Hindawi

Journal of Nanotechnology
Volume 2022, Article ID 8288585, 9 pages
[Link]

Research Article
Immobilization of Urease onto Nanochitosan Enhanced the
Enzyme Efficiency: Biophysical Studies and in Vitro Clinical
Application on Nephropathy Diabetic Iraqi Patients

Zahraa S. Al-Garawi ,1 Ali A. Taha ,2 Ahmed N. Abd,2 and Noor T. Tahir 3

1
Chemistry Department, College of Science, Mustansiriyah University, Baghdad, Iraq
2
Chemistry Department, College of Science, University of Diyala, Baqubah, Diyala, Iraq
3
Department of the National Diabetes Center for Treatment and Research, Mustansiriyah University, Baghdad, Iraq

Correspondence should be addressed to Zahraa S. Al-Garawi; amyloid2015@[Link]

Received 24 August 2021; Revised 7 March 2022; Accepted 6 April 2022; Published 8 June 2022

Academic Editor: Marco Rossi

Copyright © 2022 Zahraa S. Al-Garawi et al. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is
properly cited.
Immobilization of enzymes is an effective method for improving the properties and applications of modern enzymes. There are
several supports for enzyme immobilization. Because of its unique features, such as inertness and high surface area, chitosan was
widely used to immobilize enzymes. Immobilization of urease onto chitosan is a promising approach to treating high urea levels in
the blood, however, the immobilization conditions for the best kinetics and enzyme efficiency are still challenging. Herein, we
tried to immobilize urease onto nanochitosan (chitosan NPs) through a cross-linker and study the kinetics (km and vmax values)
and thermodynamics (Ea, ∆H, ∆S, and ∆G) parameters of the enzyme reaction before and after immobilization at different
substrate concentration (50, 100, 150, 200, and 250 mg/dl) and incubation temperature (15, 20, 25, 30, 35, and 40°C) under selected
optimum conditions. The immobilized urease chitosan NPs was characterized in our previous work using Fourier transform
infrared infrared(FTIR), Atomic force forcemicroscopy (AFM), and andimaged here by scanning electron microscopy
microscopy(SEM). Results revealed that the highest efficiency % of immobilization (70.38%) was observed at 750 mg/ml chitosan
NPs and phosphate buffer pH 7 at 40°C. With an increase of Km value for the immobilized enzyme, however, the efficiency of the
enzyme was significantly higher than the free enzyme, p < 0.001. In addition, the activation energy of the reaction catalyzed by the
immobilized enzyme was lower than that of the free enzyme, which suggests that the active site geometry of the immobilized
enzyme was more favorable to accommodate the substrate and thus required less energy than that of the free enzyme. The reaction
was endothermic by means of positive ∆H. The immobilized urease enzyme was in vitro applied to blood samples of Iraq
nephropathy diabetic patients (n  35) to investigate the effect on serum urease activity and urea level compared to healthy
volunteers. Interestingly, the activity of serum urease significantly increased after adding the immobilized enzyme and the level of
urea significantly decreased (p < 0.0001) by ∼1.5 folds. Thus, applying an immobilized ureaseurease to remove urea from blood
could be effective in the blood detoxification or dialysis regeneration system of artificial kidney machines.

1. Introduction wastewater treatment, food, and drug analysis [3]. Urease

has been employed for the direct removal of urea from the
Urease (EC [Link]), a metalloenzyme [1], catalyzes the blood for detoxification [4]. Urease immobilization has been
hydrolysis of urea into ammonia and carbon dioxide [2]. performed on many matrices for clinical analytical purposes
Removal of urea and its ammonium ion plays an essential [5, 6]. Several strategies can be effectively used to immobilize
role in the analytical, clinical, and chemical treatments as- enzymes [7], including surface adsorption, encapsulation, or
sociated with several processes, such as blood and urine entrapment [8]. However, most procedures of immobili-
analysis, renal failure, artificial reinforcement, as well as zation cause a reduction of the enzyme activity [9, 10] or
2 Journal of Nanotechnology

alter the urease structure (e.g., when immobilization hap- chosen for this study was diabetes patients with nephropathy
pens on the nanoceria surface) [11]. There is no single diagnosed with high level of blood urea. The immobilized
approach or support that retains the enzymatic activity to be enzyme polymer was characterized using scanning electron
suitable for applications, yet. This is due to the different microscopy (SEM), Fourier transform infrared (FTIR), and
chemical properties and compositions of the enzymes, as atomic force microscopy (AFM). Kinetics and thermody-
well as the diverse qualities of the substrates and products. namic behaviors of the enzyme were investigated before and
The major advantages of immobilization techniques are the after immobilization events.
stability of the enzymatic activity after immobilization and
the reversibility of the process, which allows the enzyme and
support materials to be reused for multiple applications.
2. Experimental
Immobilization of enzymes is critical not just for their reuse 2.1. Materials. Urease enzyme was obtained from enzymatic
but also for their more efficient utilization. Immobilized colorimetric test with urea kit (human; Germany); chitosan
enzymes are more robust and more resistant to environ- NPs (>95% degree of deacetylation and viscosity 80 maps)
mental changes compared to free enzymes in the solution was obtained from Shaanxi Sangherb Bio-Tech, China;
[12, 13]. Chitosan, a poly-N-acetyl glucosamine, is the glutaraldehyde (25%) was from Avonchem (UK); glacial
N-deacetylated derivative of chitin (the primary component acetic acid and sodium tripolyphosphate were from ALPHA
of crustacean shells), which is the second most prevalent CHEMIKA (India); sodium phosphate buffer was from
biopolymer after cellulose [14]. Chitosan has a primary ChemicalPoint (Germany); sodium acetate were from
amine at the C-2 position of glucosamine residues. The Thomas Baker (India); and ammonium chloride and am-
presence of many high amines is a unique feature that monia were from Merck, Instruments: pH meter (TZ397A
provides chitosan with important functional properties [15]. Orion), Spectrophotometer (CRCIL. England), SEM (Philips
Chitosan has numerous important biological and chemical XL-30S FEG, Netherlands).
features due to a unique collection of traits such as bio-
compatibility, biodegradability to harmless products, non-
toxicity, physiological inertness, antibacterial activity, heavy 2.2. Methods
metal ion chelation, gel-forming ability, hydrophilicity, and
extraordinary attraction to proteins [16]. Nanoparticles 2.2.1. Immobilization of Urease onto Chitosan NPs.
(NPs) are nano-sized ultrafine colloidal drug carrier systems Preparation of chitosan NPs was carried out according to the
between 10 and 100 nm, consisting of different functional ionic gelation method described by Vaezifar [23]. Briefly,
organic and/or inorganic backbones [17]. In the past 15 750 mg of chitosan NPs (average molecular weight and
years, using of NPs becomes exponentially important. Those degree of deacetylation, 90%) were dissolved in 1% acetic
particles have been extensively explored due to their exciting acid. Sodium tripolyphosphate solution (1 mg/ml) was
unique properties compared to microparticles such as added continuously to the chitosan NPs solution and stirred
sustainability, controlling of drug release, site-specific tar- at room temperature until chitosan NPs nanoparticles
geting, and higher surface area to volume ratio than mi- formed spontaneously. After 24 h, a viscous pale-yellow
croparticles. These exceptional properties found a wide chitosan solution was formed. Glutaraldehyde solution (1%)
range of applications in biomedical and pharmaceutical was added to the viscous pale-yellow solution of chitosan
fields such as biomolecule detection, vaccines, tissue engi- NPs and stirred for 24 h at room temperature to become
neering, drug delivery, cancer therapy, and high and quick increasingly viscous. The enzyme 1.2 IU (0.1 mL solution in
accuracy diagnosis [18]. Moreover, chitosan NPs are more 0.1 mM phosphate buffer, pH 7) was immobilized on chi-
stable, soluble in aqueous acidic solution, biodegradable, less tosan NPs. After that, the solutions were combined and
toxic, and fully biocompatible natural polymer. These chi- agitated for 1 h at room temperature.
tosan NPs are approved by GRAS (generally recognized as
safe by the United States Food and Drug Administration (US
FDA)) [19]. Functionalization of NPs surface is to improve 2.2.2. Urease Activity (Free and Immobilized). Urease ac-
and/or add extra properties to enable those NPs to be ap- tivity was measured before and after immobilization using a
plicable in medical or pharmaceutical fields. In this regard, spectrophotometric method [24]. Briefly, urea (30 μl, 50 mg/
different types of nanomaterials with characteristic prop- dL) was incubated with 0.25 ml of urease solution (1.2 μl for
erties and specific functional groups located on the surface free and 1.56 μl for immobilized enzyme) at 37°C for 10 min.
are the priority of the functionalization process. Some A volume of 0.3 ml of Berthelot’s reagent A (5.0 gm phe-
molecules are used to functionalize the NPs surface such as nol + 25 mg of sodium nitroprusside in 500 mL of phosphate
small proteins, fragments of peptides, antibodies, DNA, buffer) was added and mixed well, and then 0.15 ml of
enzymes, and oligosaccharides [20]. Interestingly, this bio- Berthelot’s reagent B (2.5 g sodium hydroxide + 4.2 mL of
chemical modification of the surface of NPs is often caused sodium hypochlorite in 500 mL of phosphate buffer) was
less toxicity and high stability in biological fluids [21, 22]. added; the mixture was mixed well and incubated at dark for
Herein, we aimed to increase the efficiency of the urease 30 min to allow the enzymatic hydrolysis of urea into am-
enzyme by immobilizing onto chitosan NPs and thus monia and water. The resulted ammonia was measured at
functionalize their surfaces and deliver the enzyme into the 670 nm. The activities of free and immobilized urease were
site-cell target without degradation. The population target expressed as μmol·min−1 [25]. One unit of enzyme activity
Journal of Nanotechnology 3

was defined as the amount of enzyme that converts 1 μmol The relationship that links the change of enthalpy (∆H)
ammonia into product per minute at pH 7 and 37 °C. to the change of entropy (∆S), the Eyring–Polanyi equation
(equation (5)), was used to determine these parameters [27]:
V KB ΔS ΔH 1
2.2.3. Effect of pH and Temperature on the Enzyme Activity. 􏼒Ln 􏼓 � Ln + − . (5)
The effect of pH on immobilized urease activity was studied T h R R T
at different pH values ranging from 3.6 to 10.9, using 0.1 M The fraction (Ln V/T) was plotted versus [1000/T(k)] to
sodium acetate (pH 3.6–6.0), 0.1 M sodium phosphate (pH obtain the slope (−ΔH)/R) and the intercept was (Ln KB/h +
6.0–8.0), and 0.1 M ammonium chloride-NaOH (pH ΔS/R), where KB � Boltzmann constant (1.38 × 10−23 J.K-1).
8.0–11.0). The activity of the enzyme was measured as R � General gases constant (8.314 J.K-1 mol-1). H � Planck
previously described [24] to indicate the optimum pH that constant (6.62 × 10−34 J.s).
gives the highest activity. The free Gibbs relationship (equation (6)) was then used
To determine the optimum temperature of the enzymatic to calculate the change in Gibbs energy:
reaction, the reaction mixture (enzyme, urea, and reagents A
and B) was incubated under different temperatures ΔG � ΔH − TΔS. (6)
(20–60°C) and the optimum pH. The activity of the enzyme where the temperature (T) was measured in Kelvin at 25°C.
was measured as previously described [24].

2.2.6. Characterization of the Free and Immobilized Enzyme.

2.2.4. Effect of Concentration of Chitosan NPs on the Enzyme Chitosan NPs and the immobilized enzyme were well
Activity. To determine the best concentration of chitosan characterized using FTIR and AFM [28]. They were also
NPs to carry the enzyme efficiently, different weights of characterized here using SEM.
chitosan NPs (0.25, 0.5, 0.75, and 1.0 gm) were used to
immobilize the enzyme using the suitable conditions for the
immobilization event. The activity of the immobilized urease 2.2.7. Scanning Electron Microscopy (SEM). SEM was used
was measured as previously described [24]. to characterize the morphology of chitosan NPs and
immobilized urease on chitosan NPs. Spraying gold powder
was used to apply the gold coating. To create a dilute sus-
2.2.5. Kinetic and Thermodynamic Parameters. Kinetics and pension, samples were sonicated in water for 3 minutes.
thermodynamics were determined by using five different After drying, a drop of the original chitosan NPs diluted
concentrations of urea substrate (50, 100, 150, 200, and suspension was put on a glass slide and examined.
250 mg/dL) at different incubation times (10, 20, and 30 min)
and temperatures (15, 20, 25, 30, 35, 40, and 45°C). The
Michalis–Menten equation (equation (1)) was linearized by 2.3. Clinical Application of Immobilized Enzymes. For the
the Lineweaver–Burk equation (equation (2)) and used to best utilization of the immobilized enzyme polymer, it was in
determine the kinetics parameters Vmax, Km, and Vmax/Km vitro applied to the serum of diabetic nephropathy patients
[26]: who are supposed to have high urea levels. The results were
compared with those obtained from control volunteers. The
Vmax [S] enzyme activity and urea level were measured before and
V� , (1)
Km +[S] after adding the immobilized enzyme. Some other param-
eters such as sex, age, weight, height, and fasting blood sugar
where V° � initial velocity, μg·N·g−1·min−1; [S] � urea con- (FBS) were collected and compared.
centration, mM; Vmax � maximum velocity, μg·N·g−1·min−1;
and Km � Michaelis constant, mM.
[S] [S] K 2.3.1. The Study Groups. This study included 70 volunteers
� + m. (2) in total who visited the National Diabetes Center of Mus-
V Vmax Vmax
tansiriyah University in Baghdad. The participants were
The linearized version (equation (3)) of the Arrhenius separated into two groups: the first group consists of 35
equation (equation (4)) was used by assuming K≈V to de- patients with diabetic nephropathy aged between 23 and 60
termine Ea and A parameters for the immobilized urease: years, and the second group was a control group consisting
of 35 healthy participants aged between 18 and 50 years.
Ea
K � Ae − , (3) Research ethics of human rights were followed according to
RT the standard ethics of the National Diabetes Center of
Ea 1 Mustansiriyah University. All participants were diagnosed
ln K � ln A − , (4) by specialists of the center.
R T
where K � velocity constant, min−1; Ea � activation energy,
KJ·mol−1; A � pre-exponential factor, μg·g−1·min−1; and 2.3.2. Blood Samples. A volume of 5 ml blood was collected
R � 8.314 K−1·mol−1. in the morning, left at room temperature for half an hour to
coagulate, centrifuged at 3,000 rpm for 10 minutes, and then
4 Journal of Nanotechnology

the serum was collected. The serum samples were stored at demonstrated that the optimum concentration of chitosan
−4°C until further enzyme analyses. NPs (750 mg·w/w) and that of urease (1.2 mg/ml) were ef-
ficient for the immobilization process. The loading capacity
of urease on chitosan NPS decreased with increasing the
2.3.3. Exclusion Criteria. This study included patients free of amount of chitosan NPs at a constant concentration of the
pregnancy, chronic complications of the kidney, any kind of enzyme (Figure 1(a)). The high amount of chitosan NPs over
tumors, any participants out of the required age limit (18–60 750 mg may distribute urease throughout the chitosan NPs
years old), and animals. particles. Therefore, it is likely that 750 mg w/w of chitosan
NPs provides a significant surface area occupied by urease
2.3.4. Statistical Analysis. Unpaired Student t-test (with [31].
Welch’s correction, two-tailed) and ordinary one-way
ANOVA with Brown–Forsythe and Bartlett’s tests were 3.1.2. Effect of pH on Immobilization Process. The effect of
performed to indicate statistical descriptive and analyses and pH on the activity of immobilized urease enzyme is shown in
significant differences between the studied groups at p (Figure 1(b)). The optimum pH of the immobilized urease
significant level <0.05. For statistical analysis, GraphPad was 7.0. The activity of the enzyme showed a sharp im-
Prism 9 software was used. provement from pH 3.6 to 7.0 due to the production of
ammonia, which modulated the acidity of the solution and
2.3.5. Determination of Serum Urea Concentration. The enhanced the activity [32]. Figure 1(b) also showed that the
immobilized urease was used to hydrolyze urea in the serum activity dropped in the basic solution, which may be due to a
of patients and compare the results with those of control in possible distribution of the protonation status of side chains
vitro. The immobilized urease was incubated in phosphate of the amino acid residues on the surface of the enzyme. This
buffer 50 mM (pH 7.0) at 37°C. The reaction was initiated by deprotonation in an alkaline medium could negatively affect
adding 90 μl of serum. After 10 minutes, the reaction the intramolecular electrostatic interactions, cause desatu-
mixture was taken out; the precipitated proteins were re- ration of the 3D architecture of the enzyme, and reduce its
moved by centrifugation; and the urea was estimated from activity [33].
the supernatant [29, 30]. A standard curve was used for the
known concentration. The urea estimated in serum samples 3.1.3. Effect of Temperature on Immobilization Urease
was also compared with measurements done through Activity. The effect of temperature on immobilized urease
®
Autoanalyzer (Genolab TEK, USA). activity is shown in Figure 1(c). The activity of the enzyme
increased as the temperature increased to 40°C due to urease
2.3.6. Determination of Serum Urease Activity. Before loading capacity as a large number of potential binding sites
adding the immobilized enzyme, serum urease activity was on its surface, allowing it to spread more easily on the
calculated by incubating 90 μl of serum sample with 10 μl nanoparticle surface [29, 34]. Over 40°C, there was a decline
urea (8.33 mMol) at 37°C for 10 minutes. The reaction was in the urease activity due to the denaturation of the enzyme.
started by adding 100 μl of Berthlos reagent (reagent A). A The velocity of the enzyme and the kinetics was deter-
volume of 50 μl of Berthlos reagent B was then added to the mined using Michaelis–Menten as follows:
reaction mixture and incubated at 37°C in the dark. The V max[S]
optical density was detected at wavelength 670 nm. V� , (7)
Km +[S]
After adding immobilized urease, the activity of the
enzyme was calculated by incubating 90 μl of serum sample where Km is the Michaelis–Menten constant and Vmax is the
with 90 μl of immobilized enzyme and 10 μl of urea maximal reaction rate. Lineweaver–Burk plot (Figure 2(a))
(8.33 mMol) at 37°C for 10 minutes. The reaction was started was used to obtain Km values. The Km for free and immo-
by adding adding 100 μl of Berthlos reagent (reagent A). A bilized urease were 10.01 and 11.36 mM, respectively. This
volume of 50 μl of Berthlos reagent B was then added to the result indicated that the free urease enzyme has a higher
reaction mixture and incubated at 37°C in the dark. The affinity for its substrate urea when compared to the urease
optical density was detected at wavelength 670 nm. immobilized on chitosan NPs. Increased Km of urease en-
zyme after immobilization has similarly been reported by
3. Results and Discussion [29, 30, 35]. Furthermore, Vmax for free and immobilized
urease on chitosan NPs were 61.34 μmol·min−1 and
3.1. Kinetic Parameters 63.69 μmol·min−1, respectively (Figure 2(a)).
3.1.1. Effect of Concentration of Chitosan NPs on the
Immobilization. At a constant urease enzyme concentra- 3.2. Thermodynamic Parameters. Arrhenius graph between
tion, the activity of immobilized urease displayed a typical ln K and 1/T was used to calculate Ea of free and immobilized
saturation curve with the increase of the amount of chitosan urease activity [36], where the relationship between ln (K/T)
NPs (Figure 1(a)). When the concentration of chitosan NPS and 1/T was used to calculate the enthalpy (∆H) and entropy
increased up to 1 g·w/w, a gradual increase in the activity of (∆S). The results in Figure 2(b) indicated that the activation
immobilized urease was indicated up to 2701 U/L. This result energy of immobilized urease was 27.295 KJ/mol, which was
Journal of Nanotechnology 5

3000
2500

Activity U (I)
Activity U (I)
Activity U (I)
2000
1500
1000
500
0
0 0.5 1 1.5 3.6 6 7 8 10.9 20 30 40 50 60
[nanochitosan] PH Temperature
(a) (b) (c)

Figure 1: Kinetics of immobilized enzyme. Effect of amount of chitosan NPs on immobilization urease activity (a), the effect of pH on the
enzyme activity (b), and the effect of temperature on the activity of immobilized enzyme (c).

0.06 y = 0.1785x + 0.0157 5 y = -3283.1x+14.447

y = 0.1632x + 0.0163 4.5 y = -4968.6x+20.247
0.05
4
0.04
3.5
1 (v) Mm/min

0.03 3

In (V)
0.02 2.5
2
0.01 1.5
1/[S] mM
0 1
-0.2 -0.1 0 0.1 0.2 0.3 0.5
-0.01
0
-0.02 0.0031 0.0032 0.0033 0.0034 0.0035
1/T (K2)
free enzyme
Immobilized enzyme Immobilized enzyme
free enzyme
(a) (b)

Figure 2: (a) Lineweaver–Burk plots of free enzyme and immobilized enzyme and (b) Arrhenius relationships of free enzyme and
immobilized enzyme.

lower than that of free enzyme 41.303 KJ/mol, which con- enzyme forms, which means that the conversion of the [E − S]
firmed the previous study [37]. Lower Ea for the immobi- complex to the product was more spontaneous [40].
lized enzyme could indicate that the conformation of the
active site was more favorable to bind most of the [S]
molecules in the reaction mixture, and thus, more [E − S] 3.3. Characterization of the Immobilized Enzyme Copolymer.
complexes could reach the energy barrier [38]. The polymer chitosan NPs loaded enzyme was well char-
The thermodynamic parameters of the urease hydrolysis acterized in our previous work by atomic force microscope
reaction, enthalpy ∆H, entropy ∆S, and Gibbs free energy ∆G AFM and Furious transfer infrared FTIR.
were calculated for immobilized urease enzyme and com-
pared with the standard values of urease (see Figure 3).
Results of the immobilized enzyme showed ∆H � 24.96 KJ/ 3.3.1. Scanning Electron Microscopy (SEM). The result of
mol, ∆S � −0.132 KJ/mol, and ∆G � 64.29 KJ/mol. The pa- SEM in Figure 4 showed the morphology of the immobilized
rameters of free enzyme are ∆H � 38.97 KJ/mol, enzyme before and after immobilization. Figure 4(a) showed
∆S � −0.084 KJ/mol, and ∆G � 64.0 KJ/mol) [39]. The lower that the morphology of chitosan NPs was uniform, spherical,
enthalpy ∆H value of the immobilized enzyme could indicate and well dispersed. The morphology of the urease enzyme
the efficiency of the transition state or activated complex showed a heterogeneous surface (Figure 4(b)); however, the
[E − S], when compared with that of the free enzyme. The morphology of the immobilized enzyme was uniform sur-
entropy ∆S values of immobilized and free form were neg- face when compared to chitosan NPs (Figure 4(c)). This
ative. It suggests that the structure of [E − S] at the transition might imply that the enzyme’s interaction with chitosan NPs
state is more ordered than that of the ground state. The ∆G is is more efficient. As a result, the compact and spherical form
the best parameter to estimate the feasibility and extent of the of chitosan NPs may become fluffy in the presence of the
chemical reaction. Results indicated low ∆G values for both urease enzyme.
6 Journal of Nanotechnology

0
0.0031 0.0032 0.0033 0.0034 0.0035
-0.5

-1

Lin V (T)
-1.5

-2

-2.5

-3

-3.5 1/T (k)

Immobilized enzyme
Free enzyme
Figure 3: The thermodynamic curve of free urease immobilized urease enzyme.

50 um 200 nm 500 nm

(a) (b) (c)

Figure 4: SEM images of urease enzyme (a), chitosan NPs particles (b), and immobilized urease-chitosan NPs (c). Scale bars � (a) 50 μm,
(b) 200 μm, and (c) 500 nm.

Table 1: A description of the studied groups. Values were expressed by mean ± SD.
Sex Age (year) Weight (Kg) BMI (Kg/m2) Duration of disease (year) FBS (mg/mL)
N 35 35 35 35 35 35
17 F
Mean ± SD (patients) 40.34 ± 8.468 80.69 ± 15.08 28.64 ± 4.710 6.343 ± 3.81 165.8 ± 9.55∗∗∗∗ ∝
18 M
19 F
Mean ± SD (control) 30.43 ± 7.81 67.80 ± 10.70 24.19 ± 2.61‡ — 89.00 ± 9.97 ∝
16 M
‡ ∗∗∗∗
Calculated for 10 participants only. P < 0.0001, refers to the difference in the significance level between FBS of patients compared to that of control.

3.4. Clinical Application of Immobilized Enzyme. The (p < 0.0001; Table 1; Figure 5(a)). Nephropathy diabetic
immobilized urease was in vitro applied to serum of ne- patients suffer from hyperglycemia perhaps due to injury to
phropathy diabetic Iraqi patients who suffered hyperaemia the small blood vessels in the body due to their high levels of
to investigate the effect of the released enzyme on the serum sugar, which reduces the ability of the small blood vessels in
urea level. Results were compared with control volunteers. the kidneys to carry out their function in purifying the blood
The serum concentration of urea hydrolyzed by the and filtering it from wastes, causing the accumulation of
immobilized urease enzyme was compared with those ob- these wastes in the blood.
®
tained by the Autoanalyzer . Table 1 showed that the mean
age of the studied population ranged from 30 to 40 years old,
Table 2 indicates that applying the embedded enzyme
has significantly enhanced the serum activity of urease for
patients were overweight (BMI � 28.64 ± 4.710 Kg/m2, patients by ∼2-folds and by ∼3.5-folds for controls
<29 Kg/m2) compared with normal-weight controls (p < 0.0001, see Figure 5). This enhancement could imply the
(24.19 ± 2.61 Kg/m2), and they were suffered from ne- successful release of urease from the platform (chitosan) into
phropathy diabetes for ∼6 years ± 3.81. Levels of fasting the serum within 30 min incubation time. Interestingly,
blood sugar FBS were significantly higher in patients Table 2 also showed that the urea level in patients was
Journal of Nanotechnology 7

200 ****

Patients-Immbeded urease
150
Concentration mg (dL)
****
Patients-Free urease

100

Control-Immbeded urease

50 ****

Control-Free urease

0
0 20 40 60 80
FBS-Patients

FBS-Control

Enzyme activity (IU)

(a) (b)

Control-After addition

****
Control-Befor addition

Patients-After addition

****
Patients-Before addition

0 20 40 60 80
Urea Concentration (mg/mL)
(c)

Figure 5: Effect of the immobilized urease enzyme on the activity of serum urease enzyme and urea concentration of nephropathy diabetic
patients and control volunteers: (a) levels of FBS in patients compared to that in control, (b) effect of the immobilized urease enzyme on the
enzyme activity, and (c) effect of the immobilized urease enzyme on the urea level. All data were collected in triplicates. The black bars refer
to the standard error of means SEM. The significance level of differences p was <0.0001.

Table 2: Levels of free and embedded urease in patients compared with controls. Values were expressed by mean ± SD.
Free urease (IU) Embedded urease (IU) Urea (mg/dL) § (before addition) Urea (mg/dL) § (after addition)
Mean ± SD
20.15 ± 2.28 ∝ 37.88 ± 9.32∗∗∗∗ ∝ 63.11 ± 7.95 ∝ , ξ 42.97 ± 3.40∗∗∗∗ ∝
(patients)∗∗∗ ξ
Mean ± SD (control)∗∗∗ ξ 14.34 ± 0.96 ∝ 51.32 ± 7.53∗∗∗∗ ∝ 29.80 ± 5.00 ∝ , ξ 16.34 ± 3.22∗∗∗∗ ∝
§ ∗∗∗∗ ∗∗∗
Before and after adding the immobilized enzyme. p < 0.0001. P < 001, ξ refers to the significance level of blood urea in patients before adding the
embedded enzyme compared to that of control. ∝ refers to the significance level of blood urea in patients after adding the embedded enzyme compared to that
of control or to that before the addition.

significantly elevated over that of the control adding the embedded enzyme and therefore lowered the
(63.11 ± 7.95 mg/dL vs. 29.80 ± 5.00 mg/dL). However, the urea concentration in serum (Table 2). Thus, applying the
urea level of patients was reduced after adding the embedded embedded urease enzyme onto chitosan showed for the first
enzyme by around ∼−1.5-fold and ∼−2-fold for controls time a successful acceleration of the original serum urease
(p < 0.001; Figure 5(b)). The reduction of urea level enzyme. Mechanistically, the availability of a larger number
explained that the catalyzing action of urease to hydrolyze of enzyme molecules increased the chance for the available
urea into bicarbonate and water was highly activated after substrate molecules to bind and then be hydrolyzed.
8 Journal of Nanotechnology

4. Conclusion edited the final manuscript. Ali A Taha has collected and
analyzed the samples and data and wrote the manuscript.
This study explored the immobilization efficiency of urease Ahmed N Abed has contributed to writing, revising, and
enzyme onto nanochitosan particles, for improving the enzyme editing the manuscript. Noor T has helped with collecting
prosperities in biotechnological and biomedical applications. the clinical data.
The immobilization conditions were optimized. Accordingly,
the urease enzyme was successfully immobilized onto chitosan Acknowledgments
NPs as a support material, confirmed by results of FTIR, AFM,
and SEM. The kinetic and thermodynamic parameters revealed The authors would like to thank Chemistry Department at
that the affinity of urea to the active site of the immobilized Diyala University for valuable assistance in FTIR analysis
enzyme was lower than that of the free enzyme. The enzymatic and Chemistry Department at Baghdad University for useful
efficiency after immobilization was higher than the free en- discussion about AFM results.
zyme, and the reaction was endothermal. Additionally, in vitro
application of the immobilized urease on sera of nephropathy Supplementary Materials
diabetic patients significantly enhanced the enzyme activity of
the serum urease and decreased the urea level (p < 0.0001) by Graphical abstract. (Supplementary Materials)
∼1.5-folds. To our knowledge, this is the first time to show the
effect of immobilized urease enzyme on reducing the blood References
urea level of nephropathy diabetic patients. Thus, applying an
immobilized urease to remove urea from blood could be ef- [1] B. Zerner, “Recent advances in the chemistry of an old en-
fective in the blood detoxification or dialysis regeneration zyme, urease,” Bioorganic Chemistry, vol. 19, no. 1,
system of artificial kidney machines. pp. 116–131, 1991.
[2] J. B. Sumner, A Source Book in Chemistry, 1900–1950, Harvard
University Press, Cambridge, MA, USA, pp. 322–327, 2013.
Data Availability [3] S. Yabuki, “Polyelectrolyte complex membranes for immo-
bilizing biomolecules, and their applications to bio-analysis,”
All data have been included in the manuscript. Analytical Sciences, vol. 27, no. 7, p. 695, 2011.
[4] K. B. Lee, D. K. Boadi, and R. J. Neufeld, “Blood urea clearance
Additional Points with microencapsulated urease,” Journal of Theoretical Biol-
ogy, vol. 175, no. 3, pp. 295–303, 1995.
No animals were used in this research. All human research [5] N. Das, P. Prabhakar, A. M. Kayastha, and R. C. Srivastava,
procedures were in accordance with the ethical standards of “Enzyme entrapped inside the reversed micelle in the fabri-
the committee responsible for human experimentation cation of a new urea sensor,” Biotechnology and Bioengi-
(institutional and national) authorized by the National neering, vol. 54, pp. 329–332, 1997.
Diabetes Center for Treatment and Research. Although this [6] A. S. Hoffman, “Hydrogels for biomedical applications,”
was an in vitro study, the authors have obtained permission Advanced Drug Delivery Reviews, vol. 64, pp. 18–23, 2012.
from all participants. [7] L. Cao, “Immobilised enzymes: science or art?” Currurrent
Opinion in Chemical Biology, vol. 9, pp. 217–226, 2005.
[8] D. E. Anderson, S. Balapangu, H. N. A. Fleischer et al.,
Ethical Approval “Investigating the influence of temperature on the kaolinite-
base synthesis of zeolite and urease immobilization for the
Ethical approval was given by the ethics committee of potential fabrication of electrochemical urea biosensors,”
Mustansiriyah University (Iraq). Sensors, vol. 17, no. 8, p. 1831, 2017.
[9] L. Zhang, Y. Du, J. Song, and H. Qi, “Biocompatible magnetic
Consent nanoparticles grafted by poly(carboxybetaine acrylamide) for
enzyme immobilization,” International Journal of Biological
Not applicable. Macromolecules, vol. 118, pp. 1004–1012, 2018.
[10] Y. Q. Almulaiky, R. M. El-Shishtawy, M. Aldhahri et al.,
Disclosure “Amidrazone modified acrylic fabric activated with cyanuric
chloride: a novel and efficient support for horseradish per-
This study was registered in the research annual plan of the oxidase immobilization and phenol removal,” International
Chemistry Department at Mustansiriyah University, 2020 Journal of Biological Macromolecules, vol. 140, pp. 949–958,
[Link] 2019.
[11] H. K. Al-Hakeim, M. K. Khudhair, and E. A. Grulke, “Im-
mobilization of urease enzyme on nanoceria modifies sec-
Conflicts of Interest ondary and tertiary protein structures,” Acta Chimica
Slovenica, vol. 9, pp. 44–53, 2016.
The authors declare that they have no conflicts of interest. [12] N. Alegret, A. Dominguez-Alfaro, and D. Mecerreyes, “3D
scaffolds based on conductive polymers for biomedical ap-
Authors’ Contributions plications,” Biomacromolecules, vol. 20, no. 1, pp. 73–89, 2018.
[13] S. A. Mohamed, M. H. Al-Harbi, Y. Q. Almulaiky et al.,
Zahraa Al-Garawi designed the project, analyzed and “Immobilization of Trichoderma harzianum α-amylase on
interpreted the data, wrote the first draft, and reviewed and PPyAgNp/Fe3O4-nanocomposite: chemical and physical
Journal of Nanotechnology 9

properties,” Artificial Cells, Nanomedicine, and Biotechnology, low loading,” Journal of the American Oil Chemists’ Society,
vol. 46, pp. 201–206, 2018. vol. 74, no. 2, pp. 107–111, 1997.
[14] R.-S. Juang, F.-C. Wu, and R.-L. Tseng, “Solute adsorption and [32] J. M. Bibby and D. W. L. Hukins, “Measurement of pH to
enzyme immobilization on chitosan beads prepared from quantify urease activity,” Journal of Biochemical and Bio-
shrimp shell wastes,” Bioresource Technology, vol. 80, no. 3, physical Methods, vol. 25, no. 4, pp. 231–236, 1992.
pp. 187–193, 2001. [33] A. Kumari and A. M. Kayastha, “Immobilization of soybean
[15] S. A. Agnihotri, N. N. Mallikarjuna, and T. M. Aminabhavi, (glycine max) α-amylase onto chitosan and amberlite MB-150
“Recent advances on chitosan-based micro- and nanoparticles beads: optimization and characterization,” Journal of Mo-
in drug delivery,” Journal of Controlled Release, vol. 100, no. 1, lecular Catalysis B: Enzymatic, vol. 69, no. 1-2, pp. 8–14, 2011.
pp. 5–28, 2004. [34] H. C. Tekedar and G. Şanlı-Mohamed, “Molecular cloning,
[16] K. Yang, N.-S. Xu, and W. W. Su, “Co-immobilized enzymes over expression and characterization of thermoalkalophilic
in magnetic chitosan beads for improved hydrolysis of esterases isolated from Geobacillus sp.” Extremophiles, vol. 15,
macromolecular substrates under a time-varying magnetic no. 2, pp. 203–211, 2011.
field,” Journal of Biotechnology, vol. 148, no. 2-3, pp. 119–127, [35] M. A. J. Al-Khafaji and M. J. Ewadh, “Immobilization of
2010. urease in gelatin beads for urea estimation,” Iraqi National
[17] J. Jeevanandam, A. Barhoum, Y. S. Chan, A. Dufresne, and Journal of Chemistry, vol. 331, pp. 131–137, 2009.
M. K. Danquah, “Review on nanoparticles and nano- [36] K. D. Rasch, R. W. Siegel, and H. Schultz, Quenching and
Recovery Experiments on Tungsten, Argonne National Labo-
structured materials: history, sources, toxicity and regula-
ratory, Lemont, IL, USA, 1976.
tions,” Beilstein Journal of Nanotechnology, vol. 9, no. 9,
[37] R. L. Oliveira, M. F. Silva, A. Converti, and T. S. Porto,
pp. 1050–1074, 2018.
“Biochemical characterization and kinetic/thermodynamic
[18] G. R. Rudramurthy and M. K. Swamy, “Potential applications
study of Aspergillus tamarii URM4634 β-fructofuranosidase
of engineered nanoparticles in medicine and biology: an
with transfructosylating activity,” Biotechnology Progress,
update,” Journal of Biological Inorganic Chemistry, vol. 23, vol. 35, no. 6, Article ID e2879, 2019.
2018. [38] M. R. Javed, M. H. Rashid, H. Nadeem, M. Riaz, and
[19] R. Pangestuti and S. K. Kim, “Neuroprotective properties of R. Perveen, “Catalytic and thermodynamic characterization of
chitosan and its derivatives,” Marine Drugs, vol. 8, no. 7, endoglucanase (CMCase) from Aspergillus oryzae cmc-1,”
pp. 2117–2128, 2010. Applied Biochemistry and Biotechnology, vol. 157, no. 3,
[20] J. Yoo, C. Park, G. Yi, D. Lee, and H. Koo, “Active targeting pp. 483–497, 2009.
strategies using biological ligands for nanoparticle drug de- [39] R. L. de Oliveira, O. S. da Silva, A. Converti, and T. S. Porto,
livery systems,” Cancers, vol. 11, no. 5, p. 640, 2019. “Thermodynamic and kinetic studies on pectinase extracted
[21] L. Guerrini, R. A. Alvarez-Puebla, and N. Pazos-Perez, from Aspergillus aculeatus: free and immobilized enzyme
“Surface modifications of nanoparticles for stability in bio- entrapped in alginate beads,” International Journal of Bio-
logical fluids,” Materials, vol. 11, no. 7, p. 1154, 2018. logical Macromolecules, vol. 115, pp. 1088–1093, 2018.
[22] A. Borowik, K. Butowska, K. Konkel et al., “The impact of [40] M. A. Abdel-Naby, S. A. Ahmed, H. R. Wehaidy, and S. A. El-
surface functionalization on the biophysical properties of Mahdy, “Catalytic, kinetic and thermodynamic properties of
silver nanoparticles,” Nanomaterials, vol. 9, no. 7, p. 973, 2019. stabilized bacillus stearothermophilus alkaline protease,” In-
[23] S. Vaezifar, S. Razavi, M. A. Golozar, S. Karbasi, M. Morshed, ternational Journal of Biological Macromolecules, vol. 96,
and M. Kamali, “Effects of some parameters on particle size pp. 265–271, 2017.
distribution of chitosan nanoparticles prepared by ionic ge- [41] A. A. T. A. N. Abd, Z. S. Al-Garawi, AIP Conference Pro-
lation method,” Journal of Cluster Science, vol. 24, no. 3, ceeding 2021.
pp. 891–903, 2013.
[24] R. L. Sinsabaugh, H. Reynolds, and T. M. Long, “Rapid assay
for amidohydrolase (urease) activity in environmental sam-
ples,” Soil Biology and Biochemistry, vol. 32, no. 14,
pp. 2095–2097, 2000.
[25] G. Arabaci and A. Usluoglu, J. Chem.
[26] P. W. Atkins and J. De Paula, 1998.
[27] M. E. El-Hefnawy, M. Sakran, A. I. Ismail, and
E. F. Aboelfetoh, “Extraction, purification, kinetic and ther-
modynamic properties of urease from germinating Pisum
Sativum L. seeds,” BMC Biochemistry, vol. 15, pp. 15–18, 2014.
[28] A. A. T. A. N. Abd, Zahraa S. A. L., 2021.
[29] S. Kumar, A. Dwevedi, and A. M. Kayastha, “Immobilization
of soybean (glycine max) urease on alginate and chitosan
beads showing improved stability: analytical applications,”
Journal of Molecular Catalysis B: Enzymatic, vol. 58, no. 1-4,
pp. 138–145, 2009.
[30] A. M. Kayastha and N. Das, “A simple laboratory experiment
for teaching enzyme immobilization with urease and its ap-
plication in blood urea estimation,” Biochemical Education,
vol. 27, no. 2, pp. 114–117, 1999.
[31] J. A. Bosley and A. D. Peilow, “Immobilization of lipases on
porous polypropylene: reduction in esterification efficiency at