Molekul, Vol. 20. No. 2, July 2025: 268 – 278

Articles

MOLEKUL

eISSN: 2503-0310

https://doi.org/10.20884/1.jm.2025.20.2.13134

Optimization and Characterization of Urease Immobilization from Red Lentil Seeds
(Lens culinaris) Using Chitosan
Zusfahair1*, Dian Riana Ningsih1, Bilalodin2, Amin Fatoni1, Adilla Luthfia2, Purwati1,
Niken Istikhari Muslihah1, Inessa Putri Apriliadina1
1

Department of Chemistry, Faculty of Mathematics and Natural Sciences, Jenderal Soedirman University,
Purwokerto 53123, Indonesia
2
Department of Physics, Faculty of Mathematics and Natural Sciences, Jenderal Soedirman University,
Purwokerto 53123, Indonesia
*Corresponding author email: zusfahair@gmail.com
Received September 21, 2024; Accepted July 07, 2025; Available online July 20, 2025

ABSTRACT. Urease is an enzyme that plays a vital role in catalyzing the hydrolysis of urea into ammonia (NH 3) and carbon
dioxide (CO2). This study focuses on the isolation of urease from red lentil seeds, followed by its immobilization. The objective
of this research is to optimize and characterize urease that has been immobilized using chitosan and activated with
glutaraldehyde. Red lentil seeds were processed with a mortar and pestle at low temperatures (4 °C) to obtain a crude
enzyme extract, which was then concentrated using 50% acetone (P50) prior to immobilization. The optimization process for
P50 urease immobilization involved assessing various factors, including chitosan concentration, glutaraldehyde
concentration, temperature, and the immersion duration in glutaraldehyde. The findings revealed that the optimal conditions
for immobilizing P50 urease were achieved at a chitosan concentration of 0.75%, with a 2% glutaraldehyde soak at 25 °C
for 2 hours, resulting in an enzyme activity of 7.042 U/g. The immobilized P50 urease demonstrated the ability to be reused
up to 7 times while maintaining 51% of its initial activity. Scanning Electron Microscopy (SEM) analysis indicated
morphological changes in the beads after the addition of glutaraldehyde and the enzyme, shifting from a rounded to an
irregular shape. Additionally, Fourier Transform Infrared Spectroscopy (FTIR) analysis identified C-N and C=N peaks,
confirming the successful incorporation of glutaraldehyde.
Keywords: immobilization, red lentil seeds, glutaraldehyde, chitosan, urease

INTRODUCTION
Urease is an enzyme that plays a crucial role in
catalyzing the hydrolysis of urea into ammonia (NH3)
and carbon dioxide (CO2) (Singh et al., 2017). This
process is a key component of the nitrogen cycle, with
significant implications in various biological,
agricultural, and industrial applications. Urease plays
some important role in the decomposition of organic
matter and the nitrogen cycle in the soil for plant
growth (Koçak, 2020). The results of urease enzyme
hydrolysis can help the biocementation process by
forming calcium carbonate (Xu et al., 2021). Urease
has been isolated from multiple sources, including
legumes such as pea seeds (Pisum sativum L.), broad
beans (Vicia faba L.) (Bedan, 2020), winged bean
seeds (Psophocarpus tetragonolobus (L.) DC.)
(Zusfahair et al., 2023), and mung beans (Vigna
radiata L.) (Muslihah et al., 2024). Previous research
has successfully isolated urease from red lentils,
resulting in the production of free enzymes.
Free enzymes are generally more fragile than
inorganic catalysts. Their application in industrial
processes is often limited by high costs and inherent

instability. Urease, for example, is particularly unstable
due to its sensitivity to temperature and pH, as well as
its inability to be reused. To overcome these
limitations, enzyme immobilization—using solidphase biocatalysts—has emerged as a promising
solution. Consequently, there is increasing interest in
this approach within bio-based industries (Verma et
al., 2020).
Free enzymes tend to be more fragile compared to
inorganic catalysts. Their use in industrial applications
presents challenges, such as high costs and instability,
which can limit their practicality. Urease, for example,
is particularly unstable due to its sensitivity to
temperature, pH, and inability to be reused. To
overcome these limitations, enzyme immobilization
using solid-phase has emerged as a promising
solution. Consequently, there is increasing interest in
this approach within bio-based industries (Verma et
al., 2020).
Immobilization generally involves attaching
enzymes to or within a solid matrix. Immobilized
enzymes display greater robustness and resistance to
environmental changes than their free counterparts for

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Zusfahair, et al.

health applications (Al-Garawi et al., 2022). Although
immobilized enzymes may exhibit lower catalytic
activity than free enzymes, they provide enhanced
stability and reusability, making them more costeffective and efficient for large-scale applications
(Kamburov & Lalov, 2014).
In practical applications, urease is typically
employed in its immobilized form. Immobilized urease
has been utilized in various scenarios, including kidney
machines for blood detoxification (Lahiri, 2015),
degradation of herbicides (Jamwal et al., 2020),
processes in the food industry to remove urea from
beverages and food products, and for reducing urea
content in agricultural waste (Lv et al., 2018). The
immobilization of urease has been investigated using
several matrices for clinical analytical applications,
including chitosan (Al-Garawi et al., 2022), sodium
alginate (Tetiker & Ertan, 2017), cellulose (Lv et al.,
2018), and polyether sulfone (Zhang et al., 2019).
The supporting matrix employed in this study is
chitosan. Chitosan has several advantages, including
good biocompatibility and biodegradability (Kurniasih
et al., 2022). In addition, its reactive amino groups
make chitosan an excellent choice for immobilization.
To improve the mechanical and thermal stability of
chitosan, crosslinking agents are necessary to create
crosslinks that strengthen its structure. Glutaraldehyde
is the most widely used crosslinking reagent (Malhotra
& Basir, 2020). It is chosen for its two aldehyde
groups, which can interact with free amino acids
present in chitosan and proteins. The immobilization
of enzymes onto chitosan occurs mainly through the
reaction of glutaraldehyde with the free amino groups
in both chitosan and the enzyme molecules, resulting
in the formation of covalent bonds. Specifically,
glutaraldehyde creates covalent imine bonds with the
amino groups of chitosan as a consequence of Schiff
base rearrangement. Its bifunctional properties,
reliability, and ease of use make glutaraldehyde a
preferred crosslinker and activating agent (Kamburov
& Lalov, 2014).
The P50 urease is subsequently immobilized using
chitosan that has been activated with glutaraldehyde.
The optimization process for the immobilized P50
urease involves determining the appropriate
concentrations of chitosan and glutaraldehyde, as well
as the immersion temperature and duration. The
resulting immobilized enzyme will be characterized in
terms of its reusability, and analyses will be conducted
using SEM-EDX and FTIR.
This research focuses on extracting urease from red
lentil seeds, followed by purification through
precipitation using 50% acetone to obtain P50 urease.
The P50 urease is then immobilized using chitosan that
has been activated with glutaraldehyde. The
optimization process for the immobilized P50 urease
involves determining the appropriate concentrations
of chitosan and glutaraldehyde, as well as the
immersion temperature and duration. The resulting

immobilized enzyme will be characterized in terms of
its reusability, and analyses will be conducted using
SEM-EDX and FTIR. The enzyme is then assessed by
detecting the ammonium produced in the reaction
using Nessler’s reagent.
EXPERIMENTAL SECTION
Materials and Methods
The equipment utilized in this study includes
standard glassware typically found in biochemistry
laboratories, an incubator, a pH meter (Hanna
Instrument), a centrifuge (Ohaus), a UV-Vis
spectrophotometer (Bel Photonic, V-M5), Fouriertransform infrared spectroscopy (FTIR), and Scanning
Electron Microscopy (JSM-6510LA). The materials
used in the experiments consist of red lentil seeds,
urea, Nessler's reagent, ammonium sulfate, distilled
water, Na2HPO4.2H2O, NaH2PO4.2H2O, CuSO4, Na2CO3, acetic acid, NaOH, and C3H6O. All chemical
materials were sourced from Merck Chemical
Company (Merck, Germany), except for chitosan
(DDA ≥ 75%) and glutaraldehyde, which were
obtained from Sigma.
Research Procedure
Extraction of urease from red lentils
Urease was isolated using the method previously
described by (Zusfahair et al., 2018). The resulting
crude extract was subsequently concentrated with
acetone.
Concentration using acetone
The concentration process adhered to the method
outlined by (Sabilla & Susanti, 2019). A total of 300
mL of crude urease extract was gradually combined
with absolute acetone (at 5 ºC) to achieve a 50%
saturation level (using a 1:1 volume ratio of 300 mL
of crude extract to 300 mL of acetone). This mixture
was gently stirred in an ice bath with a magnetic stirrer
and left to sit in a refrigerator for 12 hours to enhance
precipitation. The precipitated enzyme was then
separated by centrifugation at 10,000 rpm for 15
minutes at 4 ºC. The supernatant was carefully
discarded, and any remaining acetone on the
precipitate was removed by evaporation or absorbed
with sterilized filter paper. The precipitate obtained
from this concentration process was dissolved in 150
mL of 0.2 M phosphate buffer at pH 7
(NaH2PO4/NaHPO4,1:1 v/v), resulting in a suspension
referred to as P50 urease.
Preparation of chitosan beads
Chitosan beads were prepared following the
method described by (Malhotra & Basir, 2020). A
chitosan solution was created at a concentration of
0.5% by dissolving 0.025 g of chitosan powder in 5
mL of 2% acetic acid solution. This mixture was
homogenized using a stirrer at 60 ºC for 10 minutes.
The solution was then transferred into a syringe and
slowly pressed to form droplets in a container
containing 25 mL of 1 N NaOH solution, resulting in

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bead formation. These beads were allowed to soak for
24 hours. Chitosan beads immersed in NaOH
solution undergoes an ionotropic gelation process,
where the amino groups (–NH₃⁺) react with OH⁻ ions,
stabilizing the physical structure of the beads to
prevent them from redissolving. After immersion, the
beads were washed twice and suspended in 0.05 M
phosphate buffer at pH 7 prior to activation. The same
procedure was applied to chitosan concentrations of
0.25, 0.75, and 1%.
Immobilization of urease on chitosan beads using
glutaraldehyde crosslinker
The immobilization of urease onto chitosan beads
was conducted using glutaraldehyde as a crosslinker,
following the methodology outlined by Malhotra &
Basir (2020). In this procedure, the chitosan solution
was treated with 2% glutaraldehyde until fully
submerged, then allowed to stand at room
temperature for 2 hours. The beads were subsequently
washed five times using a repeated rinsing method
with 0.05 M phosphate buffer at pH 7 to eliminate any
residual glutaraldehyde. After washing, the beads
were incubated in a solution containing 4 mL of P50
urease at 4 °C for 24 hours, leading to the formation
of immobilized P50 urease. The immobilization
process was optimized by varying the concentration of
glutaraldehyde,
immersion
temperature,
and
immersion
duration.
The
glutaraldehyde
concentrations tested were 0.5, 1, 3, and 4%, with
immersion temperatures of 4, 35, and 40 °C, and
immersion times of 0.5, 1, 1.5, and 2.5 hours.
Activity testing of free and immobilized enzymes
The activity of urease was evaluated by adding 1
mL of a 15,000-ppm urea solution to a reaction tube,
followed by the addition of 1.95 mL of 0.2 M
phosphate buffer at pH 7 and 0.05 mL of P50 urease
solution. The control tube contained 1 mL of 15,000
ppm urea and 2 mL of 0.2 M phosphate buffer at pH
7. Both the sample and control tubes were incubated
for 15 minutes at 35 ºC. After incubation, the tubes
were cooled in a refrigerator for 5 minutes and then
heated to 80 ºC for 2 minutes. Both tubes were
centrifuged for 15 minutes at room temperature at
5,000 rpm.
For the activity test of the immobilized enzyme, 5
mL of 15,000 ppm urea solution in phosphate buffer
at pH 7 was added to the immobilized P50 urease
beads, corresponding to the number of beads
produced in the previous experiment. This mixture
was incubated for 15 minutes at 35 °C. The beads
were then separated from the solution by
decantation, and the activity was measured using
Nessler's reagent. A control solution was prepared in
the same manner, using beads without urease.
A sample volume of 1.5 mL from the resulting
solution was combined with 0.25 mL of Nessler's

reagent. The absorbance of this solution was
measured using a UV-Vis spectrophotometer at a
wavelength of 500 nm. To quantify the amount of
ammonium produced by urease, a standard curve was
prepared using ammonium sulfate solutions at
concentrations of 6, 9, 12, 15, and 18 ppm, each
treated with Nessler's reagent. The urease activity in
the red lentil sample was then calculated by
comparing its absorbance to the standard curve,
allowing determination of the amount of ammonia
released by urea hydrolysis. One unit of activity is
defined as the amount of ammonia generated from
the hydrolysis of urea by urease in the sample,
expressed as 1 µmol per minute.
Characterization of immobilized urease
The characterization of immobilized P50 urease
was performed on the beads under optimal
conditions. This characterization included assessments
of reusability and analyses using SEM-EDX and FTIR
techniques on the chitosan beads.
RESULTS AND DISCUSSION
Extraction of Urease from Red Lentil Seeds
The extraction process is designed to liberate
enzymes from the cells within plant tissues (Pramono
et al., 2018). Urease was extracted by immersion red
lentil seeds in distilled water, a process that promotes
imbibition, or the absorption of water by the dried
seeds (Pratantie et al., 2021). After immersion, the
lentils were ground and dissolved in a buffer solution
pH 7 (NaH2PO4/NaHPO4,1:1 v/v) followed by
centrifugation (10,000 rpm, 4 °C, 15 minutes). The
supernatant obtained from this centrifugation was
utilized as the crude extract, which was then
concentrated using 50% acetone (Zusfahair et al.,
2025).
Concentration Using Acetone
The concentration of the crude enzyme extract was
achieved by adding 50% acetone (designated as P50).
The results indicated that the activity of the free P50
enzyme was measured at 3.443 U/mL. This P50 was
subsequently immobilized using chitosan and
activated with glutaraldehyde.
Optimization of Immobilized P50 Urease from Red
Lentil Seeds Using Chitosan and Glutaraldehyde
Activation

Optimization of chitosan concentration

In this study, urease was immobilized using
chitosan. The presence of reactive amino groups in
chitosan makes it a suitable carrier for the enzyme.
Chitosan beads were prepared by dissolving chitosan
powder in 2% acetic acid. This dissolution resulted in
the formation of a chitosan-carboxylic acid complex,
characterized by hydrogen bonding between the
hydroxyl groups and the –NH3+ groups in chitosan,
and the carboxyl groups in acetic acid (Figure 1).

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Figure 1. Chitosan complex with carboxylic acid (Basir et al., 2017)

Figure 2. Reaction between chitosan and glutaraldehyde (Kamburov & Lalov, 2014)
The resulting chitosan solution was then transferred
to a syringe and gently pressed to form droplets in a
container filled with NaOH solution, where they were
allowed to soak for 24 hours to form immobilized
beads. These immobilized beads were then immersed
in a 2% glutaraldehyde solution and left at room
temperature. The reaction between chitosan and
glutaraldehyde is depicted in Figure 2. Glutaraldehyde
forms covalent imine bonds with the amino groups of
chitosan through Schiff base rearrangement, and it is
more likely to react with amino groups than with
hydroxyl groups (Qu & Luo, 2020).
The immobilized beads were then incubated with
the enzyme solution to promote crosslinking between
chitosan and the enzyme, leading to the formation of
immobilized P50 urease. Figure 3 illustrates the

reaction involving chitosan, glutaraldehyde, and the
enzyme. Glutaraldehyde (-CHO) interacts with various
functional protein groups, including amines, thiols,
phenols, and imidazoles, due to the high reactivity of
nucleophilic amino acid residues, primarily forming
covalent -CH=N- (schiff base) linkages with primary
amines during immobilization condition (Kamburov &
Lalov, 2014).
The protein content of immobilized P50 urease was
subsequently analyzed using the Lowry method, and
its activity was assessed at pH 7 and an incubation
temperature of 35 °C. The resulting data, which
illustrates the relationship between chitosan
concentration (%) and the relative activity of
immobilized P50 urease, is presented in Figure 4.

Figure 3. Reaction between chitosan, glutaraldehyde, and the enzyme (Kamburov & Lalov, 2014)
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Figure 4. Curve showing the effect of chitosan concentration on the activity of immobilized P50 urease
The curve in Figure 4 reveals that the activity of
immobilized P50 urease increased as chitosan
concentrations rose from 0.25 to 0.75%. However, at
chitosan concentrations exceeding 0.75%, the activity
of the immobilized urease declined. At lower chitosan
concentrations, the beads were found to be fragile and
soft, resulting in poor mechanical stability and enzyme
leaching, which can reduce catalytic activity. At higher
concentrations, the beads became harder, potentially
hindering substrate access (Malhotra & Basir, 2020).
Crosslinking can occur at various reactive sites on
chitosan, such as the –NH2 and –OH groups.
Therefore, at the optimal chitosan concentration
(0.75%), there is a potential for stable binding that
does not interfere with enzyme-substrate interactions,
resulting in the highest enzyme activity.

crosslinking between chitosan, glutaraldehyde, and
the enzyme, which resulted in lower enzyme activity
(Hegde & Veeranki, 2014). As the concentration of
glutaraldehyde increased, the activation of chitosan’s
amino groups also increased, enhancing enzyme
activity (Chen et al., 2013). At the optimal
concentration of 2%, glutaraldehyde provides
sufficient crosslinking to securely bind the enzyme
without excessive structural rigidity, which helps
maintain both catalytic activity. However, excessively
high concentrations of glutaraldehyde resulted in
binding at multiple sites, which reduced the enzyme's
flexibility and increased its brittleness (Malhotra &
Basir, 2020).

Optimization of glutaraldehyde concentration

The immersion temperature of glutaraldehyde
significantly affects chitosan, influencing both the
efficiency of the cross-linking process and the ultimate
properties of the chitosan beads. Excessively high
temperatures can cause the chitosan beads to become
brittle, which can impede effective cross-linking with
the enzyme. Figure 6 presents data on how varying
glutaraldehyde immersion temperatures impact the
relative activity of immobilized P50 urease.

The impact of glutaraldehyde concentration on the
relative activity of immobilized P50 urease is depicted
in Figure 5. The curve in Figure 5 indicates that the
activity of immobilized P50 urease increased with
glutaraldehyde concentrations between 0.5% and 2%,
but decreased at concentrations above 2%. At lower
glutaraldehyde concentrations, there were also fewer
active groups on chitosan, leading to limited

Effect of glutaraldehyde immersion temperature on
enzyme activity

Figure 5. Curve showing the effect of glutaraldehyde concentration on the activity of immobilized P50 urease

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Figure 6. Curve illustrating the effect of glutaraldehyde immersion temperature on the activity of immobilized
P50 urease
The curve in Figure 6 demonstrates that the highest
activity of immobilized P50 urease occurred in beads
immersed at a temperature of 25 °C. Enzyme activity
increased from 4 to 25 °C but experienced a sharp
decline at temperatures exceeding 25 °C. This
decrease is likely due to the rapid binding of
glutaraldehyde's aldehyde groups to the enzyme's
amino groups at elevated temperatures, which
increases steric hindrance and subsequently reduces
enzyme activity (Chen et al., 2013). This finding
contrasts with the results of Çetinus & Öztop (2003),
who reported an optimal temperature of
approximately 35 °C for catalase activity when
immobilized on chitosan beads. This suggests that
different enzymes may have unique optimal
glutaraldehyde immersion temperatures for effective
immobilization.

Optimization of glutaraldehyde immersion time

The impact of varying glutaraldehyde immersion time
on the relative activity of immobilized P50 urease is
illustrated in Figure 7. The curve in Figure 7 indicates
that the highest activity of immobilized P50 urease was
achieved when the beads were immersed in
glutaraldehyde for 2 hours. As the immersion time
increased, the enzyme activity also rose. However,
enzyme activity declined sharply when the immersion
time exceeded 2 hours. This reduction is likely due to
excessive cross-linking that occurs with prolonged
immersion, which can lead to significant alterations in
the enzyme's configuration and result in protein
denaturation (Chen et al., 2013).
Effects of Repeated Use of Immobilized P50 Urease
Repeated use is a crucial factor in enzyme
immobilization, especially for cost-effective and
economical applications. The performance of this
immobilized enzyme was evaluated through activity
tests until its activity decreased to 50%. Figure 8
illustrates the impact of repeated usage on the relative
activity of immobilized P50 urease. The data in Figure
8 shows that immobilized P50 urease can be utilized
up to seven times, retaining 51% of its initial activity.
However, with each successive use, the enzyme's

activity declined. This reduction is likely due to the
weakening of the bond between the enzyme and its
supporting matrix. The decrease in enzyme activity
upon reuse may result from distortions caused by
frequent interactions between the active site and the
substrate, which can diminish catalytic efficiency (Zhou
et al., 2013).
The findings in Figure 8 are further supported by
the results of the SEM analysis presented in Figure 9.
The SEM analysis of chitosan beads (a) reveals a
spherical morphology with an average pore size of
0.22 µm. In contrast, the chitosan-enzyme beads (b)
show a reduced spherical shape due to the enzyme
coating the chitosan surface. This change in
morphology may suggest enzyme attachment. The
chitosan-glutaraldehyde-enzyme beads (c) exhibit a
transformation in surface shape from round to
irregular as a result of coverage by the
glutaraldehyde-enzyme solution, indicating enhanced
surface cross-linking that could help maintain enzyme
activity under operational conditions. In the chitosanglutaraldehyde-enzyme beads used five times (d), the
loss of enzyme due to repeated use allows the
spherical shape to re-emerge more clearly, with an
average pore size of 0.31 µm, although the beads still
retain enzyme activity, indicating that the immobilized
enzyme maintains stability despite repeated cycles.
(Gür et al., 2018) noted changes in the morphology
of activated and immobilized enzyme beads, reporting
that the surface roughness increased following
glutaraldehyde treatment and enzyme immobilization.
Additionally, surface porosity was found to increase
after the application of glutaraldehyde (Gilani et al.,
2016).
SEM analysis is utilized to investigate the surface
morphology of materials at high magnification, while
EDX analysis is employed to assess the elemental
composition of the samples (Kustomo, 2022). The
results of the EDX analysis can be found in Figure 10
and Table 1.
The elements identified in immobilized P50 urease
include carbon (C) and sodium (Na). The carbon

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content in chitosan increases with the addition of the
enzyme and glutaraldehyde, rising from 21.91% to
59.67%, and then decreases to 56.11% after the
beads have been used five times. This decline is
attributed to the enzyme being released from the
surface of the immobilized beads. The increase in
carbon content reflects the presence of enzyme protein
and glutaraldehyde, both rich in carbon, nitrogen,

and
oxygen,
indicating
successful
enzyme
immobilization (Gorissen et al., 2018). The
subsequent decrease after repeated use suggests
gradual enzyme release, correlating with stability and
retained activity. In contrast, the sodium content in
chitosan decreases following the addition of the
enzyme and glutaraldehyde, as sodium dissolves in
water during the washing process.

Figure 7. Curve demonstrating the effect of glutaraldehyde immersion time on the activity of
immobilized P50 urease

Figure 8. Effect curve of repeated use on immobilized urease P50 activity.

Figure 9. SEM analysis of immobilized P50 beads at 10,000x magnification [(a) chitosan,
(b) chitosan-enzyme, (c) chitosan-glutaraldehyde-enzyme, (d) chitosan-glutaraldehyde274

Optimization and Characterization of Urease

Zusfahair, et al.

enzyme used 5 times]

Figure 10. EDX analysis of immobilized P50 beads [(a) chitosan, (b) chitosan-enzyme, (c) chitosanglutaraldehyde-enzyme, (d) chitosan-glutaraldehyde-enzyme used 5 times]
Table 1. Elements of Immobilized Beads in EDX Analysis. EDX analysis was conducted using
the standardless ZAF quantification method.
Element

Chitosan
(%)

Chitosanenzyme (%)

C
Na

21.91
15.85

53.91
5.8

Chitosanglutaraldehydeenzyme (%)
59.67
1.6

FTIR Analysis of Immobilized P50 Urease
FTIR analysis was conducted to identify the
functional groups present in the immobilized beads.
The data presented in Figure 11 show that the IR
spectra for samples A, B, and C exhibit absorbance at
the same wavelengths, indicating the presence of
similar functional groups. However, the IR spectra of
A, B, and C display different structures, resulting in
varying absorption intensities. According to Figure 11,
the percentage of transmittance follows the order C <
B < A. The increasing absorption intensity from
sample A (chitosan) to B (chitosan-enzyme) and
highest in C (chitosan-glutaraldehyde-enzyme)
corresponds to the progressive addition of enzyme
and glutaraldehyde, indicating stronger enzyme
immobilization and enhanced structural stability that
correlate with improved enzyme activity, this decrease
in transmittance can also be seen in the results
obtained by Taha et al (2020).
Upon examining the FTIR spectra, consistent bands
were identified in A, B, and C at wavelengths of 28702936, 1071-1262, and 1316-1322 cm−1. (Galan et
al., 2021) associate the bands at 2875, 1022, and
1370 cm−1 with the stretching vibrations of C–H, C–
O, and C–N, respectively, indicating the
polysaccharide structure of chitosan. Furthermore,

Chitosanglutaraldehyde-enzyme
(5x use, %)
56.11
2.28

bands at 3424 and 1651–1654 cm−1 were observed
in spectra A, B, and C, corresponding to the presence
of –OH and –NH groups in chitosan.
The FTIR spectrum revealed distinct bands
labeled A, B, and C. A notable band around 1451
cm−1 was observed in the glutaraldehyde-activated
beads (C). In the research conducted by (Hegde &
Veeranki, 2014), a band at 1495 cm−1 was identified,
which is thought to originate from the C=N bond
formed through the Schiff base reaction between
the amino groups of chitosan and the aldehyde
groups of glutaraldehyde. Additionally, in spectrum
C, a band at 1548 cm−1 was detected. (Altun &
Cetinus, 2007) indicated that the peak at 1574 cm−1
signifies the presence of C-N bonds created through
crosslinking between glutaraldehyde and chitosan,
the different demonstrating structural modifications
associated with enzyme attachment. A decrease in
transmittance at the C–N and C=N regions in
sample C compared to sample B further indicates
that the enzyme is bound to the glutaraldehyde
crosslinked
matrix. Furthermore, spectrum C
displayed a band at 2876 cm−1. (Galan et al., 2021)
linked the band at 2875 cm−1 to C-H crosslinking
that overlaps with the -CH2- groups in the
glutaraldehyde structure.

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Figure 11. FTIR analysis of immobilized beads
CONCLUSIONS
Based on the research conducted, the following
conclusions can be drawn: The production of chitosan
beads activated with glutaraldehyde for the
immobilization of urease from red lentil seeds,
concentrated with 50% acetone, demonstrates optimal
activity at a chitosan concentration of 0.75%. This
occurs with glutaraldehyde immersion at 2% and a
temperature of 25 °C for 2 hours, yielding an activity
value of 7.042 U/g. The immobilized P50 urease
exhibited a residual activity of 51% after seven uses.
SEM-EDX analysis revealed an average pore size of
0.22 µm in the chitosan beads, characterized by a
smooth, round surface texture. In contrast, the
chitosan-glutaraldehyde-enzyme beads used seven
times showed a pore size of 0.31 µm, with a distinctly
visible round surface texture. FTIR analysis confirmed
the presence of C-N and C=N peaks, indicating the
incorporation of glutaraldehyde.
ACKNOWLEDGEMENTS
We would like to express our gratitude to the
Research and Community Service Institution (LPPM) of
Jenderal Soedirman University for providing research
funding through the Public Service Agency (BLU) under
the Basic Research UNSOED scheme, contract
number: 26.573/UN23.35.5/PT.01/II/2024.
REFERENCES
Al-Garawi, Z. S., Taha, A. A., Abd, A. N., & Tahir, N.
T. (2022). Immobilization of urease onto
nanochitosan enhanced the enzyme efficiency:
Biophysical studies and in vitro clinical
application on nephropathy diabetic Iraqi
patients. Journal of Nanotechnology, 2022(1).
https://doi.org/10.1155/2022/8288585
Altun, G. D., & Cetinus, S. A. (2007). Immobilization
of pepsin on chitosan beads. Food Chemistry,
100(3), 964–971. https://doi.org/10.1016/
j.foodchem.2005.11.005
Basir, I. F., Mahatmanti, F. W., & Haryani, S. (2017).

Sintesis komposit beads kitosan/arang aktif
tempurung kelapa untuk adsorpsi ion Cu(II)
(Chitosan beads/coconut shell activated
charcoal composite for Cu(II) ion adsorption).
Indonesian Journal of Chemical Science, 6(2).
http://journal.unnes.ac.id/sju/index.php/ijcs
Bedan, D. S. (2020). Extraction, precipitation and
characterization of urease from (Vicia faba) L.
Al-Mustansiriyah Journal of Science, 31(1), 9.
https://doi.org/10.23851/mjs.v31i1.555
Çetinus, Ş. A., & Öztop, H. N. (2003). Immobilization
of catalase into chemically crosslinked chitosan
beads. Enzyme and Microbial Technology,
32(7), 889–894. https://doi.org/10.1016/
S0141-0229(03)00065-6
Chen, H., Zhang, Q., Dang, Y., & Shu, G. (2013). The
Effect of glutaraldehyde cross-linking on the
enzyme activity of immobilized β-galactosidase
on chitosan bead. Advance Journal of Food
Science and Technology, 5(7), 932–935.
https://doi.org/10.19026/ajfst.5.3185
Galan, J., Trilleras, J., Zapata, P. A., Arana, V. A., &
Grande-Tovar, C. D. (2021). Optimization of
chitosan glutaraldehyde-crosslinked beads for
reactive blue 4 anionic dye removal using a
surface response methodology. Life, 11(2), 1–
20. https://doi.org/10.3390/life11020085
Gilani, S. L., Najafpour, G. D., Moghadamnia, A., &
Kamaruddin, A. H. (2016). Stability of
immobilized porcine pancreas lipase on
mesoporous chitosan beads: A comparative
study. Journal of Molecular Catalysis B:
Enzymatic, 133, 144–153. https://doi.org/
10.1016/j.molcatb.2016.08.005
Gorissen, S. H. M., Crombag, J. J. R., Senden, J. M.
G., Waterval, W. A. H., Bierau, J., Verdijk, L. B.,
& van Loon, L. J. C. (2018). Protein content and
amino acid composition of commercially
available plant-based protein isolates. Amino
Acids, 50, 1685–1695. https://doi.org/10.
1007/s00726-018-2640-5

276

Optimization and Characterization of Urease

Zusfahair, et al.

Gür, S. D., İdil, N., & Aksöz, N. (2018). Optimization
of enzyme co-immobilization with sodium
alginate and glutaraldehyde-activated chitosan
beads.
Applied
Biochemistry
and
Biotechnology,
184(2),
538–552.
https://doi.org/10.1007/s12010-017-2566-5
Hegde, K., & Veeranki, V. (2014). Studies on
immobilization of cutinases from thermobifida
fusca on glutaraldehyde activated chitosan
beads. British Biotechnology Journal, 4(10),
1049–1063.
https://doi.org/10.9734/bbj/
2014/12558
Jamwal, S., Ranote, S., Dautoo, U., & Chauhan, G. S.
(2020). Improving activity and stabilization of
urease by crosslinking to nanoaggregate forms
for herbicide degradation. International Journal
of Biological Macromolecules, 158, 521–529.
https://doi.org/10.1016/j.ijbiomac.2020.04.2
24
Kamburov, M., & Lalov, I. (2014). Preparation of
chitosan beads for trypsin immobilization.
Biotechnology & Biotechnological Equipment,
26(1),(1), 156–163. https://doi.org/10.5504/
50YRTIMB.2011.0029
Koçak, B. (2020). Importance of urease activity in soil.

International Scientific and Vocational Studies
Congress – Science and Health, 12(December),

12–15. www.bilmescongress.com
Kurniasih, M., Purwati, Dewi R. S., Budiarni K. (2022).
Synthesis and Antimicrobial Activity of Silver NMethyl Chitosan. Molekul, 17(2), 145–155,
2022, doi: 10.20884/1.jm.2022.17.2.5059.
Kustomo. (2022). Uji karakterisasi dan mapping
magnetit nanopartikel terlapisi asam humat
dengan scanning-electron-microscope–energy
dispersive x-ray(SEM-EDX) [Characterization
and mapping test of humic acid-coated
magnetite–energy dispersive x-ray(SEM-EDX)]
nanoparticles. Indonesian Journal of Chemical
Science, 9(3), 148–153.
Lahiri, P. (2015). Effective immobilization of jack bean
(Canavalia ensiformis) urease onto chitosan.
Journal of Chemistry and Chemical Sciences,
5(7), 2319–7625.
Lv, M., Ma, X., Anderson, D. P., & Chang, P. R. (2018).
Immobilization of urease onto cellulose spheres
for the selective removal of urea. Cellulose,
25(1), 233–243. https://doi.org/10.1007/
s10570-017-1592-3
Malhotra, I., & Basir, S. F. (2020). Application of
invertase immobilized on chitosan using
glutaraldehyde or tris (hydroxymethyl )
phosphine as cross-linking agent to produce
bioethanol.
Applied
Biochemistry
and
Biotechnology, 191(2), 838–851.
Muslihah, N. I., Zusfahair, Z., Ningsih, D. R., &
Nuradha, F. (2024). The Characteristics of
urease enzyme of green bean seeds (Vigna
radiata L.) and its activity as an antifungal

against Candida albicans. Jurnal Kimia Sains
Dan
Aplikasi,
27(3),
145–150.
https://doi.org/10.14710/jksa.27.3.145-150
Pramono, I. A., Haryadi, W., & Joko Raharjo, T.
(2018). Optimization of lipid extraction from
Spirulina platensis using osmotic pressure
assisted by ultrasonic wave and its enzymatic
production of methyl ester. Berkala MIPA, 25(2),
116–128.
Pratantie, E. M., Bintoro, V. P., & Dwiloka, B. (2021).
Isolasi enzim amilase dari kecambah kacang
tunggak (Vigna unguiculata) [Isolation of
amylase enzyme from cowpea sprouts (Vigna
unguiculata)]. Jurnal Ilmiah Teknosains, 7(1),
1–5.
Qu, B., & Luo, Y. (2020). Chitosan-based hydrogel
beads: Preparations, modifications and
applications in food and agriculture sectors – A
review. International Journal of Biological
Macromolecules,
152,
437–448.
https://doi.org/10.1016/j.ijbiomac.2020.02.240
Sabilla, I. A., & Susanti, E. (2019). Pemurnian parsial
ekstrak kasar selulase Bacillus circulans
dengan metoda pengendapan aseton (Partial
purification of crude extract of Bacillus circulans
cellulase by acetone precipitation method.
Jurnal Kimia Riset, 4(1), 40–48.E, 4(1), 40–48.
Singh, A. K., Singh, M., & Verma, N. (2017).
Extraction, purification, kinetic characterization
and immobilization of urease from Bacillus
sphaericus MTCC 5100. Biocatalysis and
Agricultural Biotechnology, 12, 341–347.
https://doi.org/10.1016/j.bcab.2017.10.020
Taha, A. A., Hameed, N. J., & Rashid, F. H. (2020).
Decolorization of phenol red dye by
immobilized laccase in chitosan beads using
laccase - Mediator - System Model. Baghdad
Science
Journal,
17(3),
720–725.
https://doi.org/10.21123/bsj.2020.17.3.0720
Tetiker, A. T., & Ertan, F. (2017). Investigation of some
properties of immobilized urease from Cicer
arietinum and its using in determination of urea
level in some animal feed. Journal of

Innovations in Pharmaceutical and Biological
Sciences (JIPBS), 4(2), 2–7.

Verma, M. L., Kumar, S., Das, A., Randhawa, J. S., &
Chamundeeswari, M. (2020). Chitin and
chitosan-based support materials for enzyme
immobilization
and
biotechnological
applications. Environmental Chemistry Letters,
18(2), 315–323. https://doi.org/10.1007/
s10311-019-00942-5
Xu, K., Peng, Y., Huang, M., Liu, Z., & Zhen, J. (2021).
Biocementation effect of high-efficiency ureaseproducing
bacteria
mutagenized
from
indigenous bacteria. IOP Conference Series:
Earth and Environmental Science, 861(7).
https://doi.org/10.1088/1755 - 1315/861/7/
072104

277

Molekul, Vol. 20. No. 2, July 2025: 268 – 278

Zhang, J., Wang, Z., He, C., Liu, X., Zhao, W., Sun,
S., & Zhao, C. (2019). Safe and effective
removal of urea by urease-immobilized,
carboxyl-functionalized PES beads with good
reusability and storage stability. ACS Omega,
4(2), 2853–2862. https://doi.org/10.1021/
acsomega.8b03287
Zhou, Y., Wang, L., Wu, T., Tang, X., & Pan, S. (2013).
Optimal immobilization of β-glucosidase into
chitosan beads using response surface
methodology.
Electronic
Journal
of
Biotechnology, 16(6). https://doi.org/10. 2225
/vol16-issue6-fulltext-5
Zusfahair, Ningsih, D. R., Fatoni, A., Bilalodin, &
Nuraini, A. N. (2023). The Isolation,
immobilization, and characterization of urease

from the seeds of winged bean ( Psophocarpus
tetragonolobus (L.) DC. Molekul, 18(1), 70–79.
Zusfahair, Z., Ningsih, D., Fatoni, A., & Santri, D.
(2018). Pemurnian parsial dan karakterisasi
urease dari biji kacang panjang (Vigna
unguiculata subsp sesquipedalis L . ) [Partial
purification and urease characterization of long
bean seeds (Vigna unguiculata subsp
sesquipedalis L.)]. Alchemy Jurnal Penelitian
Kimia, 14(1), 72–83. https://doi.org/10.
20961/alchemy.14.1.13000.72-83
Zusfahair, Z., Ningsih, D. R., Bilalodin, B., Fatoni, A.,
Setiawan, E., & Sulistyowati, A. (2025). Partial
purification and characterization of urease from
red lentils (Vicia lens (L.) Coss. & Germ.).
Molekul, 20(1), 120–128.

278