Synthesis of urease hybrid nanoflowers and their enhanced catalytic properties

Article abstract of DOI:10.1016/j.enzmictec.2015.09.005

Increasing numbers of materials have been extensively used as platforms for enzyme immobilization to enhance catalytic activity and stability. Although stability of enzyme was accomplished with immobilization approaches, activity of the most of the enzymes was declined after immobilization. Herein, we synthesize the flower shaped-hybrid nanomaterials called hybrid nanoflower (HNF) consisting of urease enzyme and copper ions (Cu²⁺) and report a mechanistic elucidation of enhancement in both activity and stability of the HNF. We demonstrated how experimental factors influence morphology of the HNF. We proved that the HNF (synthesized from 0.02 mg mL^-1 urease in 10 mM PBS (pH 7.4) at +4 °C) exhibited the highest catalytic activity of ~2000% and ~4000% when stored at +4 °C and RT, respectively compared to free urease. The highest stability was also achieved by this HNF by maintaining 96.3% and 90.28% of its initial activity within storage of 30 days at +4 °C and RT, respectively. This dramatically enhanced activity is attributed to high surface area, nanoscale-entrapped urease and favorable urease conformation of the HNF. The exceptional catalytic activity and stability properties of HNF can be taken advantage of to use it in fields of biomedicine and chemistry.

Full text of DOI:10.1016/j.enzmictec.2015.09.005

Accepted Manuscript
Title: Synthesis of Urease Hybrid Nanoflowers and Their
Enhanced Catalytic Properties
¨
Author: Burcu Somturk Ismail Yilmaz Nalan Ozdemir Ismail
Ocsoy
PII:
S0141-0229(15)30059-4
DOI:
Reference:
http://dx.doi.org/doi:10.1016/j.enzmictec.2015.09.005
EMT 8815
To appear in:
Enzyme and Microbial Technology
Received date:
Revised date:
Accepted date:
15-7-2015
9-9-2015
12-9-2015
¨
Please cite this article as: Somturk Burcu, Yilmaz Ismail, Ozdemir
Nalan, Ocsoy Ismail.Synthesis of Urease Hybrid Nanoflowers and
Their Enhanced Catalytic Properties.Enzyme and Microbial Technology
http://dx.doi.org/10.1016/j.enzmictec.2015.09.005
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Synthesis of Urease Hybrid Nanoflowers and Their Enhanced Catalytic Properties
Author names and affiliations:
1
1
1,*
2, 3,*
Burcu Somturk , Ismail Yilmaz , Nalan Özdemir and Ismail Ocsoy
1
Department of Chemistry, Faculty of Science, Erciyes University, Kayseri, 38039 Turkey
Department of Analytical Chemistry, Faculty of Pharmacy, Erciyes University, 38039
2
Kayseri, Turkey
3
Nanotechnology Research Center, Erciyes University, Kayseri, 38039 Turkey
Corresponding authors
*
Corresponding Author1
Phone: +90 352 207666-13811
Fax: +90 352 437 93 22
E-mail: ismailocsoy@erciyes.edu.tr
*
Corresponding Author2
Phone: +90 352 207666-33176
Fax: +90 352 437 93 22
E-mail: ozdemirn@erciyes.edu.tr

Graphical Abstract

HIGHLIGHTS
2+

Synthesis of Cu incorporated urease based-hybrid flower (HNF) with dramatically enhanced
catalytic activity and stability



Studies of effect of experimental conditions on formation and activity mechanism of HNF
Activity and stability measurements of HNF
The hydrolysis of urea

Abstract
Increasing numbers of materials have been extensively used as platforms for enzyme
immobilization to enhance catalytic activity and stability. Although stability of enzyme was
accomplished with immobilization approaches, activity of the most of the enzymes was
declined after immobilization. Herein, we synthesize the flower shaped-hybrid nanomaterials
called hybrid nanoflower (HNF) consisting of urease enzyme and copper ions (Cu2+) and
report a mechanistic elucidation of enhancement in both activity and stability of the HNF. We
demonstrated how experimental factors influence morphology of the HNF. We proved that
-1
the HNF (synthesized from 0.02 mg mL urease in 10 mM PBS (pH 7.4) at +4°C) exhibited
the highest catalytic activity of ~2000% and ~4000% when stored at +4°C and RT,
respectively compared to free urease. The highest stability was also achieved by this HNF by
maintaining 96.3% and 90.28% of its initial activity within storage of 30 days at +4°C and
RT, respectively. This dramatically enhanced activity is attributed to high surface area,
nanoscale-entrapped urease and favorable urease conformation of the HNF. The exceptional
catalytic activity and stability properties of HNF can be taken advantage of to use it in fields
of biomedicine and chemistry.
Keywords: Urease, urea, flower shape, hybrid nanomaterials, copper ions, enzymatic activity

1
. Introduction
Enzymes, as versatile biological materials, have been commonly utilized in a wide variety of
scientific and technical applications including production of biofuels, drug synthesis,
chemical reactions in vivo and in vitro, proteomics and industry (beverage, food and textile,
etc.). Although free enzymes have unique properties, such as high catalytic activity, reaction
specificity and low toxicity, their efficient use is very limited due to their inherent
disadvantages associated with instability during storage, loss of catalytic activity after
administration into the reaction, high cost and lack of recovery from reaction medium [1-7].
In order to overcome these limitations, several immobilization approaches have been
developed. For this intent, various supports (micro and nano size) including silica, magnetic,
zeolites, chitosan materials, molecular sieves, polymer stands, silk film and gelatin have been
widely used for enzymes immobilization via covalent, non-covalent, entrapment and cross-
linking [8-16]. The expectation with enzyme immobilization is to improve both catalytic
activity and stability, both of which are critical factors to widely and efficiently use enzyme
for many applications. Although immobilized enzymes show enhanced stability in aqueous
solution, the most of them exhibit reduced catalytic activity compared to free enzymes most
likely due to decreased mobility, unfavorable conformation and mass-transfer limitation when
attached on the solid support. [17-19]. Thus, the efficient use of immobilized enzyme is
mainly obstructed. However, Ackerman and coworkers reported one of the rare examples,
where an increase in catalytic activity was achieved after enzyme immobilization. They
simply loaded organophosphorus hydrolase enzyme into carboxylethyl-or aminopropyl
groups functionalized mesoporous silica (30 nm pore size) and demonstrated ~200%
enhancement in catalytic activity compared to free enzyme [20]. However, this enhancement
in activity and stability were not at a satisfactory level since the immobilized enzyme lost
almost 15% of its initial activity within 30 days of storage at +4°C. Furthermore, multiple,

time-consuming steps and labour-intensive synthetic procedures were also major limitations
of this work.
Therefore, there is still a high demand for a new and an alternative enzyme immobilization
strategy with improved catalytic activity and stability. Unlike conventional enzyme
immobilization methods, a recent exclusive approach in enzyme immobilization was
developed by Zare and coworkers [21]. They created organic-inorganic hybrid nanostructures
consisting of protein and metal ions, which resulted in much higher enzyme activity and
stability than free enzymes and conventional immobilized enzymes. As a following work,
Wang and coworkers reported multi-enzyme integrated hybrid nanostructures by using
glucose oxidase and horseradish peroxidase to utilize functions of these enzymes in one step
[22]. Although this system allowed them to get benefit from both enzymes, there were
disadvantages associated with long analysis time and reproducibility in synthesis of these
hybrid nanostructures. In our previous studies, we synthesized flowerlike hybrid
nanomaterials containing horseradish peroxidase (HRP) enzyme and different metal ions with
highly enhanced catalytic activity and stability. We also demonstrated that how experiential
parameters affect the morphology of flowerlike hybrid nanomaterials and explained the
enhancements in the catalytic activity and stability [23, 24].
Urease (urea amidohydrolases, EC 3.5.1.5) is a one of the most studied and attractive
enzymes in environmental, bioanalytical, and clinical applications [7, 25-27]. Urease has been
actively used as an efficient biocatalyst for hydrolysis of urea. In addition, urease also acts as
a sensor for determination of urea concentrations in several mediums including serum, blood,
urine, some alcoholic and non-alcoholic beverages, food products, and environmental wastes.
The level of urea in serum and blood is in the range of 2.5-7.5 mM, which is considered as
normal and tolerable by human body. An increase and decrease in urea level can induce very
serious diseases. High level of urea causes urinary tract obstructions, renal failures, burns and

gastrointestinal bleeding while hepatic failures and nephritic syndromes are induced by low
levels of urea [28-32].
To the best of our knowledge, herein, we present synthesis, catalytic activity and stability of
2
+
hybrid nanoflower (HNF) formed of urease enzyme and copper ions (Cu ) and a mechanistic
elucidation of enhancement in the catalytic activity and stability HNF. In this study, we also
demonstrate how reaction parameters influence the morphology of HNF and evaluate catalytic
activity and stability of HNF by hydrolysis of urea based upon their morphologies and storage
temperatures.
2
. Experimental Section
2
.1.Chemicals and Materials:
Urease from jack bean, albumin from bovine serum (BSA) (lyophilized powder), Copper (II)
sulfate pentahydrate (CuSO .5H O) and urea were purchased from Sigma-Aldrich. Salts
NaCl, KCl, Na HPO , KHPO , CaCl . 2H O, MgCl . 6H O) were also obtained from Sigma-
Aldrich. Ultrapure water (18.2 M Ω; Millipore Co., USA) was used in all experiments.
.2.Synthesis of Urease Based Hybrid Nanoflowers:
The HNF was synthesized using a modified method [21]. In a standard synthesis protocol, 60
µL aqueous CuSO from a freshly prepared stock solution (120 mM) in the biology-grade
water was added to 9 mL of 10 mM phosphate buffered saline (PBS) solution (pH 7.4)
4
2
(
2
4
4
2
2
2
2
2
4
-1
containing 0.02 mg mL of urease. The mixture was vigorously agitated and then incubated at
+
4°C for 3 days. After incubation, the blue colour precipitation (indication of HNF formation)
was observed at the bottom of reaction tube. In order to stop reaction, the mixture was
centrifuged at 10000 rpm for 15 min. The HNF was washed with water and centrifuged at at
least 3 times to remove unreacted components. Finally, collected HNF was dried under

vacuum at room temperature. Note that the HNF was also synthesized with various
2
+
concentrations of urease, Cu ion and different pH values in PBS solution to observe their
effects on the morphology of HNF. The synthesis of HNF under different experimental
parameters was occurred at only 4°C for 3 days.
2
.3.Instrumentation and Characterization:
In order to calculate encapsulation yield (EY) of urease in HNF, the urease concentration in
the supernatant was measured with Bradford protein method using BSA as a standard. The
weight of HNF powder and EY value was utilized to calculate the weight percentage (WP) of
-1
2+
enzyme in HNF. The EY of HNF (from 0.02 mg mL urease and 0.8 mM Cu ions at +4°C)
was calculated as ~99.2% while WP was found as ~19%. The EY and WP of each HNF were
calculated accordingly prior to activity and stability measurements. First, the EY of HNFs
-1
2+
synthesized (from 0.02 mg mL urease and 0.8 mM Cu ions at +4°C) at pH 6, 8 and 9 are
almost the same as ~98.15%, ~99.1% and ~99.4%, respectively while WP of all HNFs is
2
+
around ~19%. Second, EY and WP of HNFs synthesized (from 0.8 mM Cu ions in pH 7.4 at
-1
+
~
~
4°C) using 2.5 , 0.5, 0.05 and 0.01 mg mL urease were calculated as ~99.2%, ~99.4%,
99.3% and ~99.5%, respectively while WP of HNFs are determined to be ~29%, ~24%,
21% and ~16%, respectively. The activity and stability of the HNF formed as a function of
the pH of the PBS solution and concentrations of urease enzyme were determined by UV-VIS
spectrophotometer (HITACHI).
The dry HNF powders were put on carbon tape covered stub, then it was coated with gold by
a sputter coater. Finally, images of HNF were generated by scanning electron microscopy
(
SEM) (ZEISS EVO LS10). In addition to SEM, elemental analysis of the HNF was carried
out using energy-dispersive X-ray (EDX) (ZEISS EVO LS10) to determine weight and
atomic percentage of Cu in the HNF. The crystal pattern of Cu (PO . 3H O was identified
3
4
)
2
2

with X-ray diffraction analysis (XRD) (BRUKER AXS D8). Firstly, the HNF was dried at
8
2
0°C; XRD was applied to the dry HNF for crystal analysis.
.4.Urease Enzyme Activity Measurement:
-1
The HNF and free urease (1 mg mL identical amount) were dissolved in PBS solution (pH
-1
7
.0) in two different reaction tubes. To the each tube, 0.8 mL urea (3 mg mL ) was added and
then the resulting both tubes were incubated for 30 min. After incubation, 4 mL deionized
water and 100 µL Nessler’s reagent were added to the each tube and then NH produced
3
through hydrolysis of urea form each tube was spectrophotometrically determine at 425 nm.
In the study, one unit of enzyme activity was defined as the amount of enzyme that changes
the absorbency 0.01 unit per min. It is known that KM (Michaelis constant) is an important
characteristic for an enzyme which shows the affinity of the enzyme to the substrate and the
robustness of the enzyme-substrate complex [33]. For this reasons, the KM values of free
urease and HNF were determined. To determine the KM, a certain concentration of free
urease and HNF were incubated with five different concentrations of urea (0.3, 0.45, 0.60,
0
.75, 1.05 mM). Thus, the KM values were calculated from the plot of 1/V versus 1/[S] by the
method of Lineweaver and Burk [34].
3
. Results and Discussion
In general, hybrid nanomaterials composed of two or more different nano components
have attracted great attention due to their enhanced properties compared to their individual
components [35-38]. Understanding the formation mechanism of the nanocrystals in solution
is a critical point for the synthesis of monodisperse both micro and nano sized materials. Two
main factors, nucleation and growth, should be studied to understand nanomaterial
production. LaMer and coworkers were the first to explain the formation of colloidal
materials [39]. In this current study, we systematically explained the changes on

morphologies of HNF formed under the various reaction parameters and on the activity and
stability of HNF based upon the morphology and storage temperature.
3
.1.Preparation and Characterization of HNF
2
+
In the synthesis strategy described above, HNF was simply prepared by mixing Cu ions and
urease enzyme in PBS solution. All HNFs were synthesized at +4°C with 3 days incubation.
2
+
Basically, there are three components, phosphate ions from the PBS solution, Cu ions and
urease enzyme, involved in the HNF formation. In order to explain the formation of HNF, two
major successive steps, the nucleation and the growth, were briefly discussed. It is first
2
+
necessary to understand the nucleation step, in which Cu ions generate copper phosphate
nanocrystals by reacting with phosphate groups. Then, the amine groups in the backbone of
2
+
urease bind to Cu ions through coordination reaction to initiate the nucleation of protein-
copper nanocrystals. In the growth step, the primary nanocrystals provide several separate
2
+
Cu biding sites for urease molecules. The key feature of the growth process is the action of
urease as “glue” to form large assemblies of urease molecules on nanocrystals. Consequently,
anisotropic growth results in complete formation of the HNF. The formation steps were
illustrated in Scheme.
Insert Scheme
The morphologies of HNF were characterized using SEM, EDX and XRD techniques.
The urease encapsulation yields in the HNF were calculated with Bradford assay and the
activity and stability of HNF were measured by UV-Vis spectrometry.
We examined the morphology of HNF as a function of (1) the pH of the PBS solution,
2
+
-
(
2) concentrations of copper (Cu ) ion, (3) urease enzyme and (4) chloride ions (Cl ). First,
effect of PBS pH values (pH 6, 7.4, 8 and 9) on the morphology of the HNF was studied as
shown in the SEM images (Fig. 1). The isoelectric point of urease used in this work is ~5.4,

where free urease net charge is expected to be neutral, whereas above and below this value,
the protein net charge is expected to be negative and positive, respectively. For all pH
-1
2+
dependent HNF formations, 0.02 mg mL urease and 0.8 mM Cu were used. The HNF
synthesized at pH 6 had spherical shapes and highly packed structure due tightly assembly of
urease molecules in the HNF (Fig. 1A). The HNF at pH 7.4 had spherical shapes as well but
less packed structure due to slightly increased charge of urease molecules (Fig. 1B). However,
when the HNF were formed at pH 8 and 9, negativity of urease molecules was increased and
thus the shapes of HNF were splayed (Fig. 1C and 1D). Note that at pH 10 and above and at
pH 5 and below, no HNF formations (even blue color precipitates) were occurred. Even the
density of negative charge on the urease molecules were sequentially increased in pH 6, 7.4, 8
2
+
2+
and 9, the positive charge of Cu ions and binding affinity between Cu ions and urease
2
+
molecules facilitate the interaction of Cu ions and urease molecules for the formation of
HNF. However, we speculate that, highly negatively charged urease molecules strongly repel
each other at pH 10 and above, which prevents the interaction between urease molecules and
Cu2+ ions. In contrast, proteins of urease were positively charged at pH 5 and below.
Therefore, no HNF formations were observed at pH 5 as well due to strong positive repulsion
2
+
among urease molecules and urease and Cu . To contribute to the explanation of HNF
formation at different pH values and their morphology evolution, zeta potential measurements
were utilized to define the charge density on the surfaces of HNF [38]. The HNF at pH 6
showed negative charge with a –3.89 mV zeta potential due to the slight deprotonation of
urease. The negativity of the HNF was increased via higher pH values. The zeta potentials of
HNF at pHs 7.4, 8, and 9 were –5.18 mV, –10.45 mV and –14.78 mV, respectively.
Insert Fig.1

Second, the effects of Cu2+ concentrations on the formation of the HNF were
-1
investigated. For these particular experiments, the HNF from 0.02 mg mL urease were
2
+
formed in PBS (pH 7.4) using various Cu concentrations. Our findings indicate that 0.8 mM
was the optimal Cu2+ concentration to create the uniform HNF. The spherical HNF with
2+
almost the same size were successfully produced when increasing Cu concentration from
2
+
0
.8 mM to 8 mM and to 80 mM (Fig. 2A, 2B and 2C). We proved that Cu ion is a key
component and a major driving force to form primary nanocrystals. For instance, no blue-
2
+
2+
color precipitates occurred without Cu or with a concentration of Cu that was below 80
2
+
µM in the reaction mixture. When the concentration of Cu was within this range, it was not
able to reach the point of supersaturation required to initiate nucleation and subsequent
growth of primary nanocrystals.
Insert Fig.2
Third, the formations of the HNF were investigated using various concentrations of
-1
urease (2.5 0.5, 0.05, 0.02 and 0.01 mg mL ). In order to observe effect of different urease
concentrations, all the HNF were synthesized under the same experimental conditions (0.8
2
+
mM Cu and pH 7.4). The remarkable differences on the morphology of HNF were observed
-1
with SEM images when urease concentration decreasing from 2.5 to 0.01 mg mL (Fig. 3).
-1
Surprisingly, a few numbers of HNF were produced via use of 2.5 mg mL urease while
-1
numerous HNF were obtained via use of 0.5 mg mL urease (Fig. 3A and 3B). However,
further observations revealed that all the petals were strictly embedded each other and no
pores but very few rifts were appeared on surfaces of HNF for both concentrations. Fig. 3A
characterized that the efficient production of HNF and pores formation on their surface were

-1
not possible with a relatively high urease concentration (2.5 mg mL ). Although the HNF was
-1
efficiently synthesized with a 0.5 mg mL urease, pores were not formed on their surface due
to this particular urease concentration (Fig. 3B). The quite uniform and spherical HNF with
-1
-1
pores was synthesized with 0.05 mg mL and 0.02 mg mL urease, respectively (Fig. 3C and
-1
3
D). When 0.01 mg mL urease was used, the HNF appeared spherical but the pores on their
surface were partially closed (Fig. 3E). Most likely, urease molecules blocked some pores on
-
the surface of HNF during the growth process. Forth is the effect of Cl ions. In a standard
HNF synthetic procedure, the certain concentrations of urease and Cu2+ were accordingly
-
mixed in 10 mM PBS solution containing ~140 mM Cl and then the proper formation of
-
HNF was observed. However, when the Cl concentration was increased to ~7 M and above
(50 fold), the blue-colored precipitate were not observed at this threshold values. We interpret
that the formation of soluble Cu (II) chloride complexes prevented the growth of nanocrystals.
However, even though blue precipitate was obtained at the bottom of the reaction tubes in the
absence of Cl‒, no HNF was formed due to the incomplete growth process (Fig. 3F).
Insert Fig.3
The elemental composition of HNF was analyzed with EDX technique (Fig. 4A).
EDX spectrum clearly showed the weight and atomic percentages of Cu in the HNF are about
1
7.65 % and 6.16 %, respectively. The peak positions and relative intensities of the HNF were
characterized with XRD analysis (4B). These results showed that all diffraction peaks of
Cu
3
(PO
4
)
2
in the HNF were consistent with those of JCPDS card (00−022−0548).
Insert Fig.4

3
.1. Enzymatic Activity and Stability Measurement of HNF
In general, urease hydrolyzes urea to the products of ammonia (NH ) and carbon
3
dioxide (CO ). The ammonia concentration, which is proportional to its absorbance intensity
2
at 425 nm, is easily measured using Nessler’s reagent [40]. Several different nanomaterials
have been used as external supports for urease immobilization in order to increase the activity
and stability. However, usually immobilized urease has improved stability but has lowered
activity compared to free enzyme [30-42]. The main potential reasons for the loss of the
activity are associated with immobilization procedure, unfavorable urease conformation, and
mass-transfer limitations between urease and substrate. However, we synthesized HNF
2
+
consisting of Cu ions and urease and tested its activity and stability. As described above, the
urea was efficiently converted to NH and CO by the HNF.
3
2
In all activity and stability experiments, the amount of HNF powder and free urease
were identical. The activity and stability of HNF presented in Fig. 1 and Fig. 3 were
determined at the following storage temperatures: (1) +4°C and (2) room temperature (RT=
2
0°C). Hence, we demonstrated the effects of morphology and storage temperature on the
catalytic activity and stability of HNF. First, four different HNFs of Fig. 1 and free urease
were dissolved in 1mL PBS (pH 6.8) in separate 5 reaction tubes and 1 mL Nessler’s reagent
and urea (0.1 mM) in were added to reaction tubes. Each tube was divided into two parts,
which were stored at two different temperatures (+4°C and RT). We believe that urease
molecules were assembled with different conformations in the HNF formed at different PBS
pHs and thus each HNF exhibited different activity and stability. We stored the first part of
reaction tubes at +4°C and then the activity experiments proved that the HNF synthesized at
pH 7.4 exhibited highest activity increase among HNF synthesized at other pH values (Fig.
5
A). It showed an increase in activity of ~2000% compared to free urease (green line in Fig.

5
C). Similarly, the HNF formed at different pH values showed the enhanced activity for pH 8
(~1430%), pH 9 (~1270%) and pH 6 (~650%) compared to free urease as shown in Fig. 5A.
However we obtained surprising results from second part of reaction tubes, where the HNF
and free urease were stored at RT (Fig. 5B and 5C). At this storage temperature, the HNF
formed in pH 7.4 achieved activity with ~3990% increase compared to free urease (purple
line in Fig. 5C). Under the same conditions, the HNF has an increase in activity for pH 8
(~2780%), for pH 9 (~2440%) and pH 6 (~950%) compare to free urease as shown in Fig. 5B.
We reach a point that the activity of free urease was drastically reduced when stored in PBS at
RT while the activity of HNF did not showed significant activity reduction under the identical
condition.
In terms of stability, these HNFs were stored at +4°C and RT for 30 days during the
activity measurements. First, stability of the HNF of Fig. 1A was determined when stored in
PBS at +4°C. The HNF for pH 7.4 showed highest stability with 3.7% loss of its original
activity while free urease lost 73.55% of its original activity (Fig. 5D). The HNFs for pH 8, 9
and 6 lost 10%, 15% and 22.55%, respectively, of their original activities (Fig. 5D).
Moreover, stability of the HNF was measured when stored at RT. Fig. 5E shows that the HNF
for pH 7.4 lost only 9.28% of its original activity while free urease lost 90.25% of its original
activity. Similarly, the HNFs for pH 8, 9 and 6 lost 13.22%, 22.34% and 35%, respectively, of
their original activities (Fig. 5E). All the facts that the activity reduction of the HNF stored at
+
4°C was smaller than that of HNF stored at RT. The hydrolysis of urea by the HNF was
illustrated in Fig. 5F.
Insert Fig.5

To gain further insight into the impact of urease concentration and storage temperature
on catalytic activity and stability of HNF, the HNFs presented in Fig. 3 and free urease with
identical amount were mixed with Nessler’s reagent and urea into separates reaction tubes
sand stored at +4°C and RT. First, the activity of HNFs in Fig. 3 stored at +4°C was
-1
evaluated. Note that the activity performance of the HNF (from 0.02 mg mL urease and at
pH 7.4) and free urease stored at +4°C and RT was already demonstrated in Fig. 5A and 5B.
-1
In addition, the HNFs from 2.5, 0.5, 0.1 and 0.01 mg mL urease stored +4°C exhibited
~840%, ~915%, ~965% and ~985%, respectively, higher activity than those concentrations of
free urease (Fig. 6A and 6C). For comparison of storage temperature, the activity of the HNF
and free urease stored at RT was also determined. The HNFs from 2.5, 0.5, 0.1 and 0.01 mg
-1
mL urease performed ~1315%, ~1430%, ~1480% and ~1960%, respectively, higher activity
than those concentrations of free urease (Fig. 6B and 6D). The results indicate that decreasing
free urease concentration caused a reduction in its activity. However, the drastic reduction in
activity of free urease was observed when stored at RT.
In terms of stability, these HNFs were stored +4°C and RT for 30 days. Note that the
-1
stability of the HNF (from 0.02 mg mL urease and at pH 7.4) and free urease stored at +4°C
and RT was already demonstrated in Fig. 6A and 6B. The HNFs from 2.5, 0.5, 0.1, and 0.01
-1
mg mL urease maintained 93.6%, 90%, 88.7% and 84%, respectively, of their original
-1
activities when stored at 4°C while 2.5, 0.5, 0.1 and 0.01 mg mL of free urease maintained
of 32.4%, 30%, 27% and 25%, respectively, of their original activities as shown in Fig. 6E.
-1
However, the HNF from 2.5 mg mL urease lost 13.3% of its original activities while the
-1
HNFs from 0.5, 0.1, and 0.01 mg mL urease 17.7%, 20%, and 22%, respectively, of their
original activities when the HNFs stored at RT for 30 days, (Fig. 6F). For comparison at this
-1
particular storage temperature (RT), 2.5, 0.5, 0.1 and 0.01 mg mL of free urease molecules
lost 88.7%, 89.3%, 89.8%, and 91%, respectively, of their original activities (Fig. 6F). These

findings with HNF indicate that: (1) urease in the HNF is much more stable compared to free
urease in solution; and (2) the storage temperature affects the activity and stability of both
HNF and free urease; (3) urease molecules are assembled in the HNF with favorable
conformation.
Insert Fig.5
Moreover, as mentioned above, determination of KM value is a key point to explain
specificity and strength of interaction between enzyme and substrate. Decreasing KM value
results in the strong the enzyme-substrate complex with high affinity while increasing Km
causes weak interaction between enzyme-substrate complexes with low affinity [43]. We
calculated the KM values of free urease and HNF as 0.11 and 0.039 mM, respectively. The
HNF had the lowest KM which dictates that they higher specificity and efficiency to react
with urea and to hydrolyze it to CO
2
and NH compared to free urease. We conclude that the
3
-1
HNF synthesized from 0.02 mg mL urease in PBS solution (pH 7.4) at +4°C and RT ranked
first in overall activity and stability assays. We attribute this to the following effects: (1) high
surface area of HNF, (2) presence of pores on HNF surface, (3) synergistic effect of
nanoscale-entrapped urease, (4) localize and favorable urease conformation in the HNF.
4
. Conclusion
In conclusion, we have reported the synthesis of HNF composed of Cu ions and
2
+
urease enzyme. The effects of reaction parameters on morphology, catalytic activity
and stability of HNF were systematically investigated. We experimentally
2
+
demonstrated how the pH values of PBS; concentrations of Cu ions, urease enzyme,
-
Cl influence the morphology of HNF. The catalytic activity and stability of HNF

synthesized under different conditions were also evaluated. The HNF exhibited
dramatically enhanced activity and stability compared to free urease. In terms of
-
1
catalytic activity, we proved that the HNF obtained (from 0.02 mg mL urease in PBS
solution (pH 7.4) at +4°C) exhibited the highest activity with ~2000% enhancement
when stored +4°C. The HNF showed the highest stability by maintaining 96.3% of its
original activity within 30 days of storage at +4°C. We conclude that this HNF has an
activity of ~200% higher than that of those HNFs from 2.5, 0.5, 0.05, and 0.01 mg mL-
1
urease. In addition to that, this particular HNF has an increase in activity of ~150%,
~
160% and ~300% compared to activities of those HNFs formed at pH 8, 9 and pH 6.
Author Contribution
B.S. and I.Y. performed experiments. I.O. and N.Ö. conceived the original idea and
designed the project. I. O and N. Ö. supervised the project. I.O. and N.Ö. mainly wrote the
manuscript.
Notes
The authors declare no financial interest
Acknowledgements
We appreciate Menekşe Sarihan at the Erciyes University Nanotechnology Research
Center for assistance with SEM and EDX measurements. We also thank Fatma Kilic Dokan at
the Erciyes University Technology Research and Implementation Center for assistance with
XRD and BET measurements. This work was supported by a grant from the Erciyes
University Scientific Research Office (FBG-2014-5376)2014-2016. We appreciate the
comments on the manuscript by Amanda Strayer as well as the suggestions.

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List of legends
Fig 1
Fig. 1. Formation of the HNF in different buffer pH values. A) pH: 6, B) pH: 7.4, C) pH: 8
and pH: 9.

Fig 2
2
+
Fig. 2. Formation of the HNF in different Cu concentrations. A) 0.8 mM, B) 8 mM, C) 80
mM.

Fig 3
Fig. 3. Formation of the HNF in different urease concentrations and with excess of chloride
-1
concentration. A) 2.5, B) 0.5, C) 0.05, D) 0.02 and E) 0.01 mg mL urease concentrations. F)
Above 7M chloride concentration.

Fig 4
Fig. 4. A) EDX analysis of the HNF, B) XRD patterns of the HNF (black line). Peak position
of the Cu
3
(PO
4
)
2
.3H O (red line, JPSCD 00−022−0548) located for comparison.
2

Fig 5
Fig. 5. Increase in catalytic activities and stabilities of the HNF synthesized at pH 6, 7.4, 8
and 9. A) Stored at +4°C, B) Stored at RT, C) Increase in catalytic activities of the free at 7.4
(stored at +4°C and RT), D) Stored at +4°C, E) Stored at RT, F) Hydrolysis of urea catalyzed
by HNF.

Fig 6
Fig. 6. Increase in catalytic activities and stabilities of the HNF synthesized from various
-1
initial urease concentrations) 2.5, 0.5, 0.1, 0.02 and 0.01 mg mL ). A) Stored at +4°C, B)
Stored at RT. Increase in catalytic activities of those concentrations of free urease. C) Stored
at +4°C, D) Stored at RT. Stability of HNF and free urease enzyme with identical conditions
towards urea. E) Stored +4°C, F) Stored at RT.

Scheme
Scheme. Schematic illustration of HNF formation with major steps: Nucleation and growth
for efficient urea hydrolysis.