Enzymatic hydrolysis of inulin by an immobilized extremophilic inulinase from the halophile bacterium Alkalibacillus filiformis

Article abstract of DOI:10.1016/j.carres.2019.107746

Bacterial inulinases are the key enzymes in the enzymatic hydrolysis of inulin and production of fructooligosaccharides (FOSs) and high fructose syrup (HFS). An extremophilic inulinase was purified from Alkalibacillus filiformis using 80% ethanol precipitation, ultrafiltration, and Q-Sepharose anion exchange chromatography. The purified inulinase was highly active in a wide range of pH, temperature, chemical reagents, and NaCl concentrations. The enzyme immobilization on cobalt ferrite magnetic nanoparticles (CoFe₂O₄ MNPs) was carried out by carrier binding method with covalent linkage and showed improved stability and reusability within a broad temperature and pH range, compared with the free enzyme. Using free and immobilized inulinases from A. filiformis, 122 g L^?1 and 160 g L^?1 fructose with 61% and 80% conversion, respectively, were obtained, with inulin as the substrate. The enzymatic properties, such as notable stability under extreme conditions, make the inulinase from A. filiformis a promising candidate for related biotechnological applications.

Full text of DOI:10.1016/j.carres.2019.107746

Carbohydrate Research 483 (2019) 107746
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Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Enzymatic hydrolysis of inulin by an immobilized extremophilic inulinase
from the halophile bacterium Alkalibacillus filiformis
a,b
b
a
a
Mahsa Yousefi-Mokri , Ali Sharafi , Shahla Rezaei , Salar Sadeghian-Abadi ,
a
a
a
Somaye Imanparast , Mehdi Mogharabi-Manzari , Yaghoub Amanzadeh ,
a,*
Mohammad Ali Faramarzi
a
Department of Pharmaceutical Biotechnology, Faculty of Pharmacy and Biotechnology Research Center, Tehran University of Medical Sciences, P.O. Box 14155–6451,
Tehran, 1417614411, Iran
b
Department of Pharmaceutical Biotechnology, School of Pharmacy, Zanjan University of Medical Sciences, Zanjan, Iran
A R T I C L E I N F O
A B S T R A C T
Keywords:
Bacterial inulinases are the key enzymes in the enzymatic hydrolysis of inulin and production of fructooligo-
saccharides (FOSs) and high fructose syrup (HFS). An extremophilic inulinase was purified from Alkalibacillus
filiformis using 80% ethanol precipitation, ultrafiltration, and Q-Sepharose anion exchange chromatography. The
purified inulinase was highly active in a wide range of pH, temperature, chemical reagents, and NaCl con-
Alkalibacillus filiformis
Halophilic bacterium
Inulinase
Immobilization
Inulin hydrolysis
Purification
centrations. The enzyme immobilization on cobalt ferrite magnetic nanoparticles (CoFe
2
O MNPs) was carried
4
out by carrier binding method with covalent linkage and showed improved stability and reusability within a
broad temperature and pH range, compared with the free enzyme. Using free and immobilized inulinases from A.
−
1
−1
filiformis, 122 g L
and 160 g L
fructose with 61% and 80% conversion, respectively, were obtained, with
inulin as the substrate. The enzymatic properties, such as notable stability under extreme conditions, make the
inulinase from A. filiformis a promising candidate for related biotechnological applications.
1
. Introduction
Inulin, the second most abundant store of polysaccharides, found
employed for industrial and biotechnological goals [4,7]. These mi-
croorganisms are proposed as one of the best sources for the commer-
cial production of inulinases, owing to the high yield of the enzymes
and their easy cultivation [8].
widely in the roots, tubers, and bulbs of plants [1]. Owing to its
abundance, inulin is considered a prominent renewable carbohydrate
source and can be used for the production of high fructose syrup (HFS),
fructooligosaccharides (FOSs), lactic acid, citric acid, ethanol, butane-
diol, and other products in the food, pharmaceutical, and bioenergy
industries, by either chemical or enzyme hydrolysis processes [2]. As
one of the major classes of industrial biocatalysts, inulinases have re-
cently received much attention for their potential use in inulin hydro-
lysis [3]. Inulinases are classified into two types: exo- and endo-in-
ulinases [4]. The endo-inulinases hydrolyze the internal linkages in
inulin to produce smaller oligosaccharides, whereas the exo-inulinases
split terminal fructose residues from the non-reducing end of inulin
Industrial utilization of enzymes generally requires enzymatic
characteristics such as stability with pH, temperature, chemicals, or-
ganic solvents, and salts. Enzymes obtained from extremophilic sources
(extremozymes) possess these characteristics and have numerous po-
tentials in versatile industrial usages, creating a great opportunity for
principal research on their structure's adaptation to function [9]. As a
result of their high stability, especially with salts, organic solvents, and
chemicals, halophilic enzymes have recently become of considerable
interest for biotechnological and industrial applications [10]. In addi-
tion, performing industrial processes under high-salt conditions can
reduce the risk of contamination and the total costs [11].
[
3,5]. Although the first inulin-hydrolyzing enzyme was isolated in
Growing interest of finding extreme enzymes has encouraged re-
searchers to isolate native strains from extreme ecological habitats for
innumerable industrial applications. Whereas knowledge has been accu-
mulating on inulinases production from mesophilic sources [4,5,8], Some
plants [6] and some plants can produce inulinases, inulin hydrolysis by
microbial inulinases sourced from filamentous fungi, yeast, and bac-
teria has been suggested as a promising approach for industrial appli-
cations, and many of the best-known microbial inulinases have been
extremophilic bacteria such as Marinimicrobium sp.,
a
marine
*
Corresponding author.
E-mail address: faramarz@tums.ac.ir (M.A. Faramarzi).
https://doi.org/10.1016/j.carres.2019.107746
Received 12 April 2019; Received in revised form 12 July 2019; Accepted 12 July 2019
Available online 13 July 2019
0008-6215/ © 2019 Elsevier Ltd. All rights reserved.

M. Yousefi-Mokri, et al.
Carbohydrate Research 483 (2019) 107746
Table 1
Purification summary of inulinase produced by A. filiformis.
Purification step
Total activity (U)
Total protein (mg)
Specific activity (U mg−1)
Yield (%)
Purification (fold)
Crude enzyme
Dialysed precipitate
Ultrafiltration
895.6
705.6
150.7
53.9
407.1
68.9
5.4
2.2
100.0
78.7
16.8
6
1.0
10.2
27.9
33.7
4.6
12.7
15.3
Ion exchange
1.6
Fig. 1. Influence of a) pH-temperature under temperatures of 25–75 °C at a pH range of 3.0–11.0; b) concentrations of salts in the range of 0.5–4.5 M on the enzyme
stability at 25 °C for 6 h. Results are shown as the mean of three independent experiments ± SD.
actinomycete, Nocardiopsis sp., Bacillus smithii, Xanthomonas oryzae, and
Sphingobacterium sp. have also been reported to produce characteristically
distinguished inulinases [12–16]. There have been few detailed studies of
the screening of halophilic bacteria, which have the ability to survive in
extreme environments and are capable of producing stable inulinases.
The main aim of this study is to introduce an extremophilic in-
ulinase from a hollophilic bacterium with considerable enzymatic
characterizations and its application in inulin degradation for
2

M. Yousefi-Mokri, et al.
Carbohydrate Research 483 (2019) 107746
Table 2
2.2. Isolation and identification of inulinase-producing bacteria
Effects of metal ions, chemical reagents, and detergents on inulinase stability.
The inulinase-producing bacteria were isolated from Aran-Bidgol
Namak) Lake (34°30′N 51°52′E), Aran-Bidgol County, Isfahan
Metal ion
Residual activity (%)
(
1
mM
5 mM
10 mM
Province, Iran. An isolate with the highest inulinase productivity was
selected for identification by 16S rDNA sequencing and phylogenetic
analysis. The selected strain was used for production of inulinase. The
More details of screening, isolation, and identification are presented in
the Supplementary material.
Control
100
100
100
3
2
+
+
+
+
Al
72.0 ± 0.9
121.2 ± 1.3
95.0 ± 1.2
75.4 ± 1.7
15.6 ± 0.7
105.0 ± 1.3
98.7 ± 1.7
101.3 ± 2.1
109.3 ± 2.4
108.4 ± 1.9
1 mM
65.7 ± 0.9
100.0 ± 2.0
88.9 ± 1.1
65.8 ± 1.5
1.7 ± 0.2
124.1 ± 1.6
107.7 ± 1.6
113.4 ± 2.1
137.7 ± 2.8
97.2 ± 1.1
2.5 mM
53.1 ± 1.0
92.1 ± 1.7
78.8 ± 1.0
36.4 ± 1.2
0
Ca
Cu
2
3
2
Fe
+
Hg
+
K
174.5 ± 2.2
128.6 ± 0.8
158.0 ± 2.3
135.5 ± 2.4
93.9 ± 1.3
5 mM
2
+
Mg
Mn
2
+
2.3. Purification and zymography of inulinase
2
2
+
+
Pb
Zn
All purification steps were performed at 4 °C. After 48 h of incuba-
tion in the fermentation medium, the bacteria were separated by cen-
trifugation at 8000×g for 15 min. The production medium containing
inulinase was concentrated with pre-chilled ethanol up to 80% (v/v)
saturation and stirred for 2 h. The dialyzed precipitate was dissolved in
buffer A (50 mM phosphate buffer, pH 7.5 with 0.5 M NaCl) and filtered
through a centrifugal ultrafiltration membrane with a cut-off value of
Chemical reagent
EDTA
105.6 ± 2.0
95.4 ± 1.5
25 mM
87.9 ± 2.1
50 mM
1
0 mM
CTAB
108.6 ± 1.9
1%
100 ± 2.1
2.5%
97.5 ± 1.2
5%
Detergents and inhibitors
Tween 80
98.3 ± 1.8
101.5 ± 2.4
81.3 ± 1.6
133.9 ± 2.6
95.2 ± 2.0
98.4 ± 1.6
78.8 ± 1.8
100.0 ± 2.2
91.7 ± 2.1
97.4 ± 1.9
74.4 ± 1.7
91.0 ± 1.6
Triton
2
-ME
5
0 kDa (Millipore, USA). The concentrated inulinase preparation was
SDS
dissolved in a minimum volume of buffer A and applied to a Q-
Sepharose XL column (16 × 130 mm), equilibrated with buffer A.
Unbound proteins were washed out with the same buffer, and then a
linear gradient of 0.5–2 mM NaCl in buffer A was subjected to a flow
Results shown are the average of three independent experiments ± S.D.
production of high fructose syrup (HFS) as a valuable alternative to
conventional sugar. This study reports the production, purification, and
immobilization of an extremophilic inulinase from the halophilic bac-
terium Alkalibacillus filiformis SR-061-HI with excellent stability in the
present of salts, detergents, and other chemical materials. To our
knowledge no literature report is found on production of inulinase of
this strain. The enzyme immobilization on cobalt ferrite magnetic na-
−1
rate of 120 mL h
and the fractions collected. The crude and purified
inulinase was electrophoresed on 12% polyacrylamide gel (SDS-PAGE)
and stained with coomassie blue. The purified inulinase was lyophilized
and stored at −20 °C for further investigations. Zymography was per-
formed by running SDS-PAGE, based on the method of Singh & Gill [3]
with some modifications.
noparticles (CoFe
2
4
O MNPs) was also performed and applied to the
enzymatic hydrolysis of inulin.
2.4. Characterization of the purified inulinase
2
. Materials and methods
2
.4.1. Optimal enzyme activity studies
The optimal pH-temperature was studied in the pH range of 6.5–8.5
2.1. Chemicals
and temperatures of 25–45 °C. The activity of the samples was de-
−
1
termined by DNS assay in a reaction containing 1 U mL
purified
Inulin (from Dahlia tubers), 3,5-dinitrosalicylic acid (DNS), tetra-
enzyme and 1% inulin. In order to establish the other optimal condi-
tions for the enzyme activity, the effects of NaCl concentration
ethyl orthosilicate (TEOS), and 2,3,5-triphenyltetrazolium chloride
were purchased from Sigma-Aldrich (St. Louis, MO, USA). Culture
media components and buffers were obtained from Merck (Darmstadt,
Germany). All other chemicals, reagents, and solvents were of analy-
tical grade from commercial sources.
(
(
0.5–3.5 M), incubation time (10–30 min), and enzyme–substrate ratio
0.1–0.5 U against 0.1–1.5%) on the inulinase were also investigated.
More details are provided in the Supplementary material.
Fig. 2. The effect of a) the amount of adsorbed inulinase on efficiency (IE %); b) the incubation time on the yield of the immobilized enzyme at 25 °C for 1–6 h.
3

M. Yousefi-Mokri, et al.
Carbohydrate Research 483 (2019) 107746
Fig. 3. Simultaneous effect of pH-temperature at 25–65 °C with pH values from 3.0 to 11.0 on the immobilized inulinase stability. The results are presented as the
mean of three independent experiments ± SD.
2
.4.2. Enzyme stability studies
(Meghnatis Kavir Kashan Co., Kashan, Iran) was employed to measure
the magnetic properties of the MNPs at room temperature.
Thermal and pH stability were examined simultaneously by pre-
incubating the purified inulinase in the pH range of 3.0–11.0 and at
temperatures of 25–75 °C for 6 h. The inhibition effect of some metal
ions, chemical reagents, and surfactants was investigated. The enzyme
osmo-stability was determined in KCl, LiCl, and NaCl at concentrations
of 0.5–4.5 M. The stability of the biocatalyst in the presence of some
organic solvent was tested by incubating inulinase in a phosphate buffer
containing the solvent at 20–80% concentrations for 6 h. Details of the
testing methods are provided in the Supplementary material.
2
.6. Immobilization of inulinase
Enzyme immobilization was performed on CoFe
2
O
4
MNPs by
covalent bonding. Briefly, 0.5 g of dry carrier was treated with 1%
TEOS in ethanol (v/v) for 6 h and dried at 40 °C overnight, followed by
activating with 5% glutaraldehyde in a phosphate buffer at 40 °C for
1
2 h. The immobilization was carried out by incubating the enzyme (5
U) with the carrier (5 mg) in 1 mL of phosphate buffer (50 mM, pH 7.5)
at 25 °C for 1–6 h. The immobilized inulinase was magnetically sepa-
rated and washed with the same buffer. The immobilized enzyme was
finally stored in a phosphate buffer at 4 °C. The immobilization effi-
ciency (IE) and yield (IY) were calculated according to the following
2
.4.3. Determination of the exo- or endo-nature of the enzyme
Hydrolytic products produced by the inulinase were qualitatively
identified by thin-layer chromatography (TLC). The TLC plates (silica
gel 60) were developed by a mixture of acetic acid-chloroform-water
(
7:6:1, v/v/v) twice, and reducing sugars were visualized by staining
equations: IE (%) = [E
] × 100, where E and E
ulinase activity, respectively, and Y
total protein and unbounded protein to the substrates, respectively.
1
/E
o
] × 100, and IY (%) = [(Y
represent the total and immobilized in-
and Y indicate the amounts of
o
− Y )/
1
reagent containing aniline in acetone (2%)-diphenylamine in acetone
2%)-phosphoric acid (85%) (5:5:1, v/v/v) [17]. Carbohydrate ap-
Y
o
o
1
(
o
1
peared as dark green spots after heating for 10 min at 70 °C. Glucose,
−1
fructose, and sucrose were used in concentration of 2.5 mg mL
sugar standards.
as
2
.6.1. Effects of temperature and pH on the immobilized enzyme stability
The effects of pH and temperature on inulinase stability were in-
2.5. Synthesis and characterization of CoFe
2
O
4
MNPs
vestigated simultaneously at 25–65 °C, using a series of buffers with pH
values from 3.0 to 11.0, including citrate (pH 3.0–5.0), phosphate (pH
CoFe MNPs were synthesized according to Sharifi et al., [18].
2 4
O
6
.0–8.0), Tris-HCl (pH 8.5–9.5), and glycine-NaOH (pH 10.0–11.0)
The preparation of the support was performed by the co-precipitating
method. Briefly, the mixed solutions of FeCl .6H O (40 mL, 0.02 M) and
CoCl .6H O (4 mL, 0.01 M) at 50 °C were added to a boiling solution of
NaOH (200 mL, 1 M) and stirred for 30 min, and then cooled at room
buffers.
3
2
2
2
2.6.2. Reusability of the immobilized inulinase
temperature. CoFe
2
O
4
MNPs were collected by an external magnet,
The reusability of the immobilized inulinase was investigated for 15
hydrolysis runs. After using the immobilized enzyme in the first cycle,
the support was collected and washed three times with phosphate
buffer (50 mM, pH 7.5), and the inulinase activity was then re-calcu-
lated. The reusability was determined by measuring the efficiency in
each reaction run. The inulinase activity was defined as 100% in the
first cycle and as relative activity during the subsequent cycles.
followed by washing several times with distilled water and thereafter
with acetone. The surface morphology study of the support was in-
vestigated by field emission scanning electron microscopy (FE-SEM).
The crystallographic pattern of nanoparticles (NPs) was recorded with
an X-ray diffraction system (XPert-MPD) using a Cu (Kα) radiation
source (λ: 1.5406 Å).
A vibrating-sample magnetometer (VSM)
4

M. Yousefi-Mokri, et al.
Carbohydrate Research 483 (2019) 107746
Fig. 4. Time-course analysis of fructose production by a) free and b) immobilized inulinase in the presence of 1–20% inulin as substrate and during 48 h of
incubation. The results are presented as mean of three independent experiments ± SD.
2.7. Enzymatic hydrolysis of inulin by the free and immobilized inulinase
2.8. Statistical analysis
The inulin hydrolysis was performed in phosphate buffer A (70%,
All the experiments were carried out in triplicate, and the results
were reported as mean ± standard deviation. One-way and two-way
ANOVA tests were used to measure the statistical significance between
the mean values (SigmaPlot version 12.0, Systat Software, Inc., San
Jose, CA). Probability values < 0.05 were considered to be significant.
w/v), the enzyme (5%, w/v) and inulin (1–20%, w/v) in shaken flasks at
1
50 rpm with a total volume of 10 mL. The content of reducing sugars,
including fructose and glucose, was investigated during 48 h of in-
cubation of the free and immobilized enzyme in the presence of 1–20%
inulin under agitation at 150 rpm. The total reducing sugar was de-
termined by the Somogyi-Nelson method with some modifications
based on Shao & Lin [19].D-glucose was measured using the Glucostat
method [20]. The amount of D-fructose was obtained by the difference
between the value of total reducing sugars and D-glucose.
3. Results and discussion
3.1. Isolation and molecular identification of the selected strain
The selected strain was identified as Alkalibacillus filiformis SR-061-
HI (Gene-Bank accession No. KY563119), based on 16S rRNA sequence
5

M. Yousefi-Mokri, et al.
Carbohydrate Research 483 (2019) 107746
analysis. To the best of our knowledge, this is the first reported study on
the isolation of a halophilic Alkalibacillus producing extracellular in-
ulinase.
3.4. Inulinase immobilization
The characterizations of support are presented in the
Supplementary material. The enzyme immobilization was performed on
the surface of the fabricated MNPs with covalent bonding. The mag-
netic properties of the support facilitate rapid separation and easy re-
covery, with minimal operational cost, using an external magnet.
Furthermore, enzyme immobilization by covalent attachment increases
the overall rigidity of the protein molecules. The results of the inulinase
3.2. Enzyme purification
The halophilic enzymes remain highly soluble in high salt media
unlike their non-halophilic proteins and they cannot easily be pre-
cipitated [21–24]. Ethanol precipitation is usually used instead of am-
monium sulfate precipitation for halophilic enzymes [20,25,26]. The
precipitated proteins by ethanol (78.7% recovery and 4.6 times of
purification fold) were subjected to ultrafiltration, followed by anion
exchange chromatography of the active fraction. Thereafter, the pur-
ified fraction was eluted at 1.5 M of NaCl. Anion exchange chromato-
graphy efficiently purified the enzyme, with a recovery yield and spe-
immobilization on CoFe
2
O MNP as an efficient and available support
4
are displayed in Fig. 2. The immobilization efficiency attained a max-
−
1
imum value (65%) at a protein concentration of 125 μg mL (Fig. 2a).
According to Fig. 2b, the maximum yield (85%) of CoFe MNP@
2 4
O
inulinase was obtained with an immobilization time of 5 h at pH 7.0.
−
1
Under these conditions, the maximum activity (equal to 502.5 U g of
−
1
−1
cific activity of 6% and 33.6 U mg
(Table 1). The results of the
CoFe
2
O MNP) was achieved with a protein loading of 20 mg g of the
4
purification, zymographiy, and exo- or endo-nature of the purified en-
zyme are showed in the Supplementary material.
carrier.
3
.4.1. Effect of pH and temperature on immobilized inulinase stability
3.3. Enzyme characteristics
The stability of the immobilized inulinase was investigated at tem-
peratures of 25–75 °C and pH range of 3.0–11.0 (Fig. 3). The free en-
zyme showed maximum relative activity at pH 7–8 and temperatures of
35–45 °C (Fig. 1a), whereas the immobilized inulinase exhibited the
maximum activity at pH 6–9 and 45 °C. The free enzyme showed 50%
3
.3.1. Optimal conditions for inulinase activity
The maximum enzyme activity was assigned at 40 °C and pH 7.0,
according to a wide range of pH-temperature trials. Similarly, the in-
ulinases produced by Arthrobacter sp. [27], Sphingobacterium sp. [28],
Xanthomonas sp. [29], and Bacillus polymyxa [30] also revealed the
highest activity at the same pH and temperature. Bacterial inulinases
usually exhibit maximal activity in the neutral pH values and at higher
temperatures [3,7]. In addition, the maximum inulinase activity was
observed in 2 M NaCl and at 30 min incubation in the presence of 0.25
maximum activity at pH 4, while CoFe
2
O MNP@inulinase revealed
4
about 70% of the relative activity. The immobilized enzyme exhibited a
remarkably wide temperature profile and maintained 80% of activity in
the temperature range of 25–55 °C and pH 6.0–9.0. Consequently, the
results showed that the immobilized inulinase demonstrated higher pH
and temperature stability compared with the free enzyme.
−
1
U mL of the enzyme and 0.75% of inulin (see more descriptions in the
Supplementary material).
3.4.2. Reusability of the immobilized inulinase
The reusability and the easy separation of immobilized enzyme from
the reaction medium are important factors in industrial processes. After
the first use of immobilized inulinase, the support was collected by an
external magnet and washed with phosphate buffer (50 mM, pH 7.0) in
order to repeat the reaction. The relative activity of the immobilized
enzyme fell below 60% after 10 repeated reactions in batch conditions.
Singh et al. [3], investigated the reutilization capacity of the im-
mobilized inulinase on Duolite A568, using glutaraldehyde, and re-
ported a 20% loss at the seventh cycle of using. In another study, the
inulinase immobilized via cross-linking and covalent methods main-
tained 80.75% and 65.45%, respectively, of its initial activities after
seven runs of hydrolysis [39].
3
.3.2. Enzyme stability
As shown in Fig. 1a, the stability of the enzymes was achieved in the
pH range of 7.0–9.0 and temperature 25–55 °C. The enzyme pH and
thermo-stability were significantly higher compared with thermophilic
inulinases from Bacillus sp. SG7 [31] and Nocardiopsis sp. DN-K15 [32]. In
addition, the inulinase was stable at NaCl concentration between 0 and
5
M, and no loss of activity was observed at a NaCl concentration of 2–3 M
for 6 h at 25 °C (Fig. 1b). However, Zhou et al., [28] reported a salt-tol-
erant inulinase from Sphingomonas sp. JB13 with high tolerance to a
broad range of NaCl concentrations, from 0.2 to 4.5 M. The addition of
metal ions showed a neutral or reinforcing effect on inulinase activity,
2+
3+
2+
3+
with the exception of Hg , Fe , Cu , and Al (Table 2). Both similar
and conflicting results have been reported for other bacterial inulinases
3.5. Enzymatic hydrolysis of inulin by free and immobilized inulinase
[27,30,33,34]. The enzyme activity was retained at more than 75% in the
presence of most chemical reagents (Table 2). Correct folding of enzymes
is mainly based on hydrophobic interactions in the core and hydrophilic
interactions on the surface of proteins. Surfactants can interact with the
hydrophobic moieties of proteins, potentially leading to an unfolding and
inactivation [35,36]. A detergent-stable enzyme indicates resistance
against unfolding caused by surfactants. Binding the enzymes to a surface
also makes them more stable and less likely to denature [37]. In this
study, an extremophilic inulinase with notable enzymatic characteristics
The obtained products of the enzymatic hydrolysis of inulin, such as
HFS and FOSs, are utilized as substitutes for conventional low-calorie
sweeteners in various industrial processes. Recently, enzyme im-
mobilization has been employed as an efficient technique for the eco-
nomical utilization of enzymes, as it suffers reduced loss of initial ac-
tivity and it improves the structural stability of the enzymes Singh et al.,
[3]. In the present study, the content of fructose was monitored during
48 h of incubation 1–20% inulin in the presence of free and im-
mobilized enzyme under agitation at 150 rpm. The results of hydrolysis
at various concentrations of the substrate and incubation times revealed
that fructose production increased rapidly during the first 24 h in the
presence of the highest amount of inulin and remained stable between
24 and 48 h in the case of free enzyme (Fig. 4). Under optimal condi-
tions, the production of fructose from the substrate (20% w/v) in the
was immobilized on cobalt ferrite magnetic nanoparticles (CoFe
2 4
O
MNPs) as a support. The immobilization of inulinase along with its con-
siderable enzymatic characterizations can be reasons for the enzyme
stability in the presence of detergents. These results are in good agree-
ment with findings from other studies [27,32]. The halophilic inulinase
indicated impressive stability in the presence of a wide range of organic
solvents at high concentrations up to 80% v/v (see more explanations in
the Supplementary material). Silva et al. [38], claimed the enzymatic
production of FOSs by inulinases from Kluyveromyces marxianus and As-
pergillus niger in an organic medium is more efficient than an aqueous
medium.
−1
−1
presence of free and immobilized enzyme was 122 g L and 160 g L
,
with 61% and 80% conversion, respectively. Numerous studies have
reported on the enzymatic hydrolysis of inulin by immobilized in-
ulinases. Kim et al. [40], reported 90% conversion of inulin (7%, w/v)
from Jerusalem artichoke extract during 20 h, with a 76% yield of
6

M. Yousefi-Mokri, et al.
Carbohydrate Research 483 (2019) 107746
fructose. In a batch system, the hydrolysis of inulin (5%) from Jer-
usalem artichoke extract has been reported at 93% in 3 h. In another
study, an immobilized inulinase was successfully applied to the pro-
duction of HFS from Asparagus racemosus raw inulin and pure inulin
exoinlinase production by semicontinuous cultivation of membrane-immobilized
Bacillus sp. 11 cells, Process Biochem. 37 (2002) 863–868.
[
[
15] N. Laowklom, R. Chantanaphan, P. Pinphanichakarn, Production, purification and
characterization of inulinase from a newly isolated Streptomyces sp. CP01, Nat.
Resour. 3 (2012) 137–144.
−
1
−1
16] J. Zhou, Y. Gao, R. Zhang, M. Mo, X. Tang, J. Li, B. Xu, J. Ding, Z. Huang, A novel
low-temperature-active exo-inulinase identified based on molecular-activity
strategy from Sphingobacterium sp. GN25 isolated from feces of Grus nigricollis,
Process Biochem. 49 (2014) 1656–1663.
(
5%), which yielded 39.2 g L and 40.2 g L of fructose, with 85.5%
and 92.6% conversion, respectively. Singh et al. [41], investigated in-
ulinase immobilization on chitosan beads, and maximum hydrolysis of
inulin, 78.2%, was obtained at 125 rpm after 4 h.
[17] B. Yuan, N. Hu, J. Sun, S.-A. Wang, F.-L. Li, Purification and characterization of a
novel extracellular inulinase from a new yeast species Candida kutaonensis sp. nov.
KRF1 T, Appl. Microbiol. Biotechnol. 96 (6) (2012) 1517–1526.
4
. Conclusion
[
18] I. Sharifi, H. Shokrollahi, M.M. Doroodmand, R. Safi, Magnetic and structural stu-
2 4
dies on CoFe O nanoparticles synthesized by co-precipitation, normal micelles and
reverse micelles methods, J. Magn. Magn. Mater. 324 (10) (2012) 1854–1861.
19] Y. Shao, A.H.-M. Lin, Improvement in the quantification of reducing sugars by
miniaturizing the Somogyi-Nelson assay using a microtiter plate, Food Chem. 240
(2018) 898–903.
Currently, HFS is produced from starch by multi-enzymatic method
[
involving action of three amylolytic enzymes with a 45% maximum
yield for fructose. Immobilized inulinases can be replaced as the cost-
effective single-step enzymatic method. The present work reports the
enzymatic hydrolysis of inulin with a 80% conversion by an im-
mobilized extremophilic endo-inulinase from the halophilic bacteria A.
[
[
20] S. Kanazawa, K. Miyashita, A modified method for determination of cellulase ac-
tivity in forest soil, J. Soil Sci. Plant Nutr. 32 (1) (1986) 71–79.
21] L. Costenaro, G. Zaccai, C. Ebel, Link between Protein− solvent and weak Protein−
protein interactions gives insight into halophilic adaptation, Biochemistry 41 (44)
(2002) 13245–13252.
−
1
filiformis with specific inulinase activity of 33.7 U mg .The purified
enzyme revealed notable characteristics of tolerance to salts, organic
solvents, metals, chemicals, and pH-temperature that could reduce the
risk of microbial contamination and the total costs. The immobilized
inulinase was demonstrated to maintain most of its activity after 10
runs, making it a promising candidate for the production of HFS.
[
[
22] C. Ebel, L. Costenaro, M. Pascu, P. Faou, B. Kernel, F. Proust-De Martin, G. Zaccai,
Solvent interactions of halophilic malate dehydrogenase, Biochemistry 41 (44)
(2002) 13234–13244.
23] C. Ebel, P. Faou, B. Kernel, G. Zaccai, Relative role of anions and cations in the
stabilization of halophilic malate dehydrogenase, Biochemistry 38 (28) (1999)
9
039–9047.
[24] R. Sinha, S.K. Khare, Protective role of salt in catalysis and maintaining structure of
halophilic proteins against denaturation, Front. Microbiol. 5 (2014) 165.
[
25] H. Onishi, T. Kobayashi, M. Kamekura, Purification and some properties of an ex-
tracellular halophilic 5′-nucleotidase from a moderate halophile, Micrococcus
varians subsp. halophilus, FEMS Microbiol. Lett. 24 (2–3) (1984) 303–306.
Acknowledgement
Research reported in this publication was supported by Elite
Researcher Grant Committee under award number 965408 from the
National Institutes for Medical Research Development (NIMAD),
Tehran, Iran to M.A.F.
[26] M. Vidyasagar, S. Prakash, V. Mahajan, Y.S. Shouche, K. Sreeramulu, Purification
and characterization of an extreme halothermophilic protease from a halophilic
bacterium Chromohalobacter sp. TVSP101, Braz. J. Microbiol. 40 (1) (2009) 12–19.
[
27] J. Zhou, Y. Gao, R. Zhang, M. Mo, X. Tang, J. Li, B. Xu, J. Ding, Z. Huang, A novel
low-temperature-active exo-inulinase identified based on Molecular-Activity
strategy from Sphingobacterium sp. GN25 isolated from feces of Grus nigricollis,
Process Biochem. 49 (10) (2014) 1656–1663.
Appendix A. Supplementary data
[
[
[
28] J. Zhou, M. Peng, R. Zhang, J. Li, X. Tang, B. Xu, J. Ding, Y. Gao, J. Ren, Z. Huang,
Characterization of Sphingomonas sp. JB13 exo-inulinase: a novel detergent-, salt-,
and protease-tolerant exo-inulinase, Extremophiles 19 (2) (2015) 383–393.
29] J. Park, J. Bae, D. You, B. Kim, J. Yun, Production of inulooligosaccharides from
inulin by a novel endoinulinase from Xanthomonas sp, Biotechnol. Lett. 21 (12)
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.carres.2019.107746.
(
1999) 1043–1046.
30] H.-J. Kwon, S.-J. Jeon, D.-J. You, K.-H. Kim, Y.-K. Jeong, Y.-H. Kim, Y.-M. Kim, B.-
W. Kim, Cloning and characterization of an exoinulinase from Bacillus polymyxa,
Biotechnol. Lett. 25 (2) (2003) 155–159.
References
[
[
[
[
[
[
[
1] K. Vijayaraghavan, D. Yamini, V. Ambika, N. Sravya Sowdamini, Trends in in-
ulinase production–a review, Crit. Rev. Biotechnol. 29 (1) (2009) 67–77.
2] C. Neagu, G. Bahrim, Inulinases-a versatile tool for biotechnology, Innov. Rom.
Food Biotechnol. 9 (2011) 1.
[31] S. Gavrailov, V. Ivanova, Isolation and characteristics of a thermophilic Bacillus
strain, producer of inulinase, J. Biosci. Biotechnol. (2014) 83–94.
[32] W.-D. Lu, A.-X. Li, Q.-L. Guo, Production of novel alkalitolerant and thermostable
inulinase from marine actinomycete Nocardiopsis sp. DN-K15 and inulin hydrolysis
by the enzyme, Ann. Microbiol. 64 (2) (2014) 441–449.
3] P. Singh, P.K. Gill, Production of inulinases: recent advances, Food Technol.
Biotechnol. 44 (2) (2006) 151.
[33] M. Warchol, S. Perrin, J.P. Grill, F. Schneider, Characterization of a purified
β‐fructofuranosidase from Bifidobacterium infantis ATCC 15697, Lett. Appl.
Microbiol. 35 (6) (2002) 462–467.
4] N. Kango, S.C. Jain, Production and properties of microbial inulinases: recent ad-
vances, Food Biotechnol. 25 (3) (2011) 165–212.
5] Z. Chi, Z. Chi, T. Zhang, G. Liu, L. Yue, Inulinase-expressing microorganisms and
applications of inulinases, Appl. Microbiol. Biotechnol. 82 (2) (2009) 211–220.
6] P. Rutherford, A. Deacon, β-Fructofuranosidases from roots of dandelion
[34] J. Zhou, Q. Lu, M. Peng, R. Zhang, M. Mo, X. Tang, J. Li, B. Xu, J. Ding, Z. Huang,
Cold-active and NaCl-tolerant exo-inulinase from a cold-adapted Arthrobacter sp.
MN8 and its potential for use in the production of fructose at low temperatures, J.
Biosci. Bioeng. 119 (3) (2015) 267–274.
(
Taraxacum officinale Weber), Biochem. J. 126 (3) (1972) 569.
7] P.K. Gill, A.D. Sharma, R.K. Harchand, P. Singh, Effect of media supplements and
culture conditions on inulinase production by an actinomycete strain, Bioresour.
Technol. 87 (3) (2003) 359–362.
[35] D.E.R. Otzen, Protein unfolding in detergents: effect of micelle structure, ionic
strength, pH, and temperature, Biophys. J. 83 (2002) 2219–2230.
[36] D.E.R. Otzen, Protein-surfactant interactions: a tale of many states, Biochim.
Biophys. Acta 1814 (2011) 562–591.
[
[
8] H.K. Rawat, H. Soni, H. Treichel, N. Kango, Biotechnological potential of microbial
inulinases: recent perspective, Crit. Rev. Food Sci. Nutr. 57 (18) (2017) 3818–3829.
9] R. Rezaie, S. Rezaei, N. Jafari, H. Forootanfar, M.R. Khoshayand, M.A. Faramarzi,
Delignification and detoxification of peanut shell bio-waste using an extremely
halophilic laccase from an Aquisalibacillus elongatus isolate, Extremophiles 21 (6)
[37] H. Hellmuth, M. Dreja, Understanding interactions of surfactants and enzymes:
impact of individual surfactants on stability and wash performance of protease
enzyme in detergents, Tenside Surfactants Deterg. 53 (5) (2016) 502–508.
[38] M.F. Silva, D. Rigo, V. Mossi, S. Golunski, G. de Oliveira Kuhn, M. Di Luccio,
R. Dallago, D. de Oliveira, J.V. Oliveira, H. Treichel, Enzymatic synthesis of fruc-
tooligosaccharides by inulinases from Aspergillus niger and Kluyveromyces marxianus
NRRL Y-7571 in aqueous–organic medium, Food Chem. 138 (1) (2013) 148–153.
[39] M. Karimi, M. Habibi-Rezaei, K. Rezaei, A.A. Moosavi-Movahedi, J. Kokini,
Immobilization of inulinase from Aspergillus niger on octadecyl substituted nano-
porous silica: inulin hydrolysis in a continuous mode operation, Biocatalysis Agric.
Biotechnol. 7 (2016) 174–180.
(
2017) 993–1004.
[
10] N. Jafari, S. Rezaei, R. Rezaie, H. Dilmaghani, M.R. Khoshayand, M.A. Faramarzi,
Improved production and characterization of a highly stable laccase from the ha-
lophilic bacterium Chromohalobacter salexigens for the efficient delignification of
almond shell bio-waste, Int. J. Biol. Macromol. 105 (2017) 489–498.
11] V. Kumar, G.S. Bisht, S.B.S.P.G. Institu, An improved method for isolation of
genomic DNA from filamentous actinomycetes, J. Sci. Engg. Tech. Mgt 2 (2010) 2.
12] W.D. Lu, A.X. Li, Q.L. Guo, Production of novel alkalitolerant and thermostable
inulinase from marine actinomycete Nocardiopsis sp. DN-K15 and inulin hydrolysis
by the enzyme, Ann. Microbiol. 64 (2014) 441–449.
[
[
[40] W.Y. Kim, S.M. Byun, T.B. Uhm, Hydrolysis of inulin from Jerusalem artichoke by
inulinase immobilized on aminoethylcellulose, Enzym. Microb. Technol. 4 (4)
(1982) 239–244.
[
[
13] A.X. Li, L.Z. Guo, W.D. Lu, Alkaline inulinase production by a newly isolated bac-
terium Marinimicrobium sp. LS–A18 and inulin hydrolysis by the enzyme, World J.
Biol. Psychiatry 28 (2012) 81–89.
[41] R.S. Singh, K. Chauhan, J.F. Kennedy, A panorama of bacterial inulinases: pro-
duction, purification, characterization and industrial applications, Int. J. Biol.
Macromol. 96 (2017) 312–322.
14] K. Uzunova, A. Vassileva, V. Ivanova, D. Spasova, A. Tonkova, Thermostable
7