International Journal of Biological Macromolecules 126 (2019) 820–827

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International Journal of Biological Macromolecules

journal homepage: http://www.elsevier.com/locate/ijbiomac

Pectin hydrolysis in cashew apple juice by Aspergillus aculeatus URM4953

polygalacturonase covalently-immobilized on calcium alginate beads:
A kinetic and thermodynamic study
Jônatas de Carvalho Silva a,1, Pedro Renann Lopes de França b,1, Attilio Converti c, Tatiana Souza Porto a,b,⁎
a
Northeast Biotechnology Network, Federal Rural University of Pernambuco, Rua Dom Manuel de Medeiros, s/n - Dois Irmãos, Recife 52171-900, Pernambuco, Brazil
b
Federal Rural University of Pernambuco, Academic Unity of Garanhuns, Avenida Bom Pastor, s/n - Boa Vista, Garanhuns 55292-270, Pernambuco, Brazil
c
Department of Civil, Chemical and Environmental Engineering, Pole of Chemical Engineering, Genoa University, via Opera Pia 15, Genoa 16145, Italy

a r t i c l e i n f o a b s t r a c t

Article history: The kinetics and thermodynamics of pectin hydrolysis in cashew apple juice by polygalacturonase (PG) from As-
Received 10 September 2018 pergillus aculeatus URM4953 covalently-immobilized on calcium alginate beads were investigated. Immobilized-
Received in revised form 28 November 2018 PG activity in cashew apple juice was the highest at 20 °C, showing a maximum hydrolysis rate of 58.2
Accepted 25 December 2018
mg/mL·min, a catalytic constant of 166.2 s−1 and an affinity constant of 113.0 mg/mL. Since the enzyme exhibited
Available online xxxx
an allosteric behavior, the hydrolysis rate was modeled, with excellent accuracy, by the Hill Equation as function
Keywords:
of pectin concentration. The Hill coefficient increased from 3 to 5 with increasing temperature from 20 to 50 °C,
Cashew apple juice evidencing a positive cooperativity mechanism. The reaction activation energy and the standard enthalpy varia-
Pectin hydrolysis tion of enzyme unfolding were 80.3 and 16.6 kJ/mol, respectively. Consistently with the kinetic results, PG-
Polygalacturonase catalyzed pectin hydrolysis proceeded with maximum spontaneity at 20 °C, showing activation Gibbs free energy,
Thermodynamics enthalpy and entropy of 59.3 kJ/mol, 77.9 kJ/mol and 63.4 J/mol·K, respectively. Immobilized PG was successful in
Kinetics the hydrolysis of cashew apple juice pectin, requiring a low temperature to act optimally.
© 2018 Elsevier B.V. All rights reserved.

1. Introduction Polygalacturonases (EC 3.2.1.15; EC 3.2.1.67) are the most

The cashew tree (Anacardium occidentale) is native to Brazil, specif-
ically to Caatinga biome; however, nowadays, it is widespread in several
countries of Asia, Africa and Central America [1]. It produces a fruit
marketed around the world (nut), whose pseudofruit (peduncle),
known as the cashew apple, accounts for 90% of the weight and is an
added-value byproduct of cashew nut processing industry. Because of
its contents of pectin (0.11–0.16%), vitamins, sugars, minerals, amino
acids, dietary fiber, ascorbic acid, carotenoids, phenolic acids, flavonoids
and tannins, it is in fact considered a functional food with high antioxi-
dant activity [2,3].
Cashew apple juice is widely consumed in Brazil, but its increased
consumption abroad depends on the ability to technologically improve
its processing [4]. Some researchers have reported methods to clarify
juice making use of microfiltration [5] and clarifying agents [6]. How-
ever, research about enzymatic hydrolysis of juice pectin is still too
scarce.
⁎ Corresponding author at: Federal Rural University of Pernambuco, Academic Unit of
Garanhuns, Garanhuns PE 55292-270, Brazil.
E-mail address: tatiana.porto@ufrpe.br (T.S. Porto).
1
Contributed equally to the paper.
important enzymes used for breaking down pectin, a complex
heteropolysaccharide constituted of long molecules and rich in α-
D-galacturonic acid [7,8]. These enzymes are of great commercial
interest because of their hydrolytic action on the polymer chain
that breaks down the α(1–4) glycoside bonds [9]. Despite being
widely used in food industries to extract and clarify wines and
fruit juices [10,11], ferment coffee and tea [12] and extract vegeta-
ble oils [13], they can be used for fiber degumming [14], in paper in-
dustry [15] and for wastewater treatment [16].
However, free enzymes cannot be recovered for multiple use in in-
dustrial processes [17]. Immobilization makes biocatalyst application
and reuse easier and allows lowering costs. Some additional advantages
of enzyme immobilization are increased enzyme activity, easy enzyme
recovery after centrifugation or filtration, heterogeneity of the system,
rapid stop of the reaction, continuous operation, waste reduction, and
possibility to carry out several enzymatic processes [18]. Among the sev-
eral supports adopted for enzyme immobilization, alginate, a biodegrad-
able polysaccharide, is widely used because of its good biocompatibility,
low cost, easy availability and simple preparation [19]. In previous study,
immobilization of Aspergillus aculeatus polygalacturonase in calcium al-
ginate beads was optimized, which allowed preserving N90% of its initial
activity. Moreover, the use of calcium alginate as a support allowed four

https://doi.org/10.1016/j.ijbiomac.2018.12.236
0141-8130/© 2018 Elsevier B.V. All rights reserved.
 J.C. Silva et al. / International Journal of Biological Macromolecules 126 (2019) 820–827
successive reuses and high thermal stability of immobilized enzyme
[20].
Cashew apple is a nutritive fruit that needs special attention during
processing because its chemical composition and sensorial taste may
be affected by physical, chemical, biochemical and microbiological fac-
tors [3]. Then, the search for enzymes with special features such as
low optimum temperature is very important to ensure a successful pec-
tin hydrolysis without affecting the juice nutritional value.
Many researches reported the biochemical features of industrial en-
zymes, which provide information on their long-term activity [15], bio-
technological potential [21] and behavior under non-experimentally
tested conditions [22]. However, investigations on pectinases were
mostly performed using high-purity citrus pectin or galacturonic acid
as substrates. Moreover, in most kinetic studies on pectin hydrolysis,
the pectic material was preliminarily isolated from fruit juices, which
would be complex and costly in a real-life scenario [23]. Such isolation
procedures consisted of different extraction techniques using enzymes,
organic solvents, microwave or ultrasound assistance, subcritical water,
and induced electric field [24].
The enzyme-based extraction process of pectin hydrolysis can be
performed by either fungal or bacterial strains, since various of them
are known to be effective producers of pectinases with different cata-
lytic properties. Whereas the Aspergillus genus is mostly used for
pectinase production in acidic preparations such as fruit juices, bacterial
strains produce alkaline pectinases [25].
Many factors can influence and change enzyme behavior in real pro-
cesses such as inhibitors, ions and carbohydrates present in the me-
dium, which may enter the catalytic site or react with the enzyme
structure, decreasing its activity [26,27]. Moreover, knowledge of ki-
netic and thermodynamic parameters, besides reaction mechanism
and behavior, are required to design industrial reactors successfully.
Based on this background, the present work aimed to deter-
mine the kinetic and thermodynamic parameters of pectin hydro-
lysis in cashew apple juice by polygalacturonase from A. aculeatus
URM4953 covalently-immobilized in calcium alginate beads.
2. Materials and methods
2.1. Microorganism
Aspergillus aculeatus URM4953 was obtained from the URM
(University Recife Mycology) culture collection of the Federal University
of Pernambuco, Recife, Brazil, affiliated to the World Federation for
Culture Collections (WFCC), N° 604. It was maintained in mineral oil,
reactivated in nutrient solution (1% peptone, 2% glucose and 0.3% yeast
extract) and grown at 30 °C under shaking at 120 rpm for 72 h. Samples
of the fungus were transferred to 125 mL-Erlenmeyer flasks containing
Czapek medium and incubated at 30 °C for 7 days to induce sporulation.
The spores were then collected after addition of 3.0 mL of NaCl (0.9%)
and Tween 80 (0.01%) sterilized solution to the flasks. The concentration
of spores was determined using a Neubauer chamber [7].
2.2. Polygalacturonase production by submerged fermentation
Polygalacturonase (PG) was produced by A. aculeatus URM4953 in
submerged fermentation on a medium containing flour of passion
fruit peel as the main carbon source, which was prepared according to
Fontana et al. [28]. A 3.0% (w/v) suspension of flour of passion fruit
peel prepared in deionized water was autoclaved for 20 min at 121 °C
to extract pectin and reducing sugars present in the flour. The average
pectin concentration after extraction was found to be 10.48 ±
0.16 mg/mL. The mixture was then filtered to remove suspended solids,
and salts – 0.7 mM (NH4)2SO4, 0.8 mM MgSO4.7H2O and 5.0 mM
K2HPO4 – and 10 mg/mL yeast extract were added to the medium,
which was finally sterilized in autoclave under the same conditions as
above. After inoculation of 105 spores/mL, fermentations were carried
821
out at 30 °C in 250 mL-Erlenmeyer flasks containing 50 mL of medium,
under shaking at 130 rpm for 96 h. The fermented broth was then fil-
tered and centrifuged for 5 min at 4000 rpm. The supernatant was called
crude enzyme extract and stored frozen (−20 °C).
2.3. Polygalacturonase activity assay
PG activity upon immobilization was determined by the method of
Miller [29]. Briefly, 500 μL of the crude extract or 500 μg of PG-
containing calcium alginate beads were incubated with 500 μL of
10 mg/mL citrus pectin in acetate buffer, pH 4.5, at 50 °C for 40 min in
a water bath. A 100 μL-aliquot was removed and added to 1.0 mL of a so-
lution made up of 0.1 mg/mL dinitrosalicylic acid, 0.4 M NaOH and
1.55 M sodium tartrate. The mixture was boiled for 5 min and cooled
in an ice bath. Distilled water (5.0 mL) was added, and the mixture
was homogenized. The reduction of 3,5-dinitrosalicylic acid to 3-
amino-5-nitrosalicylic acid was followed by measuring the absorbance
at 540 nm with a UV–Vis spectrophotometer (SP-1105, Spectrum, Curi-
tiba, PR, Brazil). The blank was the same reaction medium described
above, where the crude extract or PG-containing calcium alginate
beads was replaced by distilled water. Data were plotted on a standard
curve (y = 0.0166 + 0.0842 x; R2 = 0.999) of absorbance (y) versus
concentration of α-D-galacturonic acid (x) as a reducing sugar
(mg/mL). One unit of pectinase activity was defined as the amount of
enzyme required to release 1 μmol of galacturonic acid per minute [7].
2.4. Covalent immobilization of polygalacturonase in Ca alginate beads
Immobilization of PG was conducted using calcium alginate as a sup-
port and glutaraldehyde as a cross-linking agent. A 0.09 M sodium algi-
nate solution was slowly syringed dropwise into a 0.3 M calcium
chloride solution under stirring. Formed beads were hardened in the
same solution for 30 min at 4 °C and washed with deionized water. To
obtain a cross-linked network, calcium alginate beads were submerged
in 0.2 M glutaraldehyde solution for 2 h at 25 ± 1 °C and stirred at
120 rpm. The cross-linked beads were then washed with deionized
water, and the complete removal of glutaraldehyde was checked spec-
trophotometrically at 245 nm. Then, they were added to a crude extract
solution (0.011 mg of protein per mL) up to a ratio of 1.0 g of beads per
5 mL of enzyme solution and mixed for 45 min at 25 ± 1 °C and
120 rpm. PG-loaded beads were washed with deionized water, dried
in sieve and conditioned at 4 °C in hermetically sealed containers. The
immobilization yield, calculated as the percentage of immobilized PG
activity compared to the extract activity determined as described in
Section 2.3, was found to be 95%.
2.5. Kinetics of pectin hydrolysis in cashew apple juice
Cashew apple juice was obtained by processing the fruits in a mixer,
followed by sieving to remove the remaining residues. The cashew
apple juice was then stored at −20 °C until use. Pectin hydrolysis was
performed by mixing in 250 mL-Erlenmeyer flasks 1.0 g of the PG-
loaded calcium alginate beads per 5.0 mL of cashew apple juice, under
stirring at 130 rpm (TE-139, Tecnal, Piracicaba, SP, Brazil) for 2 h in a
water bath. To find the optimum temperature of pectin hydrolysis in
cashew apple juice, immobilized PG activity was investigated in the
temperature range from 10 to 50 °C. For comparison purposes, the
rate of reaction was expressed as percent relative activity with respect
to its maximum value.
The rate of reaction (v) was determined by the equation:
½S f −½Si
v¼ ð1Þ
t f −t 0
where [S]i is the initial reducing sugar concentration at the start of
reaction (to) and [S]f the final one detected after 120 min of reaction
822 J.C. Silva et al. / International Journal of Biological Macromolecules 126 (2019) 820–827
(tf). Runs were performed in triplicate, and the results of substrate con-
centration expressed as means ± standard deviations.
The yield of pectin hydrolysis (Yh) was determined from the reduc-
ing sugar concentration along a period of 120 min, at time intervals of
10 min, by the equation:
½S f −½Si
Y h ð%Þ ¼  100
½S i
2.6. Physicochemical characterization
Total soluble solids (TSS) were measured using a hand-held refrac-
tometer and expressed in °Brix. pH was measured using a digital pH
meter (Tecnal, Piracicaba, SP, Brazil). Color of cashew apple juice was
determined by measuring the absorbance at 420 nm [30] using the
same UV–Vis spectrophotometer mentioned above. All the experiments
were performed in triplicate.
2.7. Determination of pectin concentration
Pectin concentration in the cashew apple juice was determined by
the method of McComb and McCready [31] with some modifications.
Aliquots of 50 μL of cashew apple juice were dissolved in 0.05 M
NaOH solution for de-esterification. Afterwards, the mixtures were dis-
solved in 6.0 mL of concentrated H2SO4 previously cooled in ice bath.
The reaction was carried out in boiling water bath for 10 min. After
that, 500 μL of 1.5 mg/mL carbazole were added. The mixture was stabi-
lized for 30 min at room temperature (25 ± 1 °C), and the reaction of
galacturonic acid with carbazole was followed measuring the absor-
bance at 520 nm using the above UV–Vis spectrophotometer. Pectin
concentration (mg/mL) was expressed in reducing sugars according to
the calibration curve y = 0.3429 x – 0.1911 (R2 = 0.996) using α-D-
galacturonic acid as a standard, where y is the absorbance and x the re-
ducing sugar concentration. All the experiments were performed in
triplicate.
3. Theory
3.1. Kinetic and thermodynamic parameters of pectin hydrolysis in cashew
apple juice
To kinetically characterize the immobilized enzyme, the kinetic pa-
rameters of pectin hydrolysis in cashew apple juice (5.0 mL) by PG-
loaded beads (1.0 g) were determined at different temperatures (20, 40
and 50 °C), using pectin concentrations in the range of 27–190 mg/mL.
Unlike multimeric enzymes, some monomeric enzymes, like that
under consideration, exhibit a non-Michaelis-Menten kinetic response,
i.e., a deviation from the hyperbolic curve leading to a sigmoidal-type
trend that can be modeled according to Hill [32]. Such a model assumes
that an enzyme can have multiple substrates or ligand binding sites and
can suffer a conformational or electrical change when bound to one sub-
strate molecule, hence altering the affinity of the other available cata-
lytic sites and leading to a so-called cooperativity [23].
According to Porter and Miller [33], cooperativity modulates differ-
ent mechanisms of regulation and influences both thermodynamic
and kinetic properties of enzymes with multiple binding sites. Specifi-
cally, thermodynamics is influenced through a ligand affinity change
due to binding of another ligand molecule to another site. Nonetheless,
cooperativity may also take place in individual binding sites of mono-
meric units when they constitute higher-order structures.
Pectin hydrolysis by PG can be described by the classical general
scheme as:
E þ nS↔ES→E þ P:
where E is the enzyme, S the substrate, n the Hill coefficient that corre-
sponds to the number of ligand sites and reflects the cooperativity, ES
the enzyme-substrate complex and P the product.
Assuming steady state, the fraction of occupied ligand-binding sites
(Ɵ) is defined as the ratio of ES concentration to the total enzyme con-
centration in free and complexed forms [34]:
ð2Þ ½ES
θ¼ ð4Þ
½ES þ ½E
while the ES association equilibrium constant (Ka) as [35]:
½ES
Ka ¼ ð5Þ
½E½Sn
Since the higher [ES] the higher Ka, such a constant can be also as-
sumed as an enzyme affinity index.
Substituting [ES] present in Eq. (4) into Eq. (5), rearranging and ex-
pressing in logarithmic form, one obtains the linearized Hill equation:


θ
log ¼ n log½S− logK d ð6Þ
1−θ
where Kd = 1/Ka is the ES dissociation equilibrium constant.
However, a modified version of the Michaelis–Menten equation,
based upon the formulation first developed by Hill, is preferred when
cooperativity occurs:
V max ½Sn
v¼ n ð7Þ
k0:5 þ ½Sn
or its linearized form:


v
log ¼ n log½S−n logk0:5 ð8Þ
V max −v
where Vmax is the maximum reaction rate and k0.5 the affinity constant,
i.e. the substrate concentration required to achieve half-maximum reac-
tion rate.
These kinetic parameters and n were calculated by fitting, using the
Origin 8.0 software (OriginLab, Northampton, MA, USA), the experi-
mental data of reaction rate versus pectin concentration according to
Eq. (7).
The total concentration of the enzyme covalently-immobilized in
calcium alginate beads (Eo) was calculated as the difference between
protein concentrations before and after immobilization, which were ex-
perimentally determined according to the Bradford [36] method.
As for any enzyme reaction [20], also pectin degradation to
galacturonic acid occurs at a rate depending on collisions between
pectinase and pectin molecules. However, according to the Arrhe-
nius theory [37], this reaction can start only if the molecules have a
minimum energy sufficient to activate it, which is called activation
energy (E*). PG reduces E* of pectin degradation, hence accelerating
the reaction, but E* does not exert any influence on the Gibbs energy
or the equilibrium constant at a given temperature. For enzyme sys-
tems like this, at temperature lower than the optimum, the Arrhe-
nius equation relates the initial specific rate of enzyme reaction
(ko), which is directly proportional to the initial enzyme activity, to
the collision frequency (Ao) and the portion of collisions that have
the minimum energy to react ðe−RT Þ:
E
ko ¼ Ao e−RT
E
ð9Þ
where R is the gas constant and T the absolute temperature.
As explained in more details elsewhere [38], any enzyme can be sup-
posedly subject to a simultaneous unfolding equilibrium, which is shifted
ð3Þ to the right side at temperatures higher than the optimum. So, the
 J.C. Silva et al. / International Journal of Biological Macromolecules 126 (2019) 820–827
contribution of the unfolded form of the enzyme becomes predominant,
and the process is governed by the equation:
! activity at only 20 °C, which would allow reducing the process costs
Ao ΔHu° −E
k0 ¼ exp ð10Þ
B RT
where ΔH°u is the standard enthalpy variation of the enzyme unfolding
equilibrium and B an additional pre-exponential factor.
E* and ΔH°u were estimated in this study from the slopes of the right
and left straight lines, respectively, which were obtained by semi-log
plots of the starting enzyme activity, detected in cashew apple juice
with pectin concentration of 163 mg/mL, versus 1/T in accordance to
the methodology described in Section 2.5.
Concepts such as activation free energy (ΔG*), enthalpy (ΔH*) and
entropy (ΔS*) are the cornerstones of understanding various biological
processes such as enzyme reaction, protein folding and interaction [39].
Eyring [40] proposed a theory based on the Arrhenius one, which al-
lows estimating these thermodynamic parameters from the so-called
catalytic constant (kcat), defined as Vmax/Eo, through the equations:
ΔH  ¼ E −RT ð11Þ
kB T −ΔG
kcat ¼ e RT ð12Þ
h
ðΔH  −ΔG Þ
ΔS ¼ ð13Þ
T
where kB and h are the Boltzmann and Plank constants, respectively.
4. Results and discussion
4.1. Optimum temperature and kinetic parameters of pectin hydrolysis
A previous characterization study showed an optimum temperature
of 50 °C for the activity of free polygalacturonase (PG) produced by As-
pergillus aculeatus URM4953 in citrus pectin [20]. Its immobilization in
calcium alginate beads lowered it to 40 °C but ensured the retention
of N90% of its initial activity after 60 min at this temperature. Moreover,
the thermodynamic parameters of immobilized PG thermoinactivation
suggested a predominating mechanism of reversible unfolding, permit-
ting four successive 40-min cycles of immobilized enzyme utilization
with only 33% of activity loss after exposition at 50 °C.
To find the best conditions for pectin hydrolysis in cashew apple
juice, the reaction was investigated at temperatures in the range of
10–50 °C, whose results are illustrated in Fig. 1 in terms of relative activ-
ity compared to the maximum one.
Fig. 1. Relative activity of pectin hydrolysis by Aspergillus aculeatus URM4953
polygalacturonase covalently-immobilized in calcium alginate beads versus temperature.
Tests were carried out in cashew apple juice at pectin concentration of 163 mg/mL and
pH 4.7.
823
Contrary to any expectation, pectin hydrolysis in cashew apple juice
by PG covalently-immobilized in calcium alginate displayed maximum
even more than in the case of citrus pectin. In addition, as mentioned
above, since cashew apple contains several thermolabile bioactive com-
pounds such as vitamins, sugars, amino acids, ascorbic acid, carotenoids,
phenolic acids, flavonoids and tannins [3], the possibility of working
practically at room temperature (25 ± 1 °C) would allow preserving
its high nutritional value and avoiding, at the same time, undesired
taste of cooked food. Moreover, the immobilization process allows re-
covering the biocatalyst for successive reuses. However, unlike citrus
pectin, cashew apple juice contains a lot of others compounds that can
influence pectin hydrolysis by PG.
Such a shift in the optimum temperature may have been due to con-
formational changes suffered by the protein on its surface during cova-
lent immobilization with glutaraldehyde [41], which may have been
responsible for alterations in catalytic site behavior, less accessibility
of inhibitors present in the crude extract, and reduced rigidity of the en-
zyme structure [42].
Polygalacturonases are known to have optimum temperature
around 50 °C [15,43,44]. However, PGs of some Aspergillus species and
strains exhibited lower optimum temperatures, e.g. Aspergillus niger
(37 °C) [45], Aspergillus flavus MTCC 7589 (40 °C) [46], Aspergillus
ibericus (40 °C) [16], or higher optimum temperature, e.g. Aspergillus
fumigatus LB-01-AP (60 °C) [47].
Moreover, since the high pectin content of cashew apple juice
may have a great influence on the hydrolysis kinetics, a series of
tests were carried out varying pectin concentration in the range of
27–190 mg/mL at the three optimal temperatures a) for pectin hy-
drolysis in cashew apple juice by immobilized PG (20 °C) and for cit-
rus pectin hydrolysis by either b) immobilized (40 °C) or c) free PG
(50 °C) [20]; the results of these tests are illustrated in Fig. 2.
It is noteworthy that the kinetic profile deviated from the typical
Michaelis-Menten kinetics, exhibiting a sigmoidal trend. Because the
Michaelis–Menten equation was unable to satisfactorily describe the
dependence of the hydrolysis rate on substrate concentration [33],
data were fitted by the Hill equation (Eq. (7)) (Fig. 2) with high values
of the coefficient of determination (Table 1).
As is well known, cooperativity of allosteric enzymes involves multi-
ple substrates or binding sites. They can suffer either structural or elec-
tronic changes, i.e., the polarization is influenced by the dielectric
constant [48], which may result in an altered substrate-binding affinity
of the remaining empty sites. However, cooperativity can also occur in
monomeric proteins with a single binding site or without macromolec-
ular oligomerization [23,49].
Fig. 2. Influence of pectin concentration on the rate of pectin hydrolysis in cashew apple
juice by polygalacturonase from Aspergillus aculeatus URM4953. Solid lines represent
fitting curves drawn according to Hill equation.
824 J.C. Silva et al. / International Journal of Biological Macromolecules 126 (2019) 820–827
Table 1
Kinetic parameters of pectin hydrolysis in cashew apple juice by polygalacturonase from
Aspergillus aculeatus URM4953 covalently-immobilized in calcium alginate beads.
Temperature Vmax (mg/mL·min)a k0.5 kcat nd R2
(°C) (mg/mL)b (s−1)c
20 58.2 113.0 166.2 3 0.999
40 34.3 101.0 98.0 4 0.995
50 11.3 89.7 32.1 5 0.999
a juice
Maximum hydrolysis rate.
b
Affinity constant.
c
Catalytic constant.
d
Hill coefficient.
Even though polygalacturonases are usually described in the litera-
ture as monomeric proteins, with a parallel β-helix and three distinct
β-sheets made up of 8 to 10 complete β-strand turns [15], many of
them from different sources have at least two substrate-binding regions
[50].
According to Ninga et al. [23], the allosteric effect of immobilized PG
can be explained by the presence of some naturally-occurring com-
pounds that can act either as activators or inhibitors with different pos-
sible mechanisms of pectin cleavage, being responsible for such a
deviation. In one case, pectin hydrolysis in different regions of the
glycose bonds can lead to various products. Alternatively, pectin may
bind to PG active site releasing a unique monomer, while the oligomer
may form either a new non-productive complex acting as an inhibitor
for other substrates or a productive one leading to the release of other
products. Another possibility is that substrate binds to the enzyme at a
given binding site forming a non-productive complex, thereby leading
to substrate inhibition, whereas its binding to another subsite may
lead to a productive complex.
The values of the affinity constant (k0.5), catalytic constant (kcat), Hill
coefficient (n) and maximum reaction rate (Vmax) of pectin degradation
by immobilized PG at different temperatures are listed in Table 1.
The Hill coefficient, whose values were estimated with excellent ac-
curacy (0.995 ≤ R2 ≤ 0.999) from Hill plots (Eq. (7)) using the experi-
mental data of Fig. 2, progressively increased from 3 to 5 with
increasing temperature from 20 to 50 °C (Table 1), highlighting increas-
ing positive cooperativity. Such a cooperativity index does in fact reflect
the number of ligand-binding sites present in the macromolecular as-
sembly that are subject to thermodynamic cooperativity [23,51].
When n = 1, the system is classified as non-cooperative, and the Hill
formalism simplifies to the standard Michaelis–Menten equation; on
the other hand, when n b 1 the system has negative cooperativity be-
cause substrate association slows down the reaction, whereas the oppo-
site takes place when n N 1 [33].
According to Cárdenas et al. [52], positive cooperativity may be due
to a mechanism involving a high- and a low-affinity conformations of
the enzyme. The equilibrium in the absence of substrate strongly favors
the low-affinity conformation, while, when substrate binds to the en-
zyme, two behaviors can be observed. At high substrate concentration
catalysis occurs releasing product(s), and the high-affinity state is re-
generated for a second catalytic step, thus a hyperbolic trend character-
istic of Michaelis–Menten kinetics is observed. On the other hand, at low
substrate concentration the enzyme slowly relaxes to the low-affinity
conformation before another substrate molecule has enough time to as-
sociate with it, thus leading to failure of substrate binding and non-
Michaelis–Menten kinetic responses. This is the case observed in
Fig. 2, where the reaction was slightly accelerated by a progressive in-
crease in pectin concentration from 27 to 86 mg/mL, giving raise to typ-
ical sigmoidal curves consistent with an allosteric behavior [33,51].
One can see that a temperature reduction from 50 to 20 °C progres-
sively increased k0.5, i.e. reduced the affinity of immobilized PG for pectin,
likely because some other saccharides present in the cashew apple juice
acted as competitive inhibitors [53] forming a non-productive complex.
On the other hand, the decrease of both kcat and Vmax with increasing
temperature can be explained by immobilized PG thermoinactivation,
which is the more pronounced the higher the temperature, affecting
the enzyme reaction after 2 h of juice processing. As a result, maximum
values of both kinetic parameters (166.2 s−1 and 58.2 mg/mL·min, re-
spectively) were obtained at 20 °C (Table 1).
4.2. Physicochemical characterization of pectin hydrolysis in cashew apple
Based on these results and to make comparison possible, the reac-
tion catalyzed by immobilized-PG was followed versus time in terms
of pH, degree of pectin hydrolysis, color and content of total soluble
solids. Whereas at 20 °C the juice pH was 4.67 ± 0.01 with negligible
variations (Fig. 3, panel A), it progressively decreased to 4.52 ±
0.02 at 40 °C and 4.48 ± 0.03 at 50 °C.
Color intensity expressed as absorbance at 420 nm progressively in-
creased along the time especially at the highest temperature (Fig. 3,
panel B), likely due to the presence of polyphenol oxidases in cashew
apple juice. In the presence of oxygen, these enzymes are in fact able
to catalyze the oxidation of ortho-diphenols to ortho-quinones, which
react forming colored substances [54] and affecting food quality in
terms of color, taste and nutritional value [55]. To face this drawback,
color formation can be prevented by a bleaching treatment before
juice clarification.
The content of total soluble solids (TSS) (Fig. 3, panel C) decreased
during the hydrolysis from 8.9 to 4.4 °Brix at 20 and 40 °C and from
8.9 to 6.6 °Brix at 50 °C, which suggests that calcium alginate beads
may have exerted a fouling action adsorbing some reducing sugars
through their pores during pectin hydrolysis [56]. The same TSS decay
was observed by Oliveira et al. [57] after clarification of umbu and
apple juices with PG-entrapped in calcium alginate beads.
On the other hand, the degree of pectin hydrolysis expressed as re-
lease of reducing sugars progressively increased (Fig. 3, panel D) and
was complementary to pectin concentration. Such a behavior was qual-
itatively similar at the three selected temperatures, but was more pro-
nounced at 20 °C, because of the highest hydrolysis rate.
4.3. Thermodynamics of pectin hydrolysis in cashew apple juice
The activation energy of pectin degradation (E* = 80.3 kJ/mol) and
the standard enthalpy variation of immobilized PG unfolding (ΔHuo =
16.6 kJ/mol) were estimated from semi-log plots of the initial enzyme
activity (Ai) versus 1/T in the temperature range of 10–50 °C (Fig. 4)
using a pectin concentration of 163 mg/mL.
Such an E* value compares with those of many other pectinases, re-
membering that small values are desired in industrial applications be-
cause they kinetically favor the enzyme-catalyzed reactions and the
process energy costs are lower. On the other hand, the ΔHuo value esti-
mated in this study is lower than that (42.5 kJ/mol) previously esti-
mated for the same immobilized-enzyme preparation employed for
citrus pectin hydrolysis at concentration of 20 mg/mL [20], which
means that the unfolded form of the immobilized enzyme is more sta-
ble, i.e. that the enzyme unfolding is less pronounced. Since the
immobilized system was the same, the higher protection of the enzyme
against unfolding observed in this study could be ascribed to the differ-
ent composition of the media, which exerts an obvious influence on en-
zyme unfolding, as well as the concentration of pectin in cashew apple
juice (163 mg/mL), which was approximately 8-fold that of citrus pectin
solution (20 mg/mL). Substrate protection, which appears to be a gen-
eral feature of different classes of enzymes, was in fact already reported
to influence the thermodynamic parameters of glucose isomerase activ-
ity and inactivation [58–61]. Unfortunately, no further comparison is
possible with literature data because most of studies report E* and
ΔHuo values estimated for isolated or partially-purified citrus pectin,
which are completely different systems.
 J.C. Silva et al. / International Journal of Biological Macromolecules 126 (2019) 820–827 825

Fig. 3. Time variations of (A) pH, (B) color, (C) content of total soluble solids and (D) yield of pectin hydrolysis during the enzymatic treatment of cashew apple juice with Aspergillus
aculeatus URM4953 polygalacturonase covalently-immobilized in calcium alginate beads.

The activation Gibbs free energy (ΔG*), enthalpy (ΔH*) and entropy
(ΔS*) of pectin hydrolysis were estimated at temperatures of 20, 40 and
50 °C (Table 2). One can see that ΔG* increased with temperature,
highlighting a higher energy barrier and then a slowing down of pectin
hydrolysis in cashew apple juice like that observed by Riaz et al. [62] for
starch hydrolysis by A. niger glucoamylase.
The same rise in temperature led to a very slight reduction in ΔH*,
which means, assuming that these variations were significant, that the
enzyme-substrate activated complex was more efficient [62], consis-
tently with the increase in the Hill coefficient from 3 at 20 °C to 5 at
50 °C. These results corroborate the intuition of Atrinson et al. [63]
that the most frequently found form of cooperativity might be ex-
plained by the thermodynamics of ligand binding, since the initial asso-
ciation of a ligand alters the affinity of a subsequent binding event.
As is well known, ΔS* is correlated to the structural rigidity of the
enzyme-substrate activated complex. An increase in temperature from
20 to 50 °C led to a remarkable decrease of this thermodynamic param-
eter, from 63.4 to 23.5 J/mol.K. Whereas such a decrease suggests a
more ordered structure of the transition state than that in the higher
temperature, the positive sign of all ΔS* values indicates that the
structure of enzyme–substrate at transition state was less ordered
than in the reacting system [64,65].
5. Conclusions
Kinetics of pectin hydrolysis in cashew apple juice by Aspergillus
aculeatus URM4953 polygalacturonase (PG) covalently-immobilized in
calcium alginate beads highlighted an allosteric behavior responsible
for positive cooperativity, which was modeled by the Hill equation. De-
spite the increase in the Hill coefficient with temperature, Vmax, kcat and
k0.5 reached their highest values at 20 °C, corresponding to the highest
hydrolysis rate but, at the same time, to the lowest enzyme affinity for
pectin. In addition, physicochemical characterization of the immobilized
enzyme did not show any significant alteration of its pH profile during
120 min of catalysis. PG-loaded calcium alginate beads suffered a small
unfolding effect during cashew apple juice hydrolysis till 50 °C. Whereas
enthalpy and entropy values decreased with increasing temperature
from 20 to 50 °C, the Gibbs free energy grew, pointing out an unfavor-
able energetic situation for pectin hydrolysis. The results of this study
taken together are quite promising because they suggest the possibility

Fig. 4. Arrhenius-type semi-log plot of initial activity of polygalacturonase from Aspergillus aculeatus URM4953 in cashew apple juice versus the reciprocal temperature.
826 J.C. Silva et al. / International Journal of Biological Macromolecules 126 (2019) 820–827
Table 2
Thermodynamic parameters of pectin hydrolysis in cashew apple juice by
polygalacturonase from Aspergillus aculeatus URM4953 covalently-immobilized in calcium
alginate beads.
Temperature ΔG* (kJ/mol)a ΔH* (kJ/mol)b ΔS* (J/mol·K)c
(°C)
20 59.3 77.9 63.4
40 64.9 77.7 40.9
50 70.0 77.6 23.5
a
Activation Gibbs free energy.
b
Activation entaphy.
c
Activation entropy.
of industrially exploiting this enzyme construct to clarify cashew apple
juice avoiding additional costs related to the use of high temperatures.
Acknowledgements
Jonatas Silva would like to thank CAPES (National Council for the Im-
provement of Higher Education, Brasilia, Brazil) for the exchange schol-
arship CSF-PVE-S- (88887.122755/2016-00) and FACEPE (Foundation
for Science and Technology support of the Pernambuco State, Recife,
Brazil) for the PhD scholarship (IBPG-0514-5,07/14).
Tatiana Porto would like to thank CNPq (National Council for Scien-
tific and Technological Development, Brazil – 478446/2011-0 and
471773/2013-1). This study was financed in part by the Coordenação
de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) –
88881062200/2014-01.
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