Inulinase immobilisation in PAA/PEG composite for efficient fructooligosaccharides production

Article abstract of DOI:10.1080/10242422.2020.1858815

Inulinase was immobilised by entrapment method in polyacrylamide/polyethylene glycol composite and evaluated for its efficiency for short-chain fructooligosaccharides (3–6 degrees of polymerisation) production in batch hydrolysis system. Aqueous two-phase

Full text of DOI:10.1080/10242422.2020.1858815

Biocatalysis and Biotransformation
ISSN: (Print) (Online) Journal homepage: https://www.tandfonline.com/loi/ibab20
Inulinase immobilisation in PAA/PEG composite for
efficient fructooligosaccharides production
Mishela Temkov, Darko Dimitrovski, Elena Velickova & Albert Krastanov
To cite this article: Mishela Temkov, Darko Dimitrovski, Elena Velickova & Albert Krastanov
(2020): Inulinase immobilisation in PAA/PEG composite for efficient fructooligosaccharides
production, Biocatalysis and Biotransformation, DOI: 10.1080/10242422.2020.1858815
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BIOCATALYSIS AND BIOTRANSFORMATION
https://doi.org/10.1080/10242422.2020.1858815
RESEARCH ARTICLE
Inulinase immobilisation in PAA/PEG composite for efficient
fructooligosaccharides production
a,b
a
a
b
Mishela Temkov , Darko Dimitrovski , Elena Velickova and Albert Krastanov
a
Faculty of Technology and Metallurgy, Ss. Cyril and Methodius University in Skopje, Skopje, Republic of North Macedonia;
Faculty of Technology, University of Food Technologies, Plovdiv, Bulgaria
b
ABSTRACT
ARTICLE HISTORY
Inulinase was immobilised by entrapment method in polyacrylamide/polyethylene glycol com-
posite and evaluated for its efficiency for short-chain fructooligosaccharides (3–6 degrees of
polymerisation) production in batch hydrolysis system. Aqueous two-phase polymerisation tech-
nique was used to synthesise the composite, where aqueous polyethylene glycol 1000 contain-
ing the enzyme was used as dispersant with ammonium persulfate as initiator. The
characteristics of free and immobilised inulinase were investigated and compared, and the
results showed shift of pH and temperature optimum and change in stability caused by the
immobilisation material. The immobilised preparation retained 50% of its initial activity after 20
successive batch cycles of 1 h each. The conversion degree of highly polymerised inulin to fruc-
tooligosaccharides (3–6 degrees of polymerisation) was 36% when using 30% PAA/PEG, w/v.
Received 4 September 2020
Revised 20 November 2020
Accepted 26 November 2020
KEYWORDS
Inulinase; immobilisation;
fructooligosaccharides; poly-
acrylamide/polyethylene
glycol composite;
characterisation
HIGHLIGHTS
ꢀ
ꢀ
ꢀ
ꢀ
ATPS extracted inulinase was effectively immobilised in PAA/PEG composite.
Immobilised enzyme showed good pH and thermal stability.
Immobilised catalyst presents good retention of activity after 20 reuses.
The entrapped enzyme is effective in producing FOS with the DP from 3 to 6 with proved
prebiotic activities.
ꢀ
The optimum conditions for batch operating mode were: 2% (w/v) inulin, 30% (w/v) PAA/PEG
composite, pH ¼ 4.4 and T ¼ 40 C.
ꢁ
Abbreviations: ATPS: aqueous-two phase systems; APS: ammonium persulfate; DP: degree of
polymerisation; FFN: fructofuranosyl nystose; FOS: fructooligosaccharides; Mw: molecular weight;
PAA: polyacrylamide; PAH BAH: 4-hydroxybenzoic acid hydrazide; PEG: polyethylene glycol; TLC:
thin-layer chromatography
Introduction
short-chain fatty acids like butyrate, propionate, and
acetate (Bali et al. 2015). Because of this, their caloric
value is estimated at 25–35% of a fully digested fruc-
tose (Roberfroid 1999). FOSs act as prebiotics by
selective stimulation of growth and sustainability of
beneficial microbiota in the gut, especially bifidobacte-
ria and lactobacilli (Oliveira et al. 2011). In addition to
low caloric value and prebiotic effect, these oligosac-
charides can induce plenty of other physiological
effects after their consumption (Flamm et al. 2001).
They can improve gut absorption of calcium and mag-
nesium; reduce the levels of serum cholesterol, phos-
pholipids, and triglycerides; increase faecal weight, act
Fructooligosaccharides (FOS) are carbohydrates con-
sisting of 2–20 polymerised fructose units linked by
b-2,1-glycosidic bonds with or without a terminal glu-
cose residue. They have a low degree of polymerisa-
tion (DP), thus low molecular weight (Mw), whereas
inulin has the same chemical composition but high
DP and therefore high molecular weight (Roberfroid
and Slavin 2000). Due to the lack of hydrolysing
enzymes in the human upper gastrointestinal tract,
these compounds are non-digestible (Mao et al. 2019).
However, they become hydrolysed and extensively fer-
mented by the microbiome in the large intestine to
CONTACT Mishela Temkov
mishela@tmf.ukim.edu.mk
Department of Food Technology and Biotechnology, Faculty of Technology and Metallurgy,
Ss. Cyril and Methodius University in Skopje, Rudjer Boskovic 16, 1000 Skopje, Republic of North Macedonia
Supplemental data for this article can be accessed here.
ß 2020 Informa UK Limited, trading as Taylor & Francis Group

2
M. TEMKOV ET AL.
as a dietary fibre and maintain glucose homeostasis
Jackson et al. 1999; Bali et al. 2015; Roberfroid 2005).
substrate concentration and only 4.97% of non-pre-
biotic saccharides in the fermentation broth. In
another study, Park et al. (1999) reported a maximum
yield of about 86% of FOS on a total sugar basis with
1% initial inulin concentration, using partially purified
enzyme from Xanthomonas sp. High yield of 84.8%
FOS was also achieved by Singh et al. 2016 (Singh
et al. 2017) when they hydrolysed 5% inulin solution
by yeast inulinase immobilised on chitosan beads after
(
These effects are revealed in a number of health bene-
fits such as: reducing gastroenteritis, colon cancer,
allergies, obesity, cardiovascular disease, and osteopor-
osis (Kleessen et al. 2001; Coxam 2005; Fernandes
et al. 2017; Nobre et al. 2018). Moreover, in recent
studies the symbiotic effects of Lactobacillus strains
and FOS have been used to modulate the immunity
and prevent or treat neonatal sepsis (Panigrahi et al.
5
h of reaction. Purified endoinulinase isolated from A.
2
017; Sch u€ ller et al. 2018). Due to above mentioned
arachidicola CBS 117610 was also used for the efficient
production of inulooligosaccharides from inulin. The
inulooligosaccharides yields with DP3 and DP4 as
major products reached 80% when 5% of inulin was
used as substrate (Jiang et al. 2019).
Immobilisation protocols have to ensure good
retention of enzyme activity, long term stability and
desired physical properties for a large scale operation.
A number of matrices and methods have been used
for inulinase immobilisation applied for FOS produc-
tion in batch systems. Enzymes were physically
adsorbed on porous support, covalently attached to
the activated surface, crosslinked into crystals or
entrapped in polyacrylamide gels (Whitesides et al.
health benefits and physiological effects, FOS and inu-
lin have been extensively applied in the food industry
as prebiotic and dietary fibres, sugar or fat replacers,
texture modifiers and stabilisers (Aidoo et al. 2014;
Salvatore et al. 2014; Krasaekoopt and Watcharapoka
2014; Tiengtam et al. 2015; Liu et al. 2016). FOS can
be obtained by enzymatic synthesis when several fruc-
tose molecules are added to the substrate sucrose
(
transfructosylation) using the fungal enzymes fructo-
syltransferase (FTase; 2.4.1.9) and b- fructofuranosidase
FFase 3.2.1.26) with maximum theoretical yield from
(
0
2
.55 to 0.60 gFOS/gSucrose (Bali et al. 2015; Han et al.
017). The resulting solution is mainly composed of 1-
kestose (GF ), nystose (GF ), and fructofuranosyl nys-
2
3
1
979; Tumturk et al. 2007; Singh et al. 2017; Temkov
tose (GF ), all of which have terminal glucose. On the
4
et al. 2019).
other hand, FOS can be also produced with a high
yield by inulin hydrolysis using endo-inulinases (EC
In this work, previously developed composite, con-
sisting of polyacrylamide (PAA) and polyethylene gly-
col (PEG) (Guorong and Zhihai 2009; Xu et al. 2010),
was for the first time employed for inulinase immobil-
isation by entrapment. The two components polymer-
ised in an aqueous two-phase system using
ammonium persulfate (APS) as initiator. Inulinase was
extracted from newly isolated Bacillus sp. 11/3 from
thermal water samples from the region of Velingrad,
Bulgaria (Gavrailov and Ivanova 2015) and was par-
tially purified in PEG rich phase from the ATPS system,
as described in our previous study (Temkov et al.
3
.2.1.7), where the product may not contain terminal
glucose (Mao et al. 2019). In both cases, glucose, fruc-
tose and sucrose are produced as non-prebiotic by-
products, which reduce FOS’s prebiotic activity, and
they should be removed from the process by nanofil-
tration, activated charcoal system and ion-exchange
chromatography or microbial treatment (Nobre et al.
2018; Picazo et al. 2018; Jiang et al. 2019). FOS with
DP 3-9 have been shown to be the most suitable
source of carbon for bacterial growth compared to
FOS with DP 10-17 and Inulin DP ꢂ 23 (Fu et al.
2
018). The separation of the enzyme from the PEG
2018). The increased use of fructooligosaccharides in
phase is a time-consuming step that can be omitted
by crosslinking the PEG phase (containing the enzyme)
with PAA, yielding an immobilised system entrapping
the entire amount of the enzyme. The immobilised
enzyme was characterised for its pH and temperature
optimum, its stability, as well as storage stability and
reusability. At the same time, the characteristics were
compared to those of the free enzyme. The immobi-
lised enzyme was used for FOS production in a batch
system. The distribution of FOS with different DP’s
was evaluated with specific attention to the produc-
tion of FOS with DP between 3 and 6.
the food industry resulted in exploring “new” micro-
bial enzymes for their production. However, industrial
production of FOS requires stable, highly active
enzyme with low-priced immobilisation preparation
under industrial conditions.
Nobre et al. (2018) synthesised FOS from sucrose
with 0.64 gFOS/ginitial sucrose yield after 38 h of fermenta-
tion, whereas Han et al. (2017) hydrolysed inulin to
FOS by using engineered Yarrowia lipolytica strain
Enop56, in which an optimised endo-inulinase gene
from Aspergillus niger was over-expressed. They
obtained a high 0.91 gFOS/gInulin yield from 6% initial

BIOCATALYSIS AND BIOTRANSFORMATION
3
Materials and methods
for solidification. The composites were left to air dry
for 48 h and ground prior to further tests. Taking into
consideration the densities of the used solutions, the
total amount of obtained solid biocatalyst was 6.38 g.
The composite was washed with distilled water twice
to remove unbound protein and the supernatant was
examined for protein and activity recovery. The
obtained immobilised biocatalyst had an enzyme load
in a concentration of 45.84 U/g. This biocatalyst was
subjected to further characterisation and used in
batch inulin hydrolysis in different amounts.
Materials
Acrylamide (AA), N,N’-methylene-bis-acrylamide (MBA),
N,N,N’,N’-tetramethyl-ethylenediamine (TEMED) and
ammonium persulfate (APS) were purchased from
Sigma Aldrich, USA. Polyethylene glycol (PEG) (Mw
¼
1000 g/mol) was supplied by Merck, Germany. Highly
polymerised inulin (DP > 25) extracted from Jerusalem
artichoke was donated by Beneo, Belgium. 4-
Hydroxybenzhydrazide (PAH-BAH) was obtained from
Alfa Aesar, Germany. Bradford reagent and bovine
serum albumin (BSA) were purchased from Bio-Rad,
USA. All chemicals were of analytical grade.
Extracellular inulinase was produced by submerged
cultivation of Bacillus sp. 11/3 and purified in an aque-
ous two-phase system.
Inulinase activity assay of free and
immobilised enzyme
Inulinase activity assay of the free enzyme was per-
formed by incubating 1 mL partially purified enzyme
solution from the PEG phase (ATPS purification
method) with 2% (w/v) inulin prepared in phosphate
2
–
ꢁ
H PO /HPO ) buffer pH 8.0 at 50 C for 20 min.
2 4– 4
Inulinase production and purification
(
After incubation, the reaction tubes were kept in a
boiling water bath for 10 min to stop the enzyme
reaction and then cooled to room temperature. In
addition, the inulinase activity assay of the immobi-
lised enzyme was performed by incubating 0.25 g
PAA/PEG-inulinase composite with 2.5 mL 2% (w/v)
The enzyme used in the study was produced by sub-
merged cultivation of Bacillus sp. 11/3 in a medium
composed of (g/L): inulin 4.0; yeast extract, 4.0; pep-
ꢁ
tone, 4.0; MgSO , 0.16; K HPO , 0.8 in a flask at 50 C
4
2
4
for 24 h at 200 rpm (Gavrailov and Ivanova 2015). The
medium was centrifuged in order to remove the
microbial cells (14,000 rpm, 3 min) and stored at
ꢃ
inulin dissolved in acetate (CH COO /CH COONa) buf-
3
3
ꢁ
fer (pH 4.4) at 50 C for 20 min. The reaction was
stopped by boiling the reaction mixture for 10 min.
After cooling to room temperature both samples were
analysed for reducing sugars with a method by Lever
ꢁ
ꢃ18 C as a crude enzyme solution.
Further, the enzyme was purified by the aqueous
two-phase system (ATPS) described in our previous
study (Temkov et al. 2018). Briefly, the enzyme was
purified in the PEG phase in a system composed of
(1972) with some modifications. For this method,
2
1
50 lL of the reaction mixture were mixed with
750 lL solution of PAH BAH dissolved in 10% 0.5 M
2
6% PEG (M ¼ 1000 g/mol) and 26% MgSO , whereas
w
4
the bulk proteins were partitioned in a salt phase.
After partitioning, the PEG phase containing the
HCl and 90% 0.5 M NaOH and were boiled for 10 min.
The absorbance was measured at 410 nm by using a
enzyme was concentrated in a vacuum evaporator at
microplate
reader
SPECTROSTAR–Nano
(BMG
ꢁ
4
0 C, until 115 lg protein/mL in the final solution
LABTECH). The concentration of fructose was calcu-
lated using a calibration curve. One unit of enzymatic
activity was defined as the amount of enzyme needed
to obtain 1 lmol of fructose per minute under the
assay conditions. Assays were performed in triplicate
and the average value was used in calculations. The
efficiency of immobilisation at different enzyme dos-
ages was calculated by using Equation (1) for the
immobilisation activity recovery:
was reached.
Inulinase immobilisation in PAA/PEG composite
A blend of acrylamide (AA), and N,N’-methylene-bis-
acrylamide was polymerised in the presence of PEG
(containing the enzyme) in ratio 1:1. In fact, 3 mL of
50% w/v diluted PEG, enriched with the enzyme at a
concentration of 97.5 U/mL, was added to 3 mL of
acrylamide/bis-acrylamide (AA). Ammonium persulfate
Activity recovery ð%Þ
Total activity of immobilised enzyme
Total activity of enzyme in PEG phase
(APS) and tetramethyl-ethylenediamine (TEMED) were
¼
ꢄ 100
used as crosslinking agents to induce AA polymerisa-
tion. The prepared solution, in a total volume of 6 mL,
was mixed thoroughly for even distribution of the
enzyme and was poured immediately into a Petri dish
(1)

4
M. TEMKOV ET AL.
ꢃ
Determination of optimal pH and temperature
reaction was performed in acetate (CH
3
COO /
ꢁ
CH COONa) buffer (pH 4.4) at 40 C for 20 min.
3
Optimal pH of the inulinase was determined by incu-
bating the reaction mixture at different pH in the
ꢁ
range between 3.6 and 10.7 at 50 C and expressed as
Operational and storage stability of immobilised
inulinase enzyme
enzymatic activity. To achieve desired pH conditions:
ꢃ
acetate (CH COO /CH COONa), phosphate (H PO /
3
3
2
4–
0
.25 g of PAA/PEG composite was added to 10 mL 2%
2–
HPO4 ), and carbonate-bicarbonate buffers (H CO /
2
3
inulin and the mixture was placed on a rotary shaker
and incubated at optimum pH and temperature as
determined in the Section “Determination of optimal
pH and temperature” for 1 hour. After each cycle, the
composite was removed by filtration, washed with dis-
tilled water and re-suspended in a new portion of 2%
inulin solution for the next cycle. After each 1 h lasting
cycle, the remaining activity was measured. The activ-
ity of the immobilised enzyme after the first cycle was
set as a control and assumed to possess a relative
activity of 100% (Mubarak et al. 2014). The experiment
was repeated 20 times using the same conditions
described above. For determining the storage stability,
the immobilised enzyme in PAA/PEG composite was
HCO ) were used, prepared and adjusted at room
temperatures. The optimal temperature of immobilised
inulinase was measured by incubating the reaction
3
–
mixture for 20 min at temperatures ranging from 20 to
ꢁ
9
0 C at optimal pH. Enzymatic activity was deter-
mined by the method described in the Section
Inulinase activity assay of free and immobi-
“
lized enzyme”.
Determination of pH and temperature stability
For the pH stability test, the free and immobilised inu-
ꢁ
linase were pre-incubated for 120 min at 50 C in the
same pH buffer solutions ranging from 3.6 to 10.7.
Temperature stability of the immobilised preparation
was measured by pre-incubating the reaction mixture
ꢁ
stored at 4 C and the residual activity was determined
after 6 months of storage.
ꢁ
for 120 min in the range of 20ꢃ90 C at optimal pH.
For both tests, after the pre-incubation, the residual
inulinase activity was measured under standard assay
conditions. The recovered activity was normalised to
the same amount of free and immobilised enzyme. In
order to estimate the inactivation rate constant of the
immobilised enzyme at a certain temperature, the
activity was measured at appropriate time intervals
during pre-incubation (Karimi et al. 2014). The inacti-
vation constant kd was determined according to
Equation (2), whereas half-lives (t1/2) and D values of
the free and immobilised enzyme were estimated
according to Equations (3) and (4).
FOS batch production
The enzymatic reaction was performed in Erlenmeyer
flasks on a shaker at optimum pH and T (defined in
the Section “Determination of optimal pH and temper-
ature”) for 24 hours. In this experiment, the amount of
immobilised preparation, initial substrate concentra-
tion and time of hydrolysis were optimised. Different
amounts of PAA/PEG/enzyme composite (1, 2 and 3 g)
obtained according to the procedure described in the
Section “Inulinase immobilization in PAA/PEG
composite” were added to 10 mL total volume of inu-
lin used as a substrate, resulting in total enzyme activ-
ity of 47, 92 and 138 U, respectively. Three different
concentrations of inulin (2, 4 and 6% w/v) were tested
for initial substrate optimisation. Samples were taken
at 0, 1, 3, 5, 7, 9, 12 and 24 h. To determine the distri-
bution of FOS in the reaction mixture at different
times, the collected samples were analysed by thin-
layer chromatography (TLC). The conversion degree of
inulin (%) was calculated by subtracting the concentra-
tion of the product FOS (DP 3–6) from the initial sub-
strate concentration.
A0
ln ¼ k ꢅ t
(2)
(3)
d
A
ln2
t1=2 ¼ k
d
ln10
D value ¼ k
(4)
d
where A and A are the initial and the residual enzym-
0
atic activities, respectively.
Determination of kinetic parameters
Kinetic parameters Km and Vmax for the immobilised
inulinase were calculated from a Hanes-Woolf plot,
using concentrations of inulin as substrate ranging
from 0.5% to 3.0% with an increment of 0.5%. The
FOS analysis by thin-layer chromatography (TLC)
coupled with ImageJ
Standard solution or samples (5 mL) from the batch
reaction were applied on chromatographic silica gel

BIOCATALYSIS AND BIOTRANSFORMATION
5
6
0 F254 plates by using an Agilent HPLC syringe. The
polyvalent ions in the immobilisation matrix (Costa et al.
2001). In addition, highly alkaline conditions cause
stretching of the protein molecule influenced by the
electrostatic repulsion which will lead to distortion of
enzyme’s active centre (Mubarak et al. 2014). Gonzalez-
Saiz and Pizarro (2001) reported a maximum activity at
pH 8.5 for the alkaline phosphatase immobilised in PAA,
which is in a more alkaline region than the maximum
activity reported for the same enzyme in free form. In
this regard, they discussed the degree of carrier’s protec-
tion over the enzyme activity which is connected to the
external – environmental pH and internal – carrier’s pH.
Since it is difficult to quantify the pH of the polyacryl-
amide matrix internal microenvironment, thus it is hard
to estimate the matrix protection degree over pH
optima. Sattar et al. (2018) reported the same optimum
pH, 5.0, for both free and immobilised protease
entrapped in PAA beads. The pH optimum reported by
Ohmiya et al. (1975) for b-galactosidase immobilised in
PAA was 4.5, while for Fusavium oxysporum inulinase
immobilised in a PAA gel, the pH was found to be 6.0
(Gupta et al. 1990).
chromatographic plates were placed in a TLC chamber
containing a mixture of n-butanol, i-propanol, acetic
acid, H O in a ratio of 7:5:4:2 as mobile phase. After
6
oven at 100 C for 20 minutes. The spots of separated
compounds were visualised with diphenylamine, anil-
ine, acetone and H PO in a ratio of 1:1:50:5 (Petkova
and Denev 2013). The plates were heated to 100 C
until the colour spots became visible. The concentra-
tions of produced FOS DP 3-6 during hydrolysis were
calculated from the standard curves by using the
ImageJ software (National Institutes of Health and
LOCI, University of Wisconsin, USA). For this purpose
calibration curves of fructose, sucrose, kestose (DP3),
nystose (DP4) and 1,1-fructofuranosylnystose (DP5) in
concentrations of 0.075, 0.1, 0.25, 0.5, 0.75 and 1.0 mg/
mL were prepared separately (see SI, Figure S1).
2
hours the plates were dried and incubated in an
ꢁ
3
4
ꢁ
Results and discussion
Bacterial inulinase partially purified in the PEG phase
(
from a system composed of polymer/salt) is immobi-
Concerning the effect of temperature, both free
and immobilised inulinase exhibited different tempera-
ture optimums as shown in Figure 1(b). From the fig-
ure, it can be noticed that the optimum temperatures
of the immobilised enzyme in PAA/PEG composite are
lised in PAA by the so-called “aqueous two-phase poly-
merisation”, which can be carried out separately and
also in two aqueous phases. This method for immobilisa-
tion where PAA can be polymerised in the presence of
PEG carrying the enzyme involves one step to obtain
the biocatalyst, while leaving out the time-consuming
and costly ultrafiltration for enzyme purification. The
enzyme is captured within the polymeric network that
allows the substrate and products to pass through, while
the diffusion of the enzyme molecules is constrained.
ꢁ
40 and 80 C for the two isoforms of the enzyme as
opposed to those of the free enzyme, which were 30
ꢁ
and 70 C. The change of the enzyme activity with
increasing temperature was as expected: the activity
rose until the critical temperature was reached and
irreversible deactivation occurred. As seen from the
data, the carrier has a mild protective effect at the
high temperatures at which the enzyme is inactivated,
due to the temperature-mediated conformational
changes within the catalytic structure of the enzyme
molecule. This protection probably comes from the
increased ability of the enzyme to renature in the
immobilised preparation, whereas the free form in the
solution is highly susceptible to damages. This was
also noticed by other authors as well. Upon immobil-
isation of alkaline phosphatase in a polyacrylamide
pH and temperature optimum
Different pH caused a significant alteration in the activity
of the immobilised preparation due to changes in the
ionisation state of the free enzyme or the enzyme-sub-
strate complex. For both, free and immobilised enzyme,
the test was performed at different pH in the range of
3
profile of the free as well as the immobilised inulinase in
the PAA/PEG composite demonstrated two peaks of
optimal pH suggesting that the inulinase is present in
two distinct forms (Figure 1(a)). Compared to the free
enzyme with pH optimums at 8.0 and 5.8, in this type of
immobilisation the optimal pH shifted to more acidic
values. The maxima of the two enzyme isoforms were at
pH 4.4 with a maximum activity of 1.6 lmol/gꢅmin and
at pH 7.6 with a maximum activity of 1.4 lmol/gꢅmin.
The pH shift is probably due to the presence of
ꢁ
.6–10.7 and at a constant temperature of 50 C. The pH
ꢁ
gel, the optimal temperature was increased by 5 C
compared to the optimal temperature for the free
enzyme (Gonzalez-Saiz and Pizarro 2001). The opti-
mum temperature of immobilised inulinase in poly-
ꢁ
ꢁ
acrylamide gel increased from 37 C to 45 C in
comparison to the free inulinase isolated from
Fusavium oxysporum (Gupta et al. 1990). Sattar et al.
ꢁ
(2018) showed 5 C higher temperature optimum for
the immobilised enzyme in polyacrylamide beads.

6
M. TEMKOV ET AL.
Figure 1. Effects of different pHs (a) and temperatures (b) on the activity of the free and entrapped inulinase in PAA/PEG com-
posite. The highest activities were taken as 100%. Optimal pH was determined by incubating the reaction mixture at different pH
ꢁ
in the range between 3.6 and 10.7 at 50 C for 20 min. Optimal temperature was measured by incubating the reaction mixture for
2
ꢁ
0 min at temperatures ranging from 20 to 90 C at pH 4.4. Free inulinase had pH optimums at 8.0 and 5.8 of both isoforms with
maximum activity of 68 U/mL and 46 U/mL respectively. The pH optimum in the immobilised form shifted to pH 4.4 with a max-
imum activity of 1.6 U/g and at pH 7.6 with a maximum activity of 1.40 U/g. Optimum temperatures of the free enzyme were 30
ꢁ
and 70 C with their maximum activities of 47 U/mL and 43 U/mL for both isoforms, respectively. The immobilised enzyme had
ꢁ
optimum temperatures at 40 and 80 C with maximum activities 1.4U/g and 0.9U/g for both isoforms, respectively. Values are
the means of three replications ± standard deviation.
These results imply protection of the enzyme against
temperature variation by the polymer matrix.
important factor for scaling up the process and
extending the life cycle of the preparation. pH stability
of the free and immobilised preparation in PAA/PEG
composites was examined by their pre-incubation in
the same pH range of 3.6–10.7 and at a constant tem-
pH and thermal stability
ꢁ
The stability of an immobilised preparation to the
extreme pH and temperature conditions is an
perature of 50 C for 2 h. After 2 h, the residual activity
was measured according to the standard inulinase

BIOCATALYSIS AND BIOTRANSFORMATION
7
assay. Free inulinase showed good pH stability in the
range between 4.6 and 7.6, retaining 96% of its initial
activity, followed by a steep decrease for higher pH
values (Figure 2(a)). On the other hand, the immobi-
lised enzyme showed greater pH stability in the alka-
line range up to pH 9.6 in which the activity sustained
over 90% of the initial activity. For higher pH values
up to 10.8 the activity diminished rapidly to only
22,030 min (367 h). This makes enzymatic hydrolysis at
high temperatures possible which prevents microbial
contamination and enable inulin to dissolve better,
thus making the process more suitable for industrial
applications (Awad et al. 2017). In the literature, there
are several reports on the thermal kinetic parameters,
but almost none for the enzyme entrapped in PAA
composites. The thermal stability reported by Awad
1
5–30% of its initial activity.
The data for the thermal stability is presented in
et al. (2017) for the inulinase immobilised in alginate-
ꢃ1
CMC beads with k , t
and D values 0.0064 min
,
d
1/2
Figure 2(b). As seen, in the range between 20 and
1
08 min and 359 min, respectively, was significantly
ꢁ
50 C both free and immobilised inulinase were very
lower than the one obtained in our study.
stable with relative activity varying from 90 to 99% of
its initial activity. The free enzyme showed high ther-
mal sensitivity for temperatures above 50 C, retaining
only about 9% of the initial activity at 60 C.
Subsequently, at 60 C the relative activity of the
ꢁ
Kinetic parameters
ꢁ
The apparent values or the kinetic parameters Km and
Vmax for the immobilised enzyme in the PAA/PEG com-
posite were calculated from the Hanes-Woolf plot (see
SI, Figure 2S). Km was calculated to be 3.1 lM, while
Vmax was 64 lM/min compared to 26 lM and 526 lM/
min for the free enzyme. These data show that the
substrate affinity significantly increases when the
enzyme is entrapped; however, its maximum rate was
reduced by around 8 times. The increased substrate
affinity was probably due to the greater distribution of
the substrate in the enzyme microenvironment than in
the external solution. On the other hand, low availabil-
ity of the enzyme’s active centres and the poor sub-
strate diffusion within the polymer matrix may be the
reason for the reduced rate. Ohmiya et al. (1975)
reported results for b-galactosidase immobilised in
polyacrylamide gels, where Km for immobilised and
free enzyme did not change and was calculated as
ꢁ
ꢁ
immobilised enzyme was 78%, at 70 C it dropped to
ꢁ
60% and 50% remained at 80 C. The superior thermal
stability of the entrapped inulinase could come from
the protective property of the PAA/PEG composite
towards the conformational changes of the enzyme’s
tertiary structure (Sattar et al. 2018). The immobilisa-
tion of inulinase in PAA/PEG has not been reported;
therefore our results were compared with enzymes
immobilised in matrices with similar composition.
b-galactosidase immobilised in polyacrylamide gel has
ꢁ
been reported to be stable at 37 C, but the relative
activity markedly decreased to 40% at 50 C (Ohmiya
ꢁ
et al. 1975). Sattar et al. (2018) also reported an
increased thermal stability for the protease immobi-
lised in polyacrylamide beads.
Figure 2(c) presents the residual activities of the
free and entrapped enzyme at optimal temperatures
over the course of time. The deactivation rate con-
2
0 mM at pH 4.5.
Moreover, the binding rate constant (kcat) was cal-
stants (k ) are calculated as inclinations of the loga-
rithms of the residual activities as a function of time
d
culated for both (free and immobilised enzyme) to
determine the number of molecules of product made
per enzyme per unit time. The specificity constant
ꢁ
ꢁ
at 40 C and 80 C (Figure 2(d)). The results in Figure
(c) show that under the same conditions, the deacti-
2
(
kcat/K ) was also calculated to compare the relative
m
vation rate of free inulinase is much higher than that
of the entrapped enzyme. The free enzyme lost more
than 50% of its initial activity after 1 h of incubation at
rates of the enzyme. The immobilised enzyme had k
cat
4
ꢃ1
0
.54 ꢄ 10 s , while this constant for the free enzyme
4
ꢃ1
ꢁ
was calculated as 2.8 ꢄ 10 s . This means that the
free enzyme can turn over more molecules of the sub-
strate into a product per second, i.e., the reaction is
faster. The catalytic potential expressed by the ratio of
80 C, while the entrapped enzyme in PAA/PEG com-
posite retained almost 100% under the same condi-
tions. The deactivation rate (k ), half-life (t1/2) and
d
decimal reduction time constants (D values) for both
temperatures of free and immobilised inulinases are
given in Table 1. We can conclude that the thermal
stability of inulinase significantly increases after the
entrapment in the PAA/PEG composite with the t1/2
increasing from 130 min (2.2 h) to 6930 min (115.5 h),
and the D-value increasing from 415 min (6.9 h) to
k
cat/K was higher for the immobilised enzyme with a
m
3
ꢃ1 ꢃ1
value 1.8 ꢄ 10 lM
s , rather than for the free
3
ꢃ1 ꢃ1
enzyme which was calculated as 1.1 ꢄ 10 lM
s .
These results show that the immobilised enzyme has
greater affinity towards the substrate of the enzyme
which is also confirmed by the lower Km.

8
M. TEMKOV ET AL.
Figure 2. Effects of different pHs (a) and temperatures (b) on the stability of the free and immobilised inulinase in PAA/PEG com-
ꢁ
ꢁ
posite, c) Thermal deactivation of free and immobilised inulinase at 40 C and 80 C; d) Determination of k of immobilised inuli-
d
ꢁ
nase in PAA/PEG composite. The effect of pH was measured by pre-incubating the enzymes (free and immobilised) at 50 C for
1
20 min in the pH buffer solutions ranging from 3.6 to 10.7. For the temperature stability test, the reaction mixture was pre-incu-
ꢁ
bated for 120 min in the range 20–90 C at optimal pH. The activities of the free and immobilised enzymes that correspond to
100% were measured as 47 U/mL and 1.4U/g respectively. The inactivation rate constant was estimated by measuring the activity
at appropriate time intervals during pre-incubation. Values are the means of three replications ± standard deviation.

BIOCATALYSIS AND BIOTRANSFORMATION
9
Figure 2. Continued.
Kikani et al. (2013) immobilised a-amylase from
Bacillus amyloliquifaciens TSWK1-1 in a polyacrylamide
gel and reported a 65% loss after 20 cycles. In con-
trast, Sattar et al. (2018) (Sattar et al. 2018) observed
approximately 72.0% retention of protease’s initial
activity entrapped in polyacrylamide beads up to the
Table 1. Kinetic parameters for thermal inactivation of free
and immobilised inulinases.
–
1
k
d
(min )
t1/2 (h)
D value (h)
ꢁ
Immobilised enzyme at 40 C
0.0001
0.0001
0.0053
0.0076
115.5
115.5
2.2
367.1
367.1
6.9
ꢁ
Immobilised enzyme at 80 C
ꢁ
ꢁ
Free enzyme at 40 C
Free enzyme at 80 C
1.5
4.8
4
th and around 24.0% activity retention at 8th cycle.
Operational and storage stability of
immobilised inulinase
The immobilised inulinase was tested after 6 months
of storage at 4 C and displayed only 43% loss of the
ꢁ
initial activity.
The operational stability of the immobilised prepar-
ation is one of the most important factors in the proc-
esses of bioconversions based on enzymes. It
determines the possible applications of the enzyme in
large processes for reducing operating costs and for
practical purposes. Therefore, that aspect is widely
studied in the present work. From the results pre-
sented in Figure 3, it can be seen that inulinase
entrapped in PAA/PEG composite has high operational
stability. With this method of immobilisation, the
enzyme maintained up to 50% of its initial activity
after 20 consecutive cycles. This significant result can
be due to the multipoint attachment of the enzyme
to the polyacrylamide gel, which creates a large num-
ber of bonds. The decrease in activity and stability
after successive cycles, on the other hand, could be
due to enzymatic denaturation or leaching out of
entrapped enzyme from the matrix. Mansour and
Dawoud (2003) (Mansour and Dawoud 2003) immobi-
lised invertase on polyacrylamide gel by an absorption
procedure and reported an exceptional 18 cycles with-
out loss of activity. They started to observe a decrease
in the initial activity of 2.85% only after the 20th cycle.
Optimisation of FOS production in a batch mode
using immobilised inulinase
The initial enzyme amount and substrate concentra-
tion were optimised for the best batch production of
FOS with DP 3-6. Three different enzyme amounts
were examined: 10%, 20% and 30% (w/v) PAA/PEG
composite with the entrapped inulinase with total
enzyme activities of 46, 92 and 138 U. In addition,
three different inulin concentrations: 2%, 4% and 6%
(w/v) were examined. The FOS production after 24 h is
shown in Figure 3S and 4S (see Supplementary
material).
The concentrations of FOS with different DP at the
end of the batch hydrolysis are presented in Figure 4.
Figure 4(a–c) shows the data when different amounts
of immobilised preparation were used, while Figure
4(d–f) shows the data with different initial substrate
concentration. Kestose, nystose, fructofuranosyl nys-
tose (FFN) and FOS with DP ¼ 6 which have prebiotic
activity were interest of our research and represent

1
0
M. TEMKOV ET AL.
Figure 3. Operational stability of immobilised inulinase in PAA/PEG composite. The reaction mixture composed of 0.25 g immobi-
ꢁ
lised enzyme and 10 mL 2% inulin solution was incubated at optimal pH and temperature (4.4 and 40 C) for 1 h. After each cycle
the catalyst was removed by filtration and the remaining activity was measured under standard assay. The activity of the immobi-
lised enzyme after the first cycle (1.7 U/g) was set as a control and assumed to possess a relative activity of 100%.
part of FOS with DP ¼ 3–6. Fructose and sucrose
are byproducts.
can be seen in Figure 4(f). A large percentage of
unconverted inulin, 77% remained in this system,
while the fructose and the sucrose were produced in
total amounts of 7% and 2%.
The total amount of obtained prebiotic FOS was
22% when 10% (w/v) of immobilised preparation was
applied (Figure 4(a)), while the unconverted inulin in
the final mixture, that is composed of FOS with DP> 6
was 68%, along with the amount of the byproducts
fructose and sucrose that were 10% together. By rais-
ing the amount of immobilised enzyme to 20% (w/v),
the percentage of products was increased to 25%
In conclusion, the best degree of conversion of
highly polymerised inulin is obtained using a 2% solu-
tion of inulin and 30% (w/v) immobilised inulinase in
PAA/PEG composite, where the yield of useful FOS is
high, while the yield of non-prebiotic monosaccharide
and disaccharide is small.
(
Figure 4(b)). However, in this system, a higher total
Comparing the efficiency of the hydrolysis with free
enzyme, added to the reaction mixture in the same
amount as the biocatalyst, it can be seen that the con-
centration of prebiotic FOS increased from 30% to
57% as the amount of enzyme increased. More pre-
cisely it was about 1.5 times higher than the product
obtained with immobilised inulinase (see SI, Figure
5S). Despite the better productivity of FOS, non-pre-
biotic sugars were also produced in higher total
amount of 30%. However, when higher initial sub-
strate concentrations were used (4 and 6%), the
amount of FOS with DP ¼ 3-6 substantially decreased
to 17 and 19%, respectively. These quantities are com-
parable or even less than the products obtained with
immobilised enzyme. The production of by-products –
fructose and sucrose in these systems was negligible.
There are many articles that deal with inulin
hydrolysis using inulinase extracted from different
microorganisms, immobilised on various supports.
Jiang et al. (2019) used endoinulinase in free form
amount of fructose and sucrose of 14% was produced.
The highest conversion of inulin (36%) to FOS with DP
¼
3-6 occurred when hydrolysis was performed with
0% (w/v) immobilised preparation (Figure 4(c)). The
3
figure shows the distribution of sugars: kestose 6%,
nystose and fructofuranosyl 9%, FOS with DP ¼ 6
1
2%, while fructose and sucrose are 4 and 7%,
respectively. The unconverted inulin composed of FOS
with DP > 6 remained about 53% in final the mixture.
In the experiment performed to study the effect of
the initial concentration of the substrate on the
degree of its conversion similar trends with other pub-
lished results were observed. The highest conversion
of 30% of the inulin to prebiotic FOS occurred when
4
% inulin (w/v) were used (Figure 4(e)). Fructose and
sucrose were also obtained in high percentages of 13
and 6%, respectively. In contrast, the degree of con-
version of inulin to useful FOS was 16%, when the ini-
tial concentration of the inulin solution was 6%. This

BIOCATALYSIS AND BIOTRANSFORMATION
11
Figure 4. Composition of the final mixture of the hydrolysis of inulin to fructose, sucrose, kestose, nystose, fructofuranosyl nystose
and FOS DP > 6 with addition of different amounts of immobilised preparation (w/v): a) 10% PAA/PEG with enzyme activity of
4
6 U, b) 20% PAA/PEG with enzyme activity of 92 U, c) 30% PAA/PEG with enzyme activity of 138 U, and different initial substrate
concentrations (w/v) d) 2% inulin, e) 4% inulin, f) 6% inulin. The enzymatic reaction was performed on a rotary shaker at opti-
ꢁ
mum pH and temperature (4.4 and 40 C). The samples were withdrawn from the reaction mixture at 0, 1, 3, 5, 7, 9, 12 and 24 h
and analysed with TLC coupled with ImageJ.
from A. arachidicola at different inulin concentrations
of 1, 3, 5 and 10%, with final inulin conversion to total
inulooligosaccharides of 88.4%, 83.5%, 80.2% and
et al. (1997) as the concentration of inulin increased
from 5% to 20% using endoinulinase from
Pseudomonas sp. No.65 for inulin hydrolysis. Their
maximum yield of 75.6% was observed when the ini-
tial inulin concentration of 5% was used with FOS DP
¼ 2, DP ¼ 3 obtained as main products. The highest
initial substrate concentration of 20% gave 67.6% of
60.1% respectively. The highest conversion was
obtained when the lowest initial concentration of sub-
strate was used, which match our results. Decrease in
total yields of oligosaccharides was observed by Yun

1
2
M. TEMKOV ET AL.
the product. Wang et al. (2016) (Wang et al. 2016)
optimised the production of FOS with endoinulinase
from A. ficuum and produced the highest yield of FOS
of 94.4% using 15% (w/v) inulin. When the inulin con-
centration of 10% was used for the reaction, endoinu-
linase from the wild microorganism Pseudomonas sp.
immobilised on ion exchange resin converted 77.6%
of it (Yun et al. 1997). Compared to our results, the
yields obtained in these studies are much higher
which might be related to the initial degree of poly-
merisation of the inulin used as a substrate. Regarding
the distribution of FOS, DP ¼ 3 and DP ¼ 4 as major
products were reported by Chen et al. (2012) (Chen
et al. 2012), using endoinulinase from A. arachidicola
in free form and recombinant endoinulinase from A.
ficuum JNSP5-06. In comparison, endoinulinase from A.
niger produces FOS with DP ¼ 2-5 as main products,
while inulinase from Pseudomonas sp. produces FOS
with DP ¼ 2 and DP ¼ 3 as main products (Yun et al.
life t1/2 at the optima was 115 h, while the D-value
was 367 h, indicating that the produced catalyst had
enhanced thermal stability and is suitable for industrial
applications. Immobilised inulinase in PAA/PEG can be
reused 20 times until the initial activity decreases to
its half, thus reducing operational costs and widening
the possible applications. Finally, the prepared catalyst
is effective in producing FOS with the DP from 3 to 6
with proven prebiotic activities. In addition, the yield
of FOS from the enzymatic hydrolysis was 36% when
initial inulin concentration was 2% (w/v) and the activ-
ity of the enzyme was 138 U. The composition of the
final mixture was composed of: kestose 6%, nystose
and fructofuranosyl 9%, FOS with DP ¼ 6-12% thus
reducing operational costs and widening the possible
applications.
Disclosure statement
1997; He et al. 2014). Interestingly, endoinulinase from
The authors have declared neither conflicts of interest nor
competing financial interests. All authors have approved
the manuscript.
Xanthomonas sp. produces only FOS with DP ¼ 5 and
DP ¼ 6 as sole products. The enzyme produced 86%
of total FOS when 8% of inulin was used as a sub-
strate and 57% total FOS when the concentration of
the substrate was increased to 10% inulin (Park et al.
Funding
This work has been funded by Bulgarian National Science
Fund under Project DN 06/11.
1999). Recombinant endoinulinase from Arthrobacter
sp. S37 produces only FOS with DP ¼ 2 as the main
product (Li et al. 2012), but interestingly the native
enzyme produced by the wild strain of Arthrobacter
sp. S37, produces FOS with DP ¼ 3, DP ¼ 4 and DP ¼
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