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Process Biochemistry 121 (2022) 45–55
Available online 27 June 2022
1359-5113/© 2022 Elsevier Ltd. All rights reserved.
Biochemical characterization of a thermally stable, acidophilic and
surfactant-tolerant xylanase from Aspergillus awamori AFE1 and hydrolytic
efciency of its immobilized form
Isaac A. Olopoda
a
, Olusola T. Lawal
a
, Oluwasegun V. Omotoyinbo
b
, Adejoke N. Kolawole
a
,
David M. Sanni
a
,
*
a
Federal University of Technology, Biochemistry Department, Enzyme and Food Biotechnology Unit, P.M.B. 704, Akure, Nigeria
b
Biological Science Department, Biochemistry Unit, Wesley University Ondo, Nigeria
ARTICLE INFO
Keywords:
Xylanase
Biochemical properties
Aspergillus awamori AFE1
Longhorn beetle
Immobilized enzyme
ABSTRACT
Capital intensiveness of bioconversion of lignocellulosic biomass can be unraveled by producing stable and
highly efcient enzyme from phytophilic microbes. The present study revealed biodegradation efciency of
immobilized and free xylanase from Aspergillus awamori AFE1 and the enzyme physicochemical properties. The
crude xylanase was subjected to three step purication followed by determination of biochemical properties. The
homogenously puried xylanase had an overall 6.86-fold purication and 5.20 % yield coupled with a molecular
weight of ~ 48 kDa. Free and immobilized xylanase exhibited optimum activity at (50 ◦C, pH 4) and (70 ◦C, pH
5) respectively while thermal and pH stability for free were achieved at a range of 30–90 ◦C and pH 2–11
respectively. Xylanase was tolerant and enhanced by organic solvents, detergents and inhibitors investigated
except ethanol and Cysteine while Mn
2+
exceptionally increased xylanase activity and its inhibition by EDTA
showed requirement for divalent cations. K
m,
0.15 mM and 1 mM and V
max
, 5 ×10
3
µM/min and 1 ×10
4
µM/
min were obtained for free and immobilized xylanase. Interestingly, immobilized xylanase showed recycling
efciency of ve cycles with higher degradation efciency over solubilized xylanase. Both free and immobilized
xylanase novel characteristics showed suitability for industrial and biotechnological applications.
1. Introduction
Enzymes have become an essential requirement for biochemical
processes which are utilized in various industrial processes [1,2] due to
their role in applied biocatalysis which is increasing virtually in all
processes globally [3]. The commencement of enzyme production for
industrial use was unveiled in the nineteenth century and has grown and
spread to other areas such as: food, beverages, pharmacy and other al-
lied processing industries [4]. One of these industrially important en-
zymes is xylanase which has extensive applications in bio-bleaching of
pulp, clarication of fruit juices, textile, food, feedstock lignocellulose
hydrolysis and enhancement of breakdown of animal feed stock [5–7].
Xylanase (EC 3.2.1.8, an endo-1,4-D-xylan xylano-hydrolase) is
known for hydrolysis of glycosidic bond cleavage in xylan, the hemi-
cellulosic polysaccharide which its content is higher compared to other
lignocellulosic materials in secondary plant cell walls and most agri-
cultural products, whose backbone is consisted of a 1,4-linked D-
xylopyranose, substituted with L-arabinofuranosyl, acetyl, and glucur-
onosyl residues [8,9]. Due to different constituent of xylan-rich agri-
cultural products, various xylo-oligosaccharides of varied chain lengths
xylobiose and xylose are released in the course of degradation of the
complexmolecules [10–12] which have been reported to be involved in
the production of value-added goods such as xylitol and biofuels [13,
14].
Agro-residues are composed of lignocellulosic compounds and are
often employed in microbial fermentation (either submerged or solid
state) especially in the production of enzymes such as xylanase owing to
their ability to enhance enzyme production and yield [15,16]. The
sources of microbial enzymes are however very crucial because they
determine their properties while they could also be used for the basis of
classication of microbes either as extremophilic (microbes which
thrives in extreme conditions), thermophilic (microbes that thrive at
50 ◦C and above), psychrophilic (microbes that thrive in cold temper-
ature), alkalophilic (the ones that thrive at alkaline pH) or acidophilic
* Corresponding author.
E-mail addresses: lawalsola2015@gmail.com (O.T. Lawal), dmsanni@futa.edu.ng, moraksanni@futa.edu.ng (D.M. Sanni).
Contents lists available at ScienceDirect
Process Biochemistry
journal homepage: www.elsevier.com/locate/procbio
https://doi.org/10.1016/j.procbio.2022.06.030
Received 22 March 2022; Received in revised form 30 May 2022; Accepted 24 June 2022
Process Biochemistry 121 (2022) 45–55
46
(The ones that thrive at acidic pH) [17–19].
Xylanase is an extracellular enzyme reported to have been produced
by lamentous fungi such as Aspergillus sp. and have been employed for
industrial production of xylanase due to their ability to hydrolyze
recalcitrant lignocellulosic materials into useful products such as bio-
fuels, thereby alleviate the burden of agro-waste on the environment
[20]. Hence, this study employed ability of xylanase-producing fungus,
Aspergillus awamori AFE1 found in the gut of long-horn beetle to degrade
crude xylan and investigate biochemical properties of the puried
xylanase including its ability to degrade agro-wastes containing xylan
using its solubilized and immobilized form.
2. Materials and methods
2.1. Materials
2.1.1. Equipment and glassware
The equipment used for this research were; portable pressure steam
sterilizer (Model YX-18LM), UV–visible spectrophotometer (Axiom 721
vis spectrophotometer UK), pH meter (Philips India), water bath shaker
(WHY-2, USA), water bath (HH-W420 thermostatic water cabinet, UK),
refrigerated centrifuge (AFI, India), analytical and top loading balance
(OHAUS Corporation, US). Other materials include glassware, spatula,
glass stirrer, inoculating loop and microliter pipette.
2.1.2. Chemicals and reagents
Chemical reagents such as, xylose, arabinoxylan, dinitrosalicylic acid
(DNSA), diethylaminoethyl (DEAE) Sephacel, Sephacryl S-200 and
bovine serum albumin (BSA) were products of Sigma-Aldrich CHEMIE
GmbH, US while chemicals such as agar agar, sodium acetate, citric acid,
distilled water, hydrochloric acid (HCl), sodium hydroxide, Tris-HCl
buffer, sodium chloride, magnesium sulphate, glycine, ethyl-
enediaminetetraacetic acid (EDTA), copper sulfate, calcium chloride,
manganese sulphate, barium chloride, cobalt chloride, calcium chloride,
iron chloride, dithiothretol, β-mercaptoethanol, sodium dodecyl sulfate,
L-cysteine, tween 20, Triton X-100, ethanol, isopropanol, butanol,
xylene, cyclohexane, methanol, n-hexane, toluene, benzene were of
analytical grade.
2.1.3. Sample collection
Three fresh xylan-rich agro-waste products such as cassava peel,
coconut husk and cocoa pod were obtained from major dump sites in
Ondo city (Lat 7.1000◦N, Long 4.8417◦E), Nigeria and brought to
Department of Biochemistry, Federal University of Technology, Akure.
2.1.4. Source of organism
Aspergillus awamori strain AFE1 which had been previously isolated
from the gut of Long-horned beetles (Cerambycidae latreille) was
collected from the Department of Biochemistry, Federal University of
Technology, Akure.
2.2. Methods
2.2.1. Sample preparation
The fresh agro-wastes were washed and allowed to drain. They were
afterwards sun dried and nely grounded in order to increase their
surface area. The resultant powdery agro-wastes were further sifted with
sieve bag in order to obtain particle sizes ranging between 75 µm and
154 µm.
2.2.2. Preparation of seed culture
Potato dextrose broth (PDB) of 3 g was dissolved in 100 mL of
distilled water and was thereafter sterilized using autoclave at 121 ◦C for
15 min after which it was allowed to cool before being inoculated with
A. awamori AFE1 followed by incubation with water bath shaker at
30 ◦C, 150 rpm for 72 h.
2.2.3. Plate screening of agro-residues degradation by Aspergillus awamori
AFE1
Xylanolytic ability of A. awamori AFE1 was carried out according to
the method of Adesina and Onilude [21] with slight modications. The
medium containing xylan 10 gl
−1
, KCl 0.5 gl
−1
, MgCl
2
0.5 gl
−1
,
(NH
4
)
2
HPO
4
2.5 gl
−1
, CaCl
2
⋅2H
2
O 0.01 gl
−1
, NaH
2
PO
4
0.5 gl
−1
, FeS-
O
4
⋅7H
2
O 0.01 gl
−1
, ZnSO
4
7H
2
O 0.002 gl
−1
was sterilized using auto-
clave and allowed to cool while 1 mL of seed culture of the fungus was
inoculated into the medium and incubated for 72 h at room temperature.
Thereafter, the plate was submerged with 1 % congo red while the stain
was removed with excess 1 M NaCl for 15 min in order to quantify the
zone of hydrolysis. However, the xylan in the medium was substituted
for cassava peel, coconut husk and cocoa pod as carbon sources in order
to investigate the capability of the fungus to degrade the agro-wastes.
The diameter of the clear zone of the xylan indicating the xylanolytic
and agro-waste degrading ability of the fungus respectively.
2.2.4. Production of xylanase in submerged fermentation by A. awamori
AFE1 using agro-wastes
Xylan as control and three agro-wastes were each used alongside the
mineral salts consisted of KCl 0.5 gl
−1
, MgCl
2
0.5 gl
−1
,
(NH
4
)
2
HPO
4
2.5
gl
−1
, CaCl
2
⋅2H
2
O 0.01gl
−1
, NaH
2
PO
4
0.5 gl
−1
, FeSO
4
⋅7H
2
O 0.01 gl
−1
,
ZnSO
4
⋅7H
2
O 0.002 gl
−1
for xylanase production. The media were
autoclaved at 121 ◦C for 15 min and then allowed to cool, after which 1
mL of the seed culture was inoculated. The media were incubated at
30 ◦C for 7 days using orbital shaker at 150 rpm while 5 mL aliquot of
the culture media were collected at 24 h interval. The resulting aliquots
of the culture media of xylan and other three agro-wastes were centri-
fuged at 10,000 g while their supernatants were used as crude enzyme.
Xylanase activity was then determined according to standard assay
procedure.
2.2.5. Production of xylanase from Aspergillus awamori AFE1 using
cassava waste as substrate
Xylanase was produced using mineral salts and cassava peel (agro-
wastes with optimum xylanase production). The production medium
was scaled up to 1 L while pH medium was adjusted to 5.3 using
NaHPO
4
. The medium was thereafter sterilized and was appropriately
inoculated with A. awamori AFE1 seed culture followed by incubation at
30 ◦C in an orbital shaker at 150 ◦C rpm for 72 h. The culture uid was
centrifuged using refrigerated centrifuged at 10,000 rpm for 15 min and
the supernatant was stored at 4 ◦C as crude enzyme for further use.
2.2.6. Determination of xylanase Activity and Sugar released
Xylanase activity was determined by mixing 0.9 mL of 1 % (w/v)
birch wood xylan (prepared in 50 mM Na-citrate buffer, pH 5.3) with
0.1 mL of aliquot enzyme. The mixture was incubated at 50 ◦C for 5 min
and the reaction was stopped by addition of 1.5 mL of 3,5-dinitrosali-
cylic acid (DNSA). The test tubes containing the reaction mixture were
boiled for 5 min and then cooled at room temperature. After cooling, the
absorbance of the colour developed was read at 540 nm. The blank was
set up without the enzyme and treated in the same condition as sample.
The amount of reducing sugar liberated was quantied using xylose as
standard. One unit of xylanase is dened as the amount of enzyme that
liberates 1 µmol of xylose equivalents per minute under the assay con-
ditions. The protocol was slightly modied for the immobilized xylanase
according to Irfan et al. [22]. Initially, 0.5 g of immobilized beads were
added to the reaction mixture containing 10 mL of 1 % xylan and
incubated at 50 ◦C for 30 min. One milliliter (1 mL) from this reaction
mixture was taken into a new test tube containing 1.5 mL DNS reagent.
The rest of the protocol remained same as that for the assay of free
enzyme. The activity was measured against agar–agar beads containing
no enzyme which was used as a control/blank at a wavelength of 540
nm.
I.A. Olopoda et al.
Process Biochemistry 121 (2022) 45–55
47
2.2.7. Determination of protein concentration
The concentration of soluble protein was estimated according to
Bradford [23] method using Bovine serum albumin standard. The
absorbance of the sample against blank was read at 595 nm with a
spectrophotometer.
2.2.8. Purication of xylanase and molecular weight determination
Xylanase was puried by Fractional Ammonium Sulphate Precipi-
tation, Dialysis, Gel-ltration Chromatography, Ion Exchange Chroma-
tography and the resulting puried xylanase was then in turn subjected
to SDS-PAGE (Sodium dodecyl sulphate polyacrylamide gel electro-
phoresis) for molecular mass determination [24].
2.2.9. Characterization of puried xylanase
2.2.9.1. Effect of temperature on the activity of both free and immobilized
xylanase. The effect of temperature on the activity of both free and
immobilized xylanase from Aspergillus awamori AFE1 was carried out by
varying the temperature condition of the reacting mixture. The reacting
mixture was incubated at different temperature ranging from 20 to 90 ◦C
at an interval of 10 ◦C. The xylanase activity was measured according to
standard assay procedure as described for both free and immobilized
xylanase above.
2.2.9.2. Thermal stability of xylanase from Aspergillus awamori AFE1.
Thermal stability was determined by pre-incubating the puried xyla-
nase at different temperature ranging from 30 to 90 ◦C with a temper-
ature interval of 10 ◦C for 3 h. One hundred microliter (0.1 mL) aliquot
of the pre-incubated xylanase was withdrawn consecutively at 30 min
interval for xylanase activity, during the 3 h pre-incubation. The xyla-
nase activity was determined according to the standard assay procedure.
2.2.9.3. Effect of pH on the activity of both free and immobilized
xylanase. The effect of pH on the activity of both free and immobilized
xylanase was investigated by carrying out xylanase assay at various pH
ranging from 2.0 to 12.0. The buffering system consisted 50 mM glycine-
HCl (2.0 and 3.0), citrate buffer (4.0 and 5.0), K
2
HPO
4
/KH
2
PO
4
buffer
(pH 6.0 and 7.0), Tris-HCl (pH 8.0, 9.0 and 10.0) and Tris-NaOH buffer
(pH 11.0 and 12.0) were employed. The enzyme assay for free and
immobilized xylanase was carried out according to the standard assay
procedure.
2.2.9.4. pH stability of xylanase from Aspergillus awamori AFE1. The pH
stability of the puried enzyme was determined by using various buffer
at pH ranging from 2.0 to 12.0 in 50 mM Glycine/HCl (2.0 and 3.0),
Citrate buffer (4.0 and 5.0), Phosphate buffer (pH 6.0 and 7.0), Tris/HCl
(pH 8.0, 9.0 and 10.0) and Tris/NaOH buffer (pH 11.0 and 12.0). One
hundred microliter (0.1 mL) aliquot of pre-incubated xylanase was
withdrawn initially and then at interval of 1 h for 6 h while enzyme
activity was determined according to assay procedure.
2.2.9.5. Effect of metal ions on the activity of the puried xylanase. Effect
of various metal ions such as Mg
2+
, Mn
2+
, Ba
2+
, Zn
2+
, Cu
2+
, Fe
2+
,
Ca
2+
and Co
2+
was investigated on xylanase activity. Different concentrations
of 1, 5 and 10 mM of the metal ions were incubated with the enzyme
solution and 50 mM of citrate buffer. The enzyme activity was deter-
mined according to standard assay procedure.
2.2.9.6. Effect of surfactants and metal chelator and other inhibitors on
xylanase activity. The effect of L-cysteine, dithiothreitol, sodium dodecyl
sulfate, EDTA were determined at 1, 5 and 10 mM while that of sur-
factants which include β-mercaptoethanol, Triton x-100 and Tween 20
were equally evaluated at 1 %, 5 % and 10 % v/v. The assay mixture
consisted each of the surfactants, chelators, other inhibitors and sub-
strate (1 % xylan in citrate buffer, pH 5.3) while enzyme solution was
also added and enzyme activity was carried out according to standard
assay procedure.
2.2.9.7. Kinetic studies of both free and immobilized xylanase. The kinetic
constants, K
m
and V
max
of both puried free and immobilized xylanase
were determined by measuring xylanase activity at different substrate
concentrations from 0.1 % to 0.8 % xylan and enzyme activity was
carried out according to assay procedure earlier described. Then, inverse
of initial velocities (V
−1
) and substrate concentrations ([S]
−1
) of each
concentration were plotted. However, V
max
and K
m
were calculated
using Lineweaver-Burk plot.
2.2.10. Hydrolysis of various xylan-richagro-wastes using free and
immobilized xylanase
Action of both free and immobilized xylanase were investigated on
hydrolysis of pure xylan and agro-wastes. The reaction mixture included
puried xylanase and 15 mL of each substrate (1 % w/v in citrate buffer,
pH 5.3) and was then incubated at 50 ◦C for 48 h. The set up was slightly
modied for hydrolysis of agro-wastes rich-xylan by immobilized xyla-
nase. The reaction mixture involved addition of 5 g of immobilized
beads to 15 mL of substrate and incubated at 50 ◦C for 48 h. Aliquots of
the reaction mixture were taken at 1 h intervals for 6 h and then
centrifuged. The supernatants of the aliquots obtained were used to
determine amount of xylose released.
2.2.11. Reusability of both free and immobilized xylanase
The immobilized enzyme was investigated for its reusability. The
immobilized enzyme assay was carried out on the xylanase-agar
immobilized beads while the beads was washed with sodium citrate
buffer for subsequent reactions. The assay was repeated until no enzyme
activity was left in the beads. The enzyme assay was carried out ac-
cording to immobilized standard assay procedure earlier stated. The
enzyme reusability after each reaction cycle was determined as:
Reusability =Xylanase activity of nth cycle ×100.
Xylanase activity of 1st cycle.
3. Result and discussion
The study revealed purication, immobilization and characteriza-
tion of xylanase from A. awamori AFE1 isolated from the gut of long-
horned beetle (cerambycidae latreille). Production and purication of
lignocellulotic enzymes (cellulose, xylanases etc.) from microorganisms
have been reported by several authors [25,26]. However, the presence
of xylanase activity in A. awamori AFE1 isolated from the gut of long-
horned beetles may not be unconnected with the presence of symbiotic
microbes lining their gut [27,28]. Several studies have clearly revealed
microbiota population inhabiting the gut of animals [29,30], ruminants
[31], snails [24] and insects [32] and further established the involve-
ment of this microora in the production of different hydrolytic enzymes
that participate in metabolic breakdown of macromolecules such as
xylan, cellulose and starch [31]. Occurrence of hydrolytic enzymes from
micro-organism harbored by termites and other phytophagous insects
have been isolated and puried [27,33]. Consequently, longhorned
beetles (adults and larvae) feed on forest trees, log of woods and plants
by boring or tunneling into the woods, thereby turning lignocellulosic
materials into absorbable forms [34]. This phytophagous mode of
feeding suggests that longhorned beetle must possess a well-developed
microbial enzyme machineries required for the degradation of ligno-
cellulose. Therefore, symbiotic microorganisms lining the gut of long-
horned beetle become a principal prospective source of xylanases - as
phytophagous insects have long been reported to harbor diversities of
microora which are metabolically relevant [35].
I.A. Olopoda et al.
Process Biochemistry 121 (2022) 45–55
48
3.1. Agro-wastes hydrolysis and xylanase optimum production by
Aspergillus awamori AFE1
Table 1 showed relative percentage of xylan and agro-wastes hy-
drolysis by A. awamori AFE1. The coconut husk was observed to be the
most preferred substrate for the fungus with 27.9 % followed by cocoa
pod with 26.5 %. Hydrolysis in the presence of cassava also showed a
high activity of 25 % while 20 % was observed when xylan (pure sub-
strate) was used. Remarkably, the resultant zone of hydrolysis exhibited
by various substrates were all considered to be signicant [36]. The
obtained hydrolysis zone revealed the ability of the A. awamori AFE1 to
use xylan-rich agricultural wastes as substrates and induce enzymes
needed for degradation of the recalcitrant wastes. Xylan, exhibiting the
least zone of hydrolysis could be attributed to specicity in the action of
xylanase - as xylan being a substrate specic for xylanase would only be
catalyzed by xylanase while other agro-waste would require synergistic
actions of lignocellulolytic enzymes for their hydrolysis.
The production of xylanase by A. awamori AFE1 grown on xylan and
agro-wastes is shown in Fig. 1. The optimum xylanase production was
observed in the presence of cassava peel with 2433.33 U/mL after 48 h
and was the agro-waste that gave the highest enzyme production of all
the four substrates. Xylan gave optimum enzyme activity at 48 h, co-
conut husk at 24 h and cocoa pod at 72 h with 1,433.33, 11,166.67 and
1,083.33 U/mL respectively. The fungus further showed decrease in
xylanase production and about 1500, 1000, 800 and 100 U/mL enzyme
production was observed after 148 h for cassava peel, xylan (the con-
trol), coconut husk and cocoa pod respectively. Comparably, Ali et al.
[37] and Ramanjaneyulu and Reddy [38] gave an account of optimum
xylanase production at 72 h incubation period for Fomes fomentarius and
Fusarium sp. BVKT R2 using wheat straw and rice straw as substrates
respectively, Irfan et al. [11] observed optimum xylanase production of
48 h for B. subtilis BS04 while B. megaterium BM07 exhibited maximum
xylanase production after 72 h of fermentation period with sugar cane
bagasse as substrate. In another studies, higher incubation period of 120
h was observed by Okeke et al. [20] with xylanase activity of 90 U/L and
359 U/L for Penicillium janthinellum and Trichoderma virens respectively.
The signicant decrease in enzyme production with time observed in
this study, might be due to the exhaustion of nutrients which tends to
reduce the performance of the fungus in synthesizing the enzyme [11].
However, cassava peel was found to be most preferred substrate for
A. awamori AFE1 for xylanase production compared to the xylan (the
standard substrate for xylanase and control) and was therefore used for
the mass production of the enzyme using its optimum incubation period.
Hence, cassava peel was found to be a better replacement for xylan as
substrate (carbon source) for xylanase production. Appropriate selection
of culture conditions such as substrate incubation period and growth
conditions usually enhance production and lower cost of enzyme [39].
3.2. Purication summary and immobilized Aspergillus awamori AFE1
xylanase
The summary of purication of xylanase obtained from A. awamori
AFE1 is presented in Table 2. The dialysate of the ammonium sulfate
precipitation of 40 %, 60 % and 80 % were observed to be 87.85, 193.10
and 282.05 U/mg specic activity and 1.31, 2.89 and 4.22- fold puri-
cation. On loading the 60 % of the concentrated dialysate against DEAE
Sephacel (Fig. 2a), one sharp peaked activity was obtained with the
purication fold of 3.24 % and 5.33 % recovery. However, subsequent
elution of DEAE Sephacel eluate on Sephadex G-100 (Fig. 2b) resulted in
one-peaked activity while a specic activity of 458.7 U/mg, 5.20 %
recovery and 6.86- fold purication were recorded for the pooled frac-
tions. Similar result was also reported by Monclaro et al. [40], where
homogenously puried xylanase from Aspergillus tamarii had 6.09
overall purication fold and 9.73 % yield. A higher purication fold and
lower yield of 27.92 % and 2.01 % were given by Seemakram et al. [41]
for xylanase from T. Dupontii KKU-CLD-E2–3. In addition, Silva et al.
[42] reported 3464 U/mg specic activity, 12 % yield and 5.3 puri-
cation fold for T. inhamatum Xyl 11. It was also shown by Deshmukh
et al. [43] that Aspergillus fumigatus R1 xylanase gave purication fold of
58, yield of 3.43 % and specic activity of 38,196.22 U/mg. Sanni et al.
[24] listed purication parameters as well as resins, purication con-
ditions as factors that could inuence purication of enzymes which
were also noted in the obtained results.
Immobilization of puried xylanase with the use of agar-agar solu-
tion resulted into cubical and regular shaped agar-agar beads as shown
in Fig. 2c. The weight of the gelly-like beads was between 0.9 and 1.1 g.
The bead gels exhibited white coloration after being immobilized with
xylanase.
3.3. Molecular weight of xylanase from Aspergillus awamori AFE1
The electropherogram of puried xylanase is shown Fig. 2d. Hence,
the resulting molecular weight (M
w
) obtained from the SDS-PAGE of the
puried xylanase was found to be ~ 48 kDa. Amir et al. [44] and
McPhillips et al. [45] obtained a comparable result of molecular weights
of 43 and 42 kDa for Aspergillus fumigatus xylanase and Remersonia
thermophila xylanase respectively while lower molecular weights of
25 kDa for T. Dupontii KKU-CLD-E2–3 [41], 33.67 kDa for Aspergillus
oryzae HML366 [46] and 35 kDa for Aspergillus oryzae LC1 [47] were
reported. However, higher molecular weight of 85 kDa for xylanase
from Humicola Insolens Y1 was observed by Xia et al. [48]. Variation in
molecular weight might not only be due to carbohydrate moiety present
as protein prosthestic group but also identity of the gene encoding the
enzyme [49]. It might also be as a result of the following conditions:
cleavage of signal peptide; post-translational modication of protein
(such as phosphorylation with phosphoryl group and O- and N- glyco-
sylation with sugar moiety) and formation of protein complexes or
protein isoform [50].
Table 1
Plate Screening of Xylan and Agro-wastes Hydrolysis by A. awamori AFE1.
S/
N
Agro-
wastes
Diameter
Whole Fungi
(mm)
Uncleared
Zone (mm)
Clear
Zone
(mm)
% Relative
Enz Activity (
%)
1 Cassava
Peel
40.00 30.00 10.00 25.00
2 Cocoa
Pod
34.00 25.00 9.00 26.47
3 Coconut
Husk
43.00 31.00 12.00 27.91
4 Xylan 59.50 47.50 12.00 20.17
Fig. 1. Effect of incubation period on xylanase production from A. wamori
AFE1. Four different carbon sources such as xylan, cassava peel, coconut husk
and cocoa pod were used as substrates in screening medium and incubated for
seven days while 5 mL fractions were collected at The data were collected in
triplicate and measured as mean ±standard deviation =Error bar.
I.A. Olopoda et al.
Process Biochemistry 121 (2022) 45–55
49
3.4. Optimum and thermal stability of free and immobilized xylanase
activity
The inuence of temperature on enzyme activity is presented in
Fig. 3a. Xylanase optimum activity was observed at 50 ◦C for free
enzyme while 70 ◦C was observed for immobilized enzyme. Free enzyme
showed over 95 % relative activity from 20 to 40 ◦C while remaining
activity of 93 % were obtained at 60–90 ◦C. The immobilized xylanase
exhibited over 92 % relative activity from 20 to 60 ◦C, while more than
92 % and 84 % relative activity were recorded at 80 and 90 ◦C respec-
tively. A shift in the optimum temperature has been commonly observed
with immobilized enzyme. Having recorded 60 ◦C optimum tempera-
ture for free xylanase, Gracida et al. [51] obtained 70 ◦C for immobilized
xylanase from his work. Likewise, Bibi et al. [52] reported a ten degrees
(10 ◦C) shift in optimum temperature from 50 ◦C to 60 ◦C for free and
immobilized xylanase respectively. There was equally a ten degrees
(10 ◦C) shift in optimum temperature of 50 ◦C for free xylanase when
immobilized unto (Alg/PEI/Na
+
) gel [53]. Optimum temperature
Table 2
Purication Table of Xylanase Obtained from A. awamori AFE1.
Purication Step/
Volume
Volume
(mL)
Enzyme Activity
(U/mL)
Protein Conc.
(mg/mL)
Total Activity
(U)
Total Protein
(mg)
Specic Activity
(U/mg)
%
Recovery
Purication
Fold
Crude Enzyme
(Volume)
300 7.10 0.106 2130 31.88 66.82 100 1
[(NH4)
2
SO4)]
Precipitation
40
%
40 8.02 0.091 320.67 3.65 87.85 15.05 1.31
60
%
40 3.50 0.018 140.00 0.73 193.10 6.57 2.89
80
%
35 3.67 0.013 128.33 0.46 282.05 6.03 4.22
DEAE Sephacel 30 3.78 0.018 113.50 0.53 216.19 5.33 3.24
Sephadex G-100 23 4.82 0.011 110.78 0.24 458.73 5.20 6.86
Fig. 2. Chromatographic separation of crude xylanase from A. awamori AFE1 isolated from Longhorn beetle, immobilization and molecular weight of the puried
xylanase. a. Elution prole of A. awamori AFE1 xylanase through DEAE Sephacel column chromatography, ow rate 50 mL/h, 2.5 cm ×30 cm column. The thick
arrow represents pooled fractions. Sodium chloride stepwise elution was used to unbind the bound protein from 0.2 to 1 M in 50 mM citrate buffer, pH 5.3. From tube
0 – 50 represent free ow protein while 51 – 130 represent bound protein eluted by sodium chloride stepwise elution. b. Elution prole of A. awamori AFE1 xylanase
through Sephadex G-100 (ow rate: 30 mL/h, column: 2.5 cm ×70 cm) column chromatography. The thick arrow in the above gure represents pooled fraction. c.
immobilization of puried xylanase using agar-agar solid matrix. Xylanase-agar agar immobilized beads (A) and Control- Agar-agar beads (B). d. Electro-
phorectogram of puried xylanase by Polyacrylamide Gel Electrophoresis using Sodium Dodecyl Sulfate (SDS-PAGE) The sample was loaded on 10 % gel along with
the standards of molecular weight ranging 25–100 kDa after which it was boiled with sodium dodecyl sulphate and mercaptoethanol at boiling temperature. Lane A
=Protein Standards and B =A. awamori molecular weight.
I.A. Olopoda et al.
Process Biochemistry 121 (2022) 45–55
50
exhibited by Talaromyces cellulolyticus Xyn30A xylanase [54] and Tri-
choderma sp. TP3–36 TXyn11A [55] has been reported to be 55 ◦C,
whereas an optimum activity at 60 ◦C and 65 ◦C were obtained by Irfan
et al. [22] and Kumar et al. [10] for agar-agar and Aluminium oxide
pellet immobilized xylanase respectively. Requirement of higher acti-
vation energy by agar-agar immobilized xylanase in the course of
forming and breaking bonds at the enzyme-substrate complex transition
stage may be responsible for variation in optimum temperature when
compared to free xylanase [22]. The higher optimum temperature
observed after immobilizing A. awamori AFE1 xylanase, showed that the
agar-agar matrix preserved xylanase activity which might be due to
enhanced stability or conformational rigidity on immobilization [10].
Therefore, A. awamori AFE1 xylanase is an exceptional thermophilic
enzyme even at an extreme temperature of 90 ◦C and thereby t for
industrial and biotechnological processes.
The puried xylanase maintained its initial activity at 50 and 60 ◦C
respectively as shown in Fig. 3b. However, at 30 and 40 ◦C, very high
residual activity of 90 % and 95 % respectively were recorded while
90.6 % residual activity was obtained at 90 ◦C. Whereas 80 ◦C and 90 ◦C
gave 87 % residual activity after 6 h of incubation. This shows
A. awamori AFE1 xylanase as a thermostable enzyme and extremophile
owing to its ability to withstand temperature as high as 90 ◦C for a long
period of time. In a similar manner, Torre et al. [56] reported an
extremely stable xylanase from Thermomyces lanuginosus PC7S1T with
remarkable residual activities of 100 % and 99.8 % at 40 – 65 ◦C and
75 ◦C for 5 and 3 h respectively while Seemakram et al. [41] observed
T. Dupontii KKU-CLD-E2–3 xylanase to be stable at 70 ◦C with over 70 %
residual activity; whereas Trichoderma sp. TP3–36 TXyn11A xylanase
displayed instability and showed remarkable loss of activity at temper-
ature above 55 ◦C [55]. Availability of additional disulde and salt
bridges, hydrophobic side chains and N-terminal proline residues could
bring about reduction in conformational freedom of the protein struc-
ture and thereby cause the protein to be more stable even at a very high
temperature [57].
3.5. pH optimum and stability of free and immobilized xylanase activity
Effect of pH on both free and immobilized xylanase is presented in
Fig. 4a. Free and immobilized xylanases were observed to be active at all
pH investigated while their pH optimum was found to be at slight acidic
region of pH 4 and 5 respectively. Interestingly, at alkaline pH 8 – 10,
over 95 % relative activity was obtained while at pH 11 and 12, about
percentage relative activity of 82 % activity was still recorded for free
xylanase. However, immobilized xylanase revealed relative activities of
82 % and 69 % at pH 8 and 9 respectively and further increase in pH
(10−12) reduced the xylanase activity to 54 %. Obviously, more than
78 % lowest relative activity obtained at various pH 3–12 for free
enzyme in this study corroborated the result of xylanase (TXyn11A)
from Trichoderma sp. TP3–36, which retained nothing less than 80 % of
its original activity at various pH values (pH 3–11) after 24 h of incu-
bation with highest activity at pH 5 [55]. Comparably, Mostafa et al.
[53] obtained a similar result where A. avus xylanase had optimum pH
at 5 and 5.5 for free and immobilized respectively. In the same vein, free
and immobilized xylanase from Bacillus pumilus VLK-1 exhibited their
optimum activity at alkaline pH 8 and 9 respectively, according to
Fig. 3. Effect of temperature on the activity and stability of free and immobi-
lized puried xylanase obtained from A awamori AFE1. a. The effect of tem-
perature was determined on the activity of puried xyalanase from 20 to 90 ◦C
at 10 ◦C interval. The activity was determined according to assay procedure and
expressed as percentage relative activity. The data were collected in triplicate
and measured as mean ±standard deviation =Error bar. b. The effect of
temperature was determined on the stability of puried xyalanase from 20 to
90 ◦C at 10 ◦C interval. The activity was measured initially at 0 min then after
30 mins interval for 3 h. The activity was determined according to assay pro-
cedure and expressed as percentage residual activity. The data were collected in
triplicate and measured as mean ±standard deviation =Error bar.
Fig. 4. Effect of pH on the activity and stability of free and immobilized
xylanase obtained from A. awamori AFE1. a. Effect of pH on the activity of
puried xylanase. The buffer systems consisted Glycine/HCl (pH 2–3), Citrate/
Citric acid (pH 4–5), K
2
HPO
4
/KH
2
PO
4
(pH 6–7), Tris/HCl (pH 8–10) and Tris/
NaOH (pH 11–12). The activity was expressed as percentage relative activity.
The data were collected in triplicate and measured as mean ±standard devi-
ation =Error bar. b. Effect of pH on stability of puried xylanase. The buffer
systems consisted Glycine/HCl (pH 2–3), Citrate/Citric acid (pH 4–5), K
2
HPO
4
/
KH
2
PO
4
(pH 6–7), Tris/HCl (pH 8–10) and Tris/NaOH (pH 11–12). The activity
was initially taken at 0 h and then after 1 h interval for 6 h. The enzyme assay
was determined according to the assay procedure while the activity was
expressed as percentage residual activity. The data were collected in triplicate
and measured as mean ±standard deviation =Error bar.
I.A. Olopoda et al.
Process Biochemistry 121 (2022) 45–55
51
Kumar et al. [10]. However, Gracida [51] observed similar optimum pH
for both free and immobilized xylanase, alkaline pH 9 was obtained by
Yadav et al. [58] and Kumar et al. [59] for Aspergillus Kamchtkensis
NASTPD13 and B. amyloliquefaciens strain SK-3 xylanase while Kumar
and Shukla [60] observed Thermomyces lanuginosus VAPS24 xylanase to
be optimum at neutral pH.
As shown in Fig. 4b, maximum enzyme stability was obtained at pH
2–4 with 91 % residual activity recorded while the enzyme still
exhibited high residual activities of 65 – 75 % at pH 5 – 10 but 36 %
remaining activity was obtained at pH 11 after 6 h of incubation. This
implies that A. awamori AFE1 xylanase was stable over a wide range of
pH. Fu et al. [55] Seemakram [41] reported highly stable TXyn11A and
T. Dupontii KKU-CLD-E2–3 xylanase at a broad pH range 3–11 and 7–10
with percentage residual activities of 80 % and 75 % respectively after
24 h, while A. kamchatkensis NASTP13 xylanase exhibited pH stability at
pH 6–9 with residual activities of 71 – 100 % [58]. Increase in amount of
amino acid such as Thr/Ser ratio and charged residues may encourage
association of polar groups and improve alpha helix and other secondary
structures steadiness while attendant low exibility including high
amount of ion pairs/aromatic residues on the surface of enzymes may be
responsible for their ability to withstand harsh conditions [57,61].
Hence, thermostable and alkaline stable xylanases are desirable for pulp
pre-bleaching and also benecial for other industrial processes espe-
cially the ones that require extreme conditions [62].
3.6. Modulatory of metal ions and organic solvent on the activity of the
puried xylanase
The effect of metal ions on enzyme activity is illustrated in Fig. 5a.
The enzyme activity was exceptionally enhanced in the presence of
Mn
2+
at all concentration investigated and Mg
2+
activated the enzyme
activity at 10 mM only. However, Ba
2+
, Zn
2+
, Ca
2+
Cu
2+
and Fe
2+
inhibited the enzyme activity at all concentration investigated while
Co
2+
only had percentage inhibition of 50 % on the enzyme activity at
10 mM. In the same vein, Silva et al. [42] reported T. inhamatum xyla-
nase (Xyl 11) to be inhibited by Ba
2+
, Zn
2+
and Ca
2+
at 10 mM but Mn
2+
stimulated the activity of the enzyme at 10 mM. It was also reported that
Mn
2+
and Mg
2+
stimulated the activity of T. lanuginosus PC7S1T xyla-
nase while Fe
2+
, Cu
2+
and Hg
2+
inhibited its activity [60]. In addition,
Mg
2+
, Co
2+
and Ba
2+
have shown to have a stimulatory effect on
B. bassiana SAN01 xylanase [63]. Metal ions regulates enzyme’s struc-
ture by either direct association with the enzyme or interact with mol-
ecules at the enzyme’s regulatory site and thereby donates or accepts
electrons (Lewis’s acids) which consequently facilitates binding of
enzyme to the substrate through ionic bonding. Through this, activa-
tion/deactivation of enzymatic activity is achieved by interaction of
metal ions with amino or carboxylic acid group of the amino acids at
either regulatory/catalytic site of the enzyme [64]. In addition, the
magnitude of ionic charge (ionic radius size) determines the extent to
which the enzyme activity and stability will be inuenced. Larger radius
tends to have minimal inuence on catalytic amino acids and vice versa,
thereby altering the enzyme’s overall conformation with impaired cat-
alytic site [65].
Effect of various organic solvents on the activity of puried xylanase
is shown in Fig. 5b. The enzyme activity was enhanced in the presence of
isopropanol, butanol, cyclohexane, n-hexane, benzene and toluene at all
concentrations investigated. However, 1 % and 5 % of xylene and
ethanol enhanced the enzyme activity, whereas 10 % concentration of
the organic solvent showed strong inhibitory effect towards the enzyme
activity with over 26 % remaining activity obtained. The obtained result
in this study is similar T. pleuroticola 08 ÇK001 xylanase [66] which was
also stimulated by the organic solvents but was only inhibited by
ethanol. Stimulatory effect of ethanol, hexane and isopropanol on
B. bassiana SAN01 xylanase has also been reported [63].
3.7. Effect of surfactants and inhibitors on xylanase activity
Effect of surfactants and inhibitors on the enzyme activity is illus-
trated in Fig. 5c. Dithiothreitol (DTT), and sodium dodecyl sulfate (SDS)
showed signicant enzymatic enhancement. Likewise, Triton X-100 and
tween 20 also enhanced the enzyme activity in a concentration depen-
dent manner. However, enzyme activity was activated at 1 mM β-mer-
captoethanol (β-ME), but 5 and 10 mM of the inhibitor inhibited the
enzyme activity. Similarly, L-cysteine and Ethylenediaminetetracetic
acid (EDTA) strongly inhibited the xylanase activity in a concentrated
Fig. 5. Effect of metal ions, organic solvents and inhibitors on xylanase activity
of A. awamori AFE1. a. Effect of metal ions on xylanase activity. The metal ions
were incubated alongside with substrate and enzyme solutions while the
enzyme activity was determined according to assay procedure. The data were
collected in triplicate and measured as mean ±standard deviation =Error bar.
b. Effect of organic solvent on enzyme activity. The organic solvents were
incubated alongside with substrate and enzyme solutions while the enzyme
activity was determined according to assay procedure The data were collected
in triplicate and measured as mean ±standard deviation =Error bar. c. Effect
of inhibitors on xylanase activity. L-cysteine, Dithiothreitol (DDT), sodium
dodecyl sulphate (SDS), Ethylenediaminetetraacetic acid (EDTA), were
included in the assay procedure in 1, 5 and 10 mM while 2-mercaptoethanol (2-
ME), Triton X-100 and Tween 20 were measured in 1 %, 5 % and 10 %. The
data were collected in triplicate and measured as mean ±standard deviation
=Error bar.
I.A. Olopoda et al.
Process Biochemistry 121 (2022) 45–55
52
dependent manner. Korkmaz et al. [66] observed β-ME enhancing Tri-
choderma pleuroticola 08 ÇK001 xylanase activity by 112 % while EDTA,
SDS and tween 20 inhibited its activity. Joshi et al. [67] observed that
SDS, tween 20 and Triton X-100 diminished the xylanase activity
whereas elevated enzyme activity was observed in the presence β-ME,
while no signicant effect was observed in the presence of EDTA. Ac-
cording to Silva et al. [42], DDT enhanced the activity of both enzyme
T. inhamatum Xyl 1 & 11 while SDS and EDTA inhibited its activity at all
concentrations examined. Amobonye et al. [63] also observed that
EDTA, SDS, Tween 20 and tween X-100 lowered the activity of
B. bassiana SAN01 xylanase.
3.8. Kinetic parameters of both free and immobilized xylanase
K
m
and V
max
for both free and immobilized xylanase were extrapo-
lated from the graph presented in Fig. 6a. The K
m
and V
max
values for
free xylanase were determined to be 0.15 mM and 5 ×10
3
µM/min
while immobilized xylanase were 1 mM and 1 ×10
4
µM/min respec-
tively. Hence, the result of kinetic study of free and immobilized xyla-
nase revealed a signicant increase in both K
m
and V
max
value from free
and immobilized enzyme. Interestingly, increase in the kinetic param-
eters observed for immobilized xylanase in this study is not unusual as
Kumar et al. [68] had earlier reported higher K
m
and V
max
value for
immobilized xylanase compared to free xylanase while agar-agar
immobilized B licheniformis S3 xylanase showed increase in K
m
when
compared to its free form [22]. Similarly, Landarani-Isfahani et al. [69]
gave a report of increase in K
m
of immobilized T. lanuginosus xylanase on
multifunctional hyper-branched polyglycerol-grafted magnetic nano-
particles while covalent immobilization of A. avus xylanase also
exhibited increase in K
m
[53]. Therefore, increase in K
m
during immo-
bilization could be as a result of steric effects or mass transfer limitations
while rise in V
max
could equally be as a result of conformation changes or
synergistic effect of enzymes [70,71].
3.9. Hydrolysis of agro-wastes by free and immobilized xylanase
The ability of both free and immobilized xylanase to hydrolyze xylan
in various agro-substrates is illustrated in Figs. 6b and 6c. Free xylanase
exhibited 38.62 %, 53.64 % and 59.68 % of xylose released on Xylan,
cassava peel, coconut husk respectively after 24 h. However, when
cocoa pod was used as substrate, the enzyme attained maximum per-
centage degradation of 51 % after 48 h. Immobilized enzyme displayed
maximum percentage xylan hydrolysis of 48.80 %, 57.14 %, 68.67 %
and 80.00 % on coconut husk, xylan, cocoa pod and cassava peel
respectively after 48 h. Higher xylan degrading ability exhibited by
immobilized xylanase is an unusual attribute as lower enzyme activity
are inherent with most immobilized enzymes compared to the free
enzyme [72]. This is also evident in the higher maximum velocity (V
max
)
of the immobilized enzyme. The size of the beads determines the activity
of the immobilized enzyme as size of beads that give optimum enzyme
activity varies. Xylanase immobilized bead size of 3.5 mM and 0.5 g
amount of beads exhibited excellent agro-waste degrading ability
compared to free enzyme. However, Demirkan et al. [73] observed
3 mM immobilized B. amyloliquefaciens
α
-amylase bead and 0.4 g
Fig. 6. Lineweaver-Burk plot of free and immobilized xylanase using xylan as substrate, Degradability of xylan in various agricultural wastes by free and Immobilized
xylanase and reusability of agar-agar immobilized puried xylanase. a. Lineweaver-Burk plot of free and immobilized xylanase using xylan as substrate. The xylan
concentrations of 0.1–0.8 % were each dissolved in citrtate buffer, pH 5.3 while the activity was determined according to assay procedure. b. Degradability of xylan
in various agricultural wastes by free puried xylanase. The data were collected three times and expressed as mean ±standard deviation =Error bar. c. Degrad-
ability of xylan in various agricultural wastes by immobilized puried xylanase. The data were collected three times and expressed as mean ±standard deviation
=Error bar. Degrdtn represents Degradation, “IX” represents Immobilized xylanase, “FX” represents Free xylanase, “Xyl” represents xylan, “Cpeel” represents
Cassava peel, “Chusk” represents Coconut husk, “Cpod” represents Cocoa. d. The reusability of agar-agar immobilized xylanase. The data were collected three times
and expressed as mean ±standard deviation =Error bar.
I.A. Olopoda et al.
Process Biochemistry 121 (2022) 45–55
53
amount of beads gave maximum starch hydrolysis while Dey et al. [74]
observed 2 mM and 2 g of B. circulans amylase beads to give efcient
starch hydrolysis, though high amylase activity of over 80 % was
observed at 3 and 4 mM bead size. The result obtained in this study may
be compared to Xu et al. [72] who linked efcient phenol removal to the
enhanced structural rigidity of the immobilized enzyme which alleviate
the extent at which enzyme was distorted during reaction with the
substrates [72]. There was also a report of protection of microenviron-
ment of immobilized enzyme from environmental factors that can bring
about reduction in enzymatic activity [75]. Efcient hydrolysis of xylan
in agro-residues such as cocoa pod, coconut husk and cassava peels by
immobilized puried xylanase from A. awamori AFE1 establishes usage
of the enzyme in industrial processes for manufacturing value-added
products from agricultural wastes.
Since, enzymes are specic in nature, puried A. awamori AFE1
xylanase would specically hydrolyze xylan in the agro-residues.
Though, complete hydrolysis of xylan (a hemicellulose) requires
concerted effort of different type of xylanases [76], however, 48 kDa
xylanase produced by A. awamori is termed endo-β-1,4 xylanase (of
which its function is to cleave internal glycosidic linkage of xylan) and
belongs to GH-10 family. This enzyme works based on the mechanism
termed double displacement (retention) [77]. It targets the xylose con-
nections closest to the side chain residues. The xylose residues connect
to sub-sites on xylanase while the bond between the monomeric residues
at the non-reducing and reducing end of the polysaccharide substrate
(xylan) is then cleaved [61].
3.10. Reusability of immobilized xylanase using agar-agar
The reusability of immobilized xylanase is shown in Fig. 6d. The
agar-agar immobilized xylanase demonstrated recycling efciency of
ve cycles with relative activity of 92.83 %, 53.72 %, 22.04 % and 9.92
% for second, third, fourth and fth cycles respectively. The result
showed a retention of more than 50 % activity till 3rd cycle after which
there was a sharp decline in activity. Similarly, Gracida et al. [51] gave a
report of six cycles of which 50 % activity of immobilized xylanase was
retained after 3rd cycle. Likewise, agar-agar and Ca
2+
-alginate immo-
bilized xylanase exhibited six recycling efciency with a retention of 62
% and 43 % reusability after 3rd cycle respectively [22]. However, more
than 70 % activity of agar-agar entrapped xylanase was observed after
3rd cycle, in a reaction of six turns obtained by Bibi et al. [52].
Observably, enzymatic activity of immobilized enzyme was decreased as
number of cycles increased and at the same time the number of cycles
was low compared to the previous studies. This could be due to inacti-
vation of enzyme resulted from enzyme denaturation or drawback
associated with entrapment as there is possibility of leakage of enzyme
from the gel matrix due to poor connection between the enzyme and
polymer matrix [10,78]. In the future study, the use of combination of
two or more entrapment methods or by cross linking (this tends to in-
crease the rigidity of the matrix) the named entrapment method is ex-
pected to prevent the leakage and thereby enhance enzyme reusability.
The obtained results have shown agar-agar immobilized xylanase to be a
good tool for cost-effective immobilization.
4. Conclusion
The results have established novel and exceptional biochemical
properties of free and immobilized extracellular xylanase puried from
A. awamori AFE1 and as efcient hydrolytic biocatalysts which could be
applied in various biotechnological processes. Interestingly, immobi-
lized form of puried xylanase exhibited obvious excellent catalytic
efciency and great afnity for substrate, improved thermal stability
and enhanced reusability. This study has established hydrolytic ef-
ciency of immobilized xylanase from A. awamori AFE1 isolated from
Long-horned beetle in releasing xylose from recalcitrant lignocellulosic-
containing agro-wastes which could be employed in industrial and
biotechnological processes with the capability of converting agro-wastes
to value-added products.
Declaration of Competing Interest
The authors declare no conict of interest.
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