus ASF4 using 18S rDNA sequencing technique. The result of
the agarose gel electrophoresis showed a single band at approximately
500bp when compared to the DNA ladder which indicates that the
isolate mostly likely belongs to Aspergillus sp (Figure 1). The
phylogenetic analysis revealed that the Aspergillus sp is very closely
related to Aspergillus fumigatus ASF4 (Figure 2). Also, from the
NCBI blast result of the query sequence and as shown on the
phylogenetic tree of evolutionary history, the organism had a bootstrap
score of 99 % with Aspergillus fumigatus strains MG991595.1,
MK719925.1, and MH378448.1.
Bootstrap score often shows the level of relatedness between the query
sequence and other homologous sequences from the NCBI Genbank.
A bootstrap score of 99 % indicates a strong relatedness of the query
sequence and Aspergillus fumigatus. Identification of fungi based on
morphology and microscopic characteristics is useful but quite
ambiguous, open-ended, and does not give accurate information about
the genus or species of the organism. The nuclear rDNA internal
transcribed spacer (ITS) region is the crucial fungal barcode marker
used to identify distinct strains and analyze fungal diversity in a
sample because the sequence (ITS) can be easily amplified from most
DNA samples using universal primers.29 This method is a precise
technique for fungal identification and has the highest probability of
identifying the widest range of fungi. GOx was produced from
Aspergillus fumigatus AFS4 under a submerged fermentation system
with a specific activity of 409 U/mg protein (Table 1). Sixty percent
(60%) ammonium sulfate saturation was suitable to precipitate GOx
with a specific activity of 602U/mg. Zia et al.30 reported 60 to 85%
ammonium sulfate saturation as suitable for commercial GOx
preparation. After dialysis, the specific activity was 647 U/mg. Two
prominent peaks indicating two different isoforms of GOx were
observed after ion-exchange chromatography (Figure 3). Simpson et
al.28 reported that the intra- and extracellular fractions of GOx
contained isoenzymes. Dialysis of enzyme solutions after ammonium
sulfate precipitation can encourage ionic scrambling leading to the
formation of aggregates with incorrect ionic bond pairing.24
Aspergillus fumigatus AFS4 GOx was purified 2.6 fold with a yield
and specific activity of 8.75% and 1062 U/mg after ion-exchange
chromatography, respectively. Gel filtration purified the enzyme 2.85
fold with a percentage yield of 6.25 and a specific activity of 1167
U/mg (Table 1). The gel filtration elution profile had a single peak of
GOx activity (Figure 4).
Figure 1: Agarose gel electrophoresis of the (M) DNA marker
and (S4) amplicons.
Figure 2: Phylogenetic tree of Aspergillus fumigatus AFS4 with other species of Aspergillus
Trop J Nat Prod Res, March 2022; 6(3):438-445 ISSN 2616-0684 (Print)
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Table 1: Purification table of GOx from Aspergillus fumigatus AFS4
Purification Step
Total Protein
(mg)
Total Activity
(U)
Specific Activity
(U/mg)
Purification Fold
Activity Yield
%
Crude Enzyme
1556
636400
409
1.00
100
(NH4)2SO4 Saturation
91.9
55330
602
1.47
13.75
Dialysis
74.6
48270
647
1.58
8.75
DEAE-Cellulose column
30.1
31990
1062
2.61
8.75
Gel column chromatography
17.0
19850
1167
2.85
6.25
Figure 3: Ion-exchange chromatogram for Aspergillus fumigatus AFS4 GOx
Figure 4: Gel filtration chromatogram for Aspergillus fumigatus AFS4 GOx
Our studies on the effect of pH on GOx activity showed that, as the
pH was increased from 3 to 5.5, the GOx activity was increased
beyond which the enzyme activity decreased making 5.5 the optimum
pH (Figure 5). As a protein, GOx has many ionizable groups such as
amino and carboxyl groups. These groups contribute to the sensitivity
of the enzyme to different pH values thereby affecting the protein
conformation, enzymes, and substrate ionization state. Jithendar et
al.31 and Belyad et al.32 reported 6.0 and 7.0 as the optima pH for
glucose oxidases produced from Aspergillus niger PIL7 and
Aspergillus niger ATCC 9029, respectively. Also, Simpson et al.28
reported that GOx activity from Penicillium sp. CBS 120262 was
maximum at pH 7 and showed a wide pH profile with more than 70%
of the highest activity between a pH range of 4.9-8.9. The result of
this study is per the result of Yuan et al.12 who reported pH optimum
of 5.5 for recombinant glucose oxidase.
Also, an increase in temperature from 30 to 40oC was accompanied by
a rise in GOx activity beyond which the enzyme activity decreased
(Figure 6). The Aspergillus fumigatus AFS4 GOx was optimally active
at 40°C and maintained a high activity over a wide range of
temperatures (30-65oC) (Figure 4b). The rapid decrease in GOx
activity beyond 40 °C could be because of subunits dissociation. The
wide range of optimal temperature of activity observed with
Aspergillus fumigatus AFS4 GOx could be attributed to the tightly
bound FAD co-factor, which held the two protein subunits together.
As a dimeric enzyme,33 the concentration of GOx may affect the
enzyme stability. Subunits dissociation may play a key role in
Aspergillus fumigatus AFS4 GOx inactivation as seen in other
multimeric enzymes.34 Yuan et al.12 also, reported a temperature
optimum of 35ºC for recombinant glucose oxidase whereas Belyad et
al.32 reported 50 ºC for GOx obtained from Aspergillus niger ATCC
9029. The Michaelis Menten constant (KM) and maximum velocity
(Vmax) of glucose oxidation obtained from the Lineweaver-Burk plot of
initial velocity values at various glucose concentrations (Figure 7)
were 24 mM and 2000 µmol/min, respectively. This indicates that
Aspergillus fumigatus AFS4 GOx had a high affinity for β D-glucose
during the oxidation process. The high affinity of Aspergillus
fumigatus AFS4 GOx for D-glucose and its wide range of optimal
temperature and pH are promising properties for its application in
0
0.2
0.4
0.6
0.8
1
1.2
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
1
4
7
10
13
16
19
22
25
28
31
34
37
40
43
46
49
52
55
58
61
64
67
70
73
76
79
82
85
88
91
94
97
100
103
106
Concentration of NaCl (M)
Absorbance at 500 nm
Fraction number
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
135810 12 14 17 19 23 26 28 30 32 34 36 38 40 44 47 49 51 53 55 58 60 64
Absorbance at 750nm
Absorbance at 500 nm
Fraction numbers
GOx activity Protein
Trop J Nat Prod Res, March 2022; 6(3):438-445 ISSN 2616-0684 (Print)
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gluconic acid production, and food industry as a bio-preservative
agent. Sukhacheva et al.35 reported that Pencillium. amagasakiense
ATCC 28686 and Pencillium. funiculosum 433 glucose oxidases
showed low KM values of 5.7 mM and 3.3 Mm, respectively.
The result on the effect of metal ions on Aspergillus fumigatus AFS4
GOx activity showed that Ag2+ and Hg2+ had a remarkably inhibitory
effect on purified GOx, whereas Ca2+, Zn2+, and Mg2+ enhanced GOx
activity. Cu2+ and Co2+ had a slight inhibitory effect on GOx (Figure
8). This is in line with the report that Ag2+ and Hg2+ are inhibitors of
GOx.10,36 These inhibitors may precipitate the enzyme making it lose
its three-dimensional structure required for catalysis. Divalent metals
bind more strongly to ribose and pyrophosphate than monovalent
metals. Therefore, enzyme inhibition by divalent metal ions is greater
than the monovalent metal ions. Inhibition of GOx by Ag2+ ions may
be due to the reaction of Ag2+ with the thiol group of the enzyme
essential for catalysis, which is in proximity to the FAD-binding
region of the enzyme.28,37 A similar result was obtained for
recombinant GOx.12 on the other hand, the increase in GOx activity by
metal ions could be attributed to the ability of the ions to act as an
electron donor or Lewis acid as they may participate directly in the
catalytic mechanism of the enzyme.37
An enzyme's thermostability is the ability of molecule of an enzyme to
withstand thermal unfolding in the absence of a substrate.
Thermostability differs from thermophilicity which is the ability of
enzymes to act optimally at increased temperatures in the presence of
substrate. Thermostability studies on Aspergillus fumigatus ASF4 GOx
were carried out by incubating the enzyme at a temperature range of
37 to 60 ᵒC for 120 min. Aspergillus fumigatus ASF4 GOx retained 85
and 90 % of its initial activity at 37 and 40 ᵒC, respectively after 120
min. At 45 and 50 ᵒC, the enzyme retained more than 50% of its
activity after 120min of incubation. 25% of the original activity was
retained after 120min of incubation at 60ᵒC (Figure 9). The plot of
residual GOx activity against time resulted in multiphasic inactivation
curves as shown in Figure 9. The enzyme appeared to be stable at 30,
40, 45, and 50°C. The multiphasic inactivation curves may be due to
the formation of thermostable aggregates, recovery, and regeneration
of activity, the existence of different isoforms of GOx.38 More so, the
rapid loss of activity in the first phase on inactivation curves might be
due to the inactivation of heat-labile isoforms of GOx.
The plots of log (% residual activity) against inactivation time (t) was
linear at all temperatures tested (Figure 10). The increase in the
inactivation rate constant (k) at higher temperatures suggests less
thermal stability at a higher temperature. The enzyme was stable at
40ᵒC with a half-life of 231.03 min. However, at 60ᵒC it was less
stable and showed a half-life of 63.01 min under similar conditions
(Figures 9 and 10). Half-life (which is the time required to lose 50%
of enzyme original activity), is a crucial economic parameter in the
industrial application of enzymes. The higher the half-life value the
more stable the enzyme.39
The D-value, the decimal reduction time is defined as the time needed
for a 90% reduction of the original activity. D-value is also, one of the
parameters used in the estimation of enzyme stability. The D-value for
Aspergillus fumigatus AFS4 GOx decreased with an increase in
temperature. The D-value was minimum (209.4 min) at 60ᵒC and
maximum (1151.5 min) at 37ᵒC. The increase in D-values suggests a
rise in the stability of Aspergillus fumigatus ASF4 GOx. The Z-value
was obtained from Figure 11. The low Z-value suggests that
Aspergillus fumigatus AFS4 GOx was stable and more sensitive to a
rise in temperature than the period of heat treatment.40 High activation
energy value (70 KJ/mol/K) (obtained from Figure 12) suggests a
greater sensitivity of GOx to change in temperature.
Thermodynamic parameters including Gibb's free energy, enthalpy,
and entropy are the tools used to analyze the heat stability of enzymes.
They give information on the secondary stabilization and
destabilization effects that may not be captured by the kinetic
parameters. These parameters provide precise proof of the unfolding
of protein during heat inactivation.41 The results showed that enthalpy
of inactivation at 37, 40, 45 50, and 60 °C were 68.06, 68.03, 67.99,
67.94, and 67.86 KJ/mol, respectively (Table 2). A slight decrease in
enthalpy from 68.06 to 67.86 KJ/mol, was observed with a
temperature increase from 37 to 60 °C.
Figure 5: Effect of pH on Aspergillus fumigatus ASF4 GOx
activity
Figure 6: Effect of temperature on Aspergillus fumigatus
ASF4 GOx activity
Figure 7: Lineweaver-Burk plot of initial velocity data at
various glucose concentrations
Figure 8: Effect of divalent metal ions on GOx activity
0.00
200.00
400.00
600.00
800.00
1000.00
1200.00
1400.00
1600.00
1800.00
0 2 4 6 8 10
Glucose oxidase (μmol/min)
pH
0.00
200.00
400.00
600.00
800.00
1000.00
1200.00
1400.00
1600.00
020 40 60 80 100
Glucose oxidase activity
(μmol/min)
Teperature (°C)
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
-10 0 10 20 30 40 50 60
1/V (µmol/min)-1
1/[S] (mM)-1
0.00
20.00
40.00
60.00
80.00
100.00
120.00
140.00
160.00
Relative GOx activity (%)
0.01M 0.02M 0.05M
Trop J Nat Prod Res, March 2022; 6(3):438-445 ISSN 2616-0684 (Print)
ISSN 2616-0692 (Electronic)
443
© 2022 the authors. This work is licensed under the Creative Commons Attribution 4.0 International License
The enthalpy of denaturation is the quantity of energy needed to
denature the enzyme. Change in enthalpy is a measure of the number
of bonds broken during inactivation and indicates whether the
inactivation process is endothermic or exothermic.42
Also, the relatively low enthalpy value reflects the enzymes' resistance
nature, while increased values represent a response to protein
denaturation.41 The positive values of ΔH obtained in this research
suggest that the oxidation of glucose by Aspergillus fumigatus ASF4
GOx was an endothermic reaction. Positive and large enthalpy values
suggest high enzyme thermal stability.39 Therefore, for Aspergillus
fumigatus ASF4 GOx to catalyze endothermic reaction suggests that it
was able to withstand the heat energy supplied into the reaction
system. Gibb's free energies were 90.04, 89.91, 90.64, 91.53, and
92.31 KJ/mol at the respective temperatures studied (Table 2). There
was an increase in ΔG with a rise in temperature, which was
maximum at 60 °C (92.31) KJmol-1. The ΔG increased with a rise in
temperature indicating that Aspergillus fumigatus ASF4 glucose
oxidase has the resistance against thermal unfolding at a higher
temperature.41 The Gibbs free energy (ΔG) tells about how spontaneity
a reaction is. The positive values of Gibb’s free energy obtained in this
research suggest that glucose oxidation to gluconolactone and H2O2 is
a non-spontaneous process as high energy is required for the process
to occur. The ability of Aspergillus fumigatus ASF4 glucose oxidase to
catalyze the oxidation process suggests that it was able to withstand
the high energy needed for the process, indicating that it is thermally
stable.39
Entropy (ΔS) shows the net enzyme and solvent disorder. Entropies
obtained at all temperatures were negative. At 60 °C, the entropy of
the system was found to be -0.071Jmol-1K-1 (Table 2). A negative ΔS°
value indicates a decrease in the disorderliness of the enzyme solution
during denaturation. The low level of disorderliness suggests that
Aspergillus fumigatus ASF4 glucose oxidase was stable at all
temperature tested.40 The negative values of ΔS obtained at all
temperatures suggest a rise in the order of the system via (a) protein
aggregation that involves the formation of few intra-/inter molecular
bonds (b) compaction of enzymes around enzyme molecules leading
to the formation of charged particles around the enzyme molecule and
the ordering of solvent molecules.43,44,45,46 Hydrophobic interactions
are the stabilizing factor that allows enzymes to retain their structures
at high temperatures. These interactions reduce the disorderliness
(entropy) of the system.13,25
Figure 9: Plot of percentage residual glucose oxidase activity
against time
Figure 10: Plot of ln(%residual glucose oxidase activity)
against time.
Figure 11: Temperature dependence of the decimal reduction
time
Figure 12: Arrhenius plot for thermal inactivation of
Aspergillus fumigatus AFS GOx
0
20
40
60
80
100
120
020 40 60 80 100 120
Residual GOx activity (%)
Time (min)
37oC 40oC 45oC
50oC 60oC
2.5
3
3.5
4
4.5
5
020 40 60 80 100 120 140
Ln (% residual activity)
Time (min)
37 ͦC 40 ͦC 45 ͦC 50 ͦC 60 ͦC
2.2
2.3
2.4
2.5
2.6
2.7
2.8
2.9
3
3.1
3.2
35 40 45 50 55 60 65
Log D
Temperature (ᵒC)
-7
-6
-5
-4
-3
-2
-1
0
0.00295 0.003 0.00305 0.0031 0.00315 0.0032 0.00325
Ln Kd
1/T
Trop J Nat Prod Res, March 2022; 6(3):438-445 ISSN 2616-0684 (Print)
ISSN 2616-0692 (Electronic)
444
© 2022 the authors. This work is licensed under the Creative Commons Attribution 4.0 International License
Table 2: Kinetics and thermodynamic parameters of thermal inactivation of Aspergillus fumigatus AFS GOx
Temperature (ᵒC)
Kd(min-1)
t1/2 (min)
D-value (min)
∆Hº(D) KJ/mol
∆Gº(D) KJ/mol
∆Sº(D) KJ/mol
37
0.002
346.55
1151.5
68.06
90.04
-0.071
40
0.003
231.03
767.7
68.03
89.91
-0.070
45
0.004
173.28
575.8
67.99
90.64
-0.071
50
0.005
138.62
460.6
67.94
91.53
-0.073
60
0.011
63.01
209.4
67.86
92.31
-0.076
Z-value (ᵒC)
0.300
Ea KJ/mol/K
70.64
Conclusion
The optimal pH and temperature for oxidation of glucose into gluconic
acid and hydrogen peroxide by GOx locally isolated from Aspergillus
fumigatus ASF4 were 5.5 and 40°C in the presence of Ca2+, Mg2+, and
Mn2+. Thermal stability studies showed that glucose oxidation by
Aspergillus fumigatus ASF4 GOx was reversible (ΔS<0), endothermic
(ΔH>0), and non-spontaneous (ΔG>0) at all temperatures tested. Also,
This paper presents for the first time, the kinetic properties of a heat-
stable glucose oxidase obtained from Aspergillus fumigatus, with a
high level of activity suitable for industrial production of gluconic acid
and hydrogen peroxide. These results would help in determining the
heat-stability and possibly economic sustainability of Aspergillus
fumigatus ASF4 glucose oxidase in the production of gluconic acid,
bio-preservation of food, and non-food systems.
Conflict of interest
The authors declare no conflict of interest.
Authors’ Declaration
The authors hereby declare that the work presented in this article are
original and that any liability for claims relating to the content of this
article will be borne by them.
Acknowledgments
I sincerely thank Prof. F. C. Chilaka of the Department of
Biochemistry, University of Nigeria, Nsukka for providing all the
material/resources used for this research.
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Immobilization process is used to facilitate the enzyme recovery and reusability. The aims of this study were to evaluate the effect of different amounts of chitosan on immobilization efficiency of Glucose Oxidase (GOX), pH effect on enzyme activity and changes of kinetic characteristics of enzyme immobilized on Magnetic Chitosan Nanoparticles (MCNP). GOX was immobilized on MCNP with cross bonds and Schiff base covalent connections. The sample with 0.3 g of chitosan had the highest immobilization efficiency (41.30%). Morphology of nanoparticles was investigated by Scanning Electron Microscopy (SEM). Immobilization of GOX on magnetic nanoparticles was verified with Fourier Transform Infrared (FTIR). The effect of pH on enzyme activity was similar on both immobilized enzyme (IE) and free enzyme (FE). The evaluation of kinetic parameters for IE and FE has showed that the effectiveness factor was 0.45. Generally, it could be concluded that magnetic nanoparticles with 0.3 g chitosan could provide a suitable medium for immobilization of GOX.
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