Urease covalently immobilized on cotton-derived nanocellulose-dialdehyde for urea detection and urea-based multicomponent synthesis of tetrahydro-pyrazolopyridines in water

Article abstract of DOI:10.1039/c9ra05240b

The urease Schiff-base covalently bonded to the designed high-content nanocellulosedialdehyde (HANCD) prepared from cotton-derived nanocellulose (NC) via tandem acid-hydrolysis and periodate-oxidation reactions was termed HANCD?urease. No change in the al

Full text of DOI:10.1039/c9ra05240b

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Urease covalently immobilized on cotton-derived
nanocellulose-dialdehyde for urea detection and
urea-based multicomponent synthesis of
tetrahydro-pyrazolopyridines in water
Cite this: RSC Adv., 2019, 9, 41893
*
Fatemeh Tamaddon
and Davood Arab
The urease Schiff-base covalently bonded to the designed high-content nanocellulosedialdehyde (HANCD)
prepared from cotton-derived nanocellulose (NC) via tandem acid-hydrolysis and periodate-oxidation
reactions was termed HANCD@urease. No change in the aldehyde content of HANCD after Schiff-base
bonding to urease and similarity in the relative enzyme activities for HANCD@urease and free enzyme
supported that the preparation conditions for HANCD-loaded urease are mild enough to prevent
denaturation of the enzyme. As the immobilized urease showed higher stability and reusability versus
free enzyme, the HANCD@urease was efficiently used to determine the urea concentration in aqueous
solutions and blood serum samples. Alternatively, the catalytic efficiency of the HANCD@urease was
demonstrated for the production of ammonia from urea in the multicomponent synthesis of 3,5-
dimethyl-4-aryl-1,4,7,8-tetrahydrodipyrazolo[3,4-b:40,30-e]pyridines (THPPs) in water. This new
environment-friendly urea sensor showed 90% preservation of the enzyme activity after the six cycles of
reuse in enzymatic reactions, while its catalytic activity in the reaction of benzaldehyde, hydrazine
hydrate, and alkyl acetoacetate with urea instead of hygroscopic ammonium salts did not change
significantly after the sixth run. Detection and production of ammonia by a bio-compatible sensor and
catalyst under mild conditions are features of this new green protocol.
Received 9th July 2019
Accepted 26th November 2019
DOI: 10.1039/c9ra05240b
rsc.li/rsc-advances
highly appropriate for biotechnological, food, cosmetic, and
bio-pharmacological uses of enzymes.
Introduction
Urease is the rst distinct Ni-containing hydrolase enzyme
for the hydrolysis of urea to ammonia and CO2 (ref. 8) to be
stabilized by chemical or physical bonding to organic9,10 and
inorganic supports.11,12 Recently, we used free urease for the in
situ bio-production of ammonia from urea in the synthesis of N-
heterocycles, though with some stability and reusability
complications for the free enzyme.13,14 Due to these draw-
backs,13–15 urease stabilization by linking to a suitable support
to preserve the enzyme activity is a challenge.16 Due to the
higher lifetime and stability of chemically bonded enzymes,
chemical immobilization (CI) of urease is more feasible17 than
physical adsorption18 or encapsulation,19–21 although CI leads to
some enzyme activity loss.22 In this context, the immobilization
of urease on cellulose,9 chitosan,23 alginate,24 gelatin,25 and
agar26 have been tried to achieve urea biosensors.27,28 Kumar
et al.24 prepared the biosensor alginate@urease and compared
it with chitosan@urease for urea detection. Cellulose is
a unique low-cost biopolymer, with amphiphilic properties and
weak hydrogen bond issues29–31 which are solvable by regio-
selective periodate oxidation (PO) of vicinal hydroxylated C2–C3
groups,32,33 for urease immobilization9 on cellulose dialdehyde
(CD). Nanocellulose dialdehyde (NCD) is an advanced version of
Due to the superior specicity and environmental benignity of
enzymes compared to traditional chemical catalysts, in vitro
enzymatic reactions have been developed in recent academic
and industrial research.1,2 However, enzyme immobilization on
proper supports is necessary due to the stability and reusability
issues of free enzymes.3 There are three types: physical
adsorption, covalent chemical bonding, and encapsulation
methods for enzyme immobilization applied for diagnostic
systems with their advantages and drawbacks.4 Physical
adsorption on supports is the simplest method for enzyme
immobilization, but is associated with uncontrolled enzyme
leaching.5 Enzymes chemically or crosslink-anchored to
supports preserve the enzyme structure more effectively in
practical use, albeit with a decrease in enzyme activity and an
increase in the price.6 Enzyme encapsulation also creates
a diffusional limitation for access to enzyme active sites and
substrates.7 Thus, the covalent chemical immobilization of
enzymes on a low-cost, biocompatible, and non-toxic support is
Department of Chemistry, Faculty of Science, Yazd University, Yazd 89195-741, Iran.
E-mail: amaddon@yazd.ac.ir; Fax: +983538210644; Tel: +983531232666
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CD and nanocellulose (NC) with the advantages of high surface
area, high diffusion power, special mechanical properties,
simple chemical access, and sustainability. Recently, low alde-
hyde content NCDs34 have been used as sorbents,35 nano-metal
templates,36 and aerogels.37,38 However, preparation of a high
dialdehyde content NC (HANCD) from low-cost cotton is very
desirable to overcome the stability and reusability issues of free
urease using Schiff-base covalent bonding.
The 3,5-dialkyl-4-aryl-1,4,7,8-tetrahydrodipyrazolo[3,4-b:40,30-
e]pyridine (THPP) motif is a key structural feature present in
naturally active compounds39 and synthetic pharmaceuticals.40
THPPs have been synthesized by multicomponent reactions
(MCRs) using heterogeneous and homogeneous acid cata-
lysts.41–45 Despite the benets, some of these pseudo-six-
component methods have drawbacks, such as long reaction
times, use of organic solvents, non-reusable catalysts, and excess
hygroscopic ammonium salts as a nitrogen source for the central
pyridine ring. To the best of our knowledge, no enzymatic green
synthesis of THPPs in water has been reported. Therefore, the
development of bio-catalytic synthesis of THPPs in water using
reusable HANCD-loaded urease and urea as a safer nitrogen
source than ammonium salts is highly anticipated. The object of
this work is simple preparation of HANCD from raw cotton,
covalent bond immobilization of urease on HANCD as HANC-
D@urease, evaluation of the aldehyde content and relative
activity/stability of the produced urea biosensor, and the catalytic
assessment of HANCD@urease in the enzymatic production of
ammonia in water for green synthesis of THPPs (Scheme 1).
Experimental section
Scheme 1 Preparation and application of HANCD@urease.
Materials
Cotton ower (obtained from Yazd, Iran) was chopped and
treated with water, ethanol, and 18% NaOH to give cellulose
microbrils (MC). H2SO4, monosodium phosphate, disodium
phosphate, Tris–acetate buffer, acetic acid, sodium chlorite
(NaClO2), sodium periodate (NaIO4), hydrazine hydrate,
hydroxylamine hydrochloride, urea, aldehydes, and ethyl ace-
toacetate were supplied by Merck (Germany). All materials were
reagent grade and used without further purication, except the
urease with absolute activity 50 U mgꢀ1 which was extracted
from soya beans and puried according to previously reported
procedures.13,46
cotton chopped and de-waxed with ethanol (5 g) in NaOH (18%,
80 mL) was reuxed. The resulting anionic micro-cellulose
(AMC) was washed with water to reach pH 7 and dried at
ꢁ
70 C. The MCD was prepared by keeping the reaction mixture
of MC in water (1 g/50 mL) and sodium periodate (1.7 g) at
neutral pH, room temperature, and dark conditions for 72 h.
To prepare NC, the MC (5 g) was added to a solution of
sulfuric acid (35% v/v, 50 mL) and the mixture was stirred
vigorously at 50 ꢁC for 3 h. Then, ice water (500 mL) was added
to stop the hydrolysis and the mixture was centrifuged and
decanted three times to get dispersed NC particles in water (1 g/
50 mL) at neutral pH. To obtain NCDs, sodium periodate
(NaIO4, 1.4 g), sodium chlorite (1.7 g), potassium periodate
(KIO4, 1.7 g), or 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO,
1.3 g) was added and the reaction mixture was kept for 1–100 h
under dark conditions, from which the HANCD was typically
prepared by PO at room temperature aer 72 h. Next, water was
added and the NCD particles were isolated aer centrifugation
and decantation.
Human blood samples
All experiments were performed in accordance with the
Guidelines of the National Institutes of Health. Experiments
were approved by the ethics committee at Shahid Sadoghi
University of Medical Science and Yazd University. Informed
consent was obtained from the voluntary human participants in
this study.
Preparation of MCD and NCDs from MC-derived cotton bres
Determination of aldehyde content for MCD and NCDs
NCDs were prepared by simultaneous preparation of MC from The aldehyde content for the as-prepared micro- and nano-CDs
cotton, conversion of MC to NC, and PO of NC under the opti- was determined by modied titration with an aqueous solution
mized conditions. To give mercerized MC, a suspension of of hydroxylamine hydrochloride (HAHC).37 Thus, NCD or MCD
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(0.25 g) was added to the solution of HAHC (0.45 g in 30 mL of a solution of free urease or HANCD@urease in sodium phos-
water) at controlled pH ¼ 4.2 and the mixture was stirred for phate buffer solution (pH ¼ 7, 0.05 M) at 4 U mLꢀ1 was incu-
24 h at room temperature to reach pH ¼ 0.33. Then NaOH (0.1 bated for 5 minutes. Then, 1 mL of urea solution (0.1 M) in
M) was added to return the pH to 4.2 and the aldehyde content phosphate buffer solution (0.05 M) with pH adjusted to between
was calculated following eqn (1):
4–9 was added and the mixture was magnetically stirred at 35 ꢁC
for 15 min until the reaction was stopped by adding 1 mL
sulfuric acid. The absolute activity was then determined spec-
trophotometrically by adding Nessler's reagent and measuring
the absorbance of the color complex with the released NH3 at
460 nm; both the relative and absolute activities were evaluated
for free urease or HANCD@urease. To determine the specic
activity for free- or immobilized-urease, a standard calibration
curve for ammonium sulfate was used. According to the enzyme
unit denition (U), one unit activity is the amount of free or
immobilized urease that causes liberation of 1 mmol ammonia
per minute at optimum pH.
V ꢂ C ꢂ 162
g ꢂ 1000 ꢂ 2
Aldehyde content ¼
ꢂ 100
(1)
where V is the volume of NaOH needed to return the pH to 4.2, C
is the NaOH concentration (mol Lꢀ1) and g is the mass of NCD
in the experiment. For example, due to the consumption of
25.7 mL NaOH to reach pH 4.2, an aldehyde content of 83% was
calculated for NCD72 (HANCD) based on two CHO groups:
25:7 ꢂ 0:1 ꢂ 162
Aldehyde content ¼
ꢂ 100 ¼ 83%:
0:25 ꢂ 1000 ꢂ 2
To check the temperature effect, the free urease or HANC-
D@urease was immersed in a 0.1 M solution of urea in various
incubated sodium phosphate buffer solutions (0.05 M) at
temperatures of 20 C, 30 C, 40 C, 50 C, 60 C, 70 C, and
80 ꢁC for 5 minutes. The released NH3 was determined as
described above, while the relative enzyme activity was evalu-
ated as the ratio of the specic activities of the HANCD@urease
and the free urease multiplied by 100. (4 U mLꢀ1).
Following the same method, an aldehyde content of 47% was
calculated for MCD.
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
ꢁ
Preparation of HANCD@urease as urea biosensor
HANCD (0.85 g) dispersed in a solution of urease (absolute
activity 50 U mgꢀ1) in phosphate buffer (0.05 M, pH ¼ 7, 100
mL) with a concentration of 1 mg urease per mL (for maximum
ꢁ
efficiency) was shacked for 24 h at 4 C. Then, the phosphate
buffer solution was added and the mixture was centrifuged,
decanted, and washed with distilled water three times to give
HANCD@urease.
HANCD@urease as a biosensor for determination of urea in
solutions or serum samples
First, three human blood serum samples with 82, 40, and 20 mg
mLꢀ1 urea (conrmed by clinical auto-analyzer) were voluntary
prepared from the blood samples of the author and coworkers
in Yazd Medical Lab in compliance with Yazd University's policy
on animal/human use and ethics. All tests on our blood serum
were assayed in agreement with the Yazd University policy on
biological samples. Alternatively, urea solutions were prepared
from urea and deionized water. To check the biosensor
HANCD@urease for urea estimation in blood serum or urꢁea
solutions, the HANCD@urease (10 mg) was incubated at 35 C
in Tris acetate buffer (0.5 mL) at pH 7. Then, 0.2 mL of urea
solution or serum sample was added, the mixture was
magnetically stirred for 5 min, acetic acid (0.5 mL, 10%) was
added to precipitate the free urease or HANCD@urease and the
mixture was centrifuged. The concentration of urea was
assessed based on the NH3 released by the enzymatic reaction
in the supernatant aer the addition of Nessler's reagent, the
absorbance of the color complex was measured at 460 nm, and
the standard calibration curves plotted for various urea
concentrations were followed.
Determination of aldehyde content for immobilized urease as
HANCD@urease
The aldehyde content for urease immobilized as HANCD@ur-
ease was determined according to a modied method used for
NCDs by titration with HAHC37 for 0.25 g of HANCD@urease.
Due to there being no change in pH aer the addition of the
HAHC and no consumption of NaOH to return the pH to 4.2,
a 0% aldehyde content was calculated for HANCD@urease
following eqn (1).
Characterization of HANCD and HANCD@urease
Fourier transform infrared (FT-IR) spectra of samples were
taken by a Bruker Equinox 55 and Bruker AC 500. The UV-Vis
spectra of NCDs for the determination of aldehyde content
were recorded by a single-beam UV-Vis (AvaSpec-2048) spec-
trophotometer. X-ray diffraction patterns (XRD) were recorded
on an X Pert Pro Panalytical. The surface morphology of
samples was viewed by eld emission electron microscopy
(FESEM) analysis model MIRA3 TE-SCAN. Thermal gravimetric
analysis (TGA) was performed by an STA 1500. HANCD and
HANCD@urease were characterized by IR-spectroscopy and
FESEM, XRD, and TGA.
Thermal stability test for HANCD@urease
To check the thermal stability, the free urease and HANC-
D@urease were separately incubated in phosphate buffer (0.05
M) at variable times at 70 ꢁC in 300 minutes. The residual
activity of the enzyme for the samples withdrawn aer each 1 h
The performance assays for urea detection by HANCD@urease
All performance assays for urea detection were repeated for ve incubation was determined following section “The performance
runs and the results were analyzed by Statistic soware version assays for urea detection by HANCD@urease”. In another test,
6 to estimate the experimental error. To check the pH effect, the response of the HANCD@urease in control of the enzyme
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activity was measured for aqueous solutions of urea (1 mM) for
a period of 35 days.
Among the prepared NCD72, NCDA72, NCDB72, NCDK48,
NCCC24, and NCCT60, the maximum 83% aldehyde content was
from the NCD72 (HANCD) obtained by PO of NC without acid/
base additive aer 72 h (entry 1). The experiments using the
other oxidants produced NCCs (entries 4–6).
HANCD@urease-catalyzed synthesis of THPPs
A mixture of hydrazine hydrate (5 mmol), alkyl acetoacetate (5
mmol), aldehyde (2.5 mmol), urea (15 mmol), and HANCD
(0.025 g) in water was magnetically stirred at 70 ꢁC as the
optimum temperature for the maximum activity of urease in
immobilized form (Section “Thermal stability test for HANC-
D@urease”). The product precipitated during the progress of
the reaction; thus, aer completion of the reaction, EtOAc was
added and the urease-loaded HANCD was ltered out. The
THPP product was obtained aer the vacuum evaporation of
EtOAc under reduced pressure and recrystallization from an
H2O/EtOH mixture (70 : 30).
Next, a close analysis of pH and reaction time was performed
at room temperature and in dark conditions to support the
optimized PO of NC (Fig. 1).
Urease immobilization and characterizations of
HANCD@urease
Due to the maximum aldehyde content of HANCD (NCD72) over
the other NCDs and MCD, it was selected for further Schiff-base
covalent immobilization of urease. The chemical bonding of
urease amino groups occurred by treatment of urease (50 U mgꢀ1
)
in phosphate buffer with HANCD under incubation conditions at
pH ¼ 7 for 24 h. Then, the yellowish solid HANCD@urease was
Reusability of HANCD@urease
ꢁ
ltered, washed with buffer solution, dried at 40 C, and char-
The reusability of HANCD@urease was evaluated over 6 cycles
of reaction runs for both the enzymatic experiment and
HANCD@urease-catalysed pseudo-six-component reaction of
hydrazine hydrate, ethyl acetoacetate, benzaldehyde, and urea.
So, aer each cycle, the separated HANCD@urease from the
reaction mixture was washed with water and phosphate buffer
solution at pH 7 and reused in a further similar reaction. All
experiments were repeated three times.
acterized by FT-IR, FESEM, XRD, TGA, and aldehyde content. The
determined aldehyde content33 for HANCD@urease was 0%,
showing the high efficacy of the urease immobilization on
HANCD. Based on the 83% aldehyde content for unloaded
HANCD, it can be concluded that all aldehyde groups are involved
in Schiff-base covalent bonding to urease.
The compared FT-IR spectra of prewashed MC, HANCD, and
HANCD@urease show the cooperative absorption bands for
O–H stretching, C–H stretching, CH2 bending, and C–O
stretching at 3300 cmꢀ1, ꢃ2900 cmꢀ1, ꢃ1460 cmꢀ1, and ꢃ1000–
Results and discussion
Preparation of biosensor and catalyst HANCD@urease under
the optimized conditions
The HANCD@urease was prepared by prewashing cotton to
cellulose-micro bers (MC), acid hydrolysis of MC to NC, PO of
NC to HANAC under the optimized conditions, and Schiff-base
bonding of urease to HANAC to get the HANCD@urease
(Scheme 1). Aer removing the hemicellulose and lignin
impurities from de-waxed cotton under mercerization condi-
tions, which improves the morphological structure and shine of
the cellulose, the NC was prepared by acid hydrolysis of MC.38
The aqueous suspension (1 g/50 mL) of the as-prepared NC was
then treated with various oxidants and acid/base additives to
give the four types of NCDs (Table 1, entries 1–4) with various
aldehyde contents or nanocellulose carboxylates (NCCs). The
aldehyde contents37 determined for NCDs are compared in the
last column of Table 1.
Fig. 1 The aldehyde content of NCDs at pHs 3 and 7 versus oxidation
times.
Table 1 Preparation of NCDs by oxidation of NC with oxidants
Entry
Oxidant/additive (g per g NC)
pH
Time (h)
NCD
Ald. content (%)
1
2
3
4
5
6
NaIO4 (1.4)/—
7
72
72
72
48
24
60
NCD72
83
52
58
—
0
NaIO4 (1.4)/HCl
NaIO4 (1.4)/NaOH
KIO4 (1.7)/—
NaClO2 (1.7)/—
TEMPO (1.3)/NaOCl
ꢃ3
ꢃ9
7
>8
10
NCDA72
NCDB72
NCDK48
NCCC24
NCCT60
47
0 (ref. 48)
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Based on the FESEM images, MC has a brillated structure
in the micro-dimension with 11–23 mm diameters, whereas NC,
HANCD, and HANCD@urease have nanostructures with
different porosities and morphologies. The difference in MC
and NC morphologies is in line with the acid cleavage of MC
bers to NC particles. Additionally, a size change from 19–
30 nm in the aggregated HANCD nanoparticles to the smoother
spherical 40–55 nm nanoparticles in the FESEM of HANC-
D@urease supports the immobilization of urease on HANCD
(Fig. 4).
As is obvious from the compared TGA curves, the mass loss
for HANCD@urease began at temperatures higher than 300 ꢁC,
whereas thermal decomposition of HANCD occurred at
ꢃ100 ꢁC. Moreover, thermal decomposition of HANCD@urease
at >300 ꢁC has a lower slope than that of HANCD. These results
support enough stability of the immobilized urease for practical
uses (Fig. 5).
Fig. 2 FT-IR spectra of MC, HANCD, and immobilized urease as
HANCD@urease.
Relative and specic enzyme activities, stability, and
reusability of the free and immobilized urease
Due to the signicant effects of pH and temperature on urease
structure and activity, the free enzyme and HANCD@urease
were compared for urea hydrolysis in solution (0.1 M) at various
temperatures (20–80 ꢁC) and buffer pHs (4–9, controlled by
1150 cmꢀ1, respectively (Fig. 2). The appearance of two new
characteristic peaks at ꢃ1740 cmꢀ1 and ꢃ890 cmꢀ1 in the
spectrum of HANCD is due to the C]O stretching and C–O–C
stretching of hydrated aldehyde32 in oxidized NC that conrms
the formation of NCD. Due to its maximum aldehyde content
(Table 1, entry 1), we termed this product HANCD. In the FT-IR
spectrum of HANCD@urease, the change in the broadness of
OH/NH2 absorption band at 2900–3500 cmꢀ1, disappearance of
the aldehyde C]O stretching, and entrance of a new peak at
1646 cmꢀ1 are due to the C]N formation by Schiff-base
bonding of HANCD aldehyde groups to amino acids of urease
(Fig. 2).
phosphate
solutions).
The reproducibility
of urea
The overlaid XRD patterns of HANCD and HANCD@urease
present similar peaks at 2q ¼ 20–23ꢁ corresponding to the (200)
diffraction plane of cellulose.49 The additional low intensity
peaks at 2q ¼ 26, 27, and 28ꢁ and the change in sharpness of
peaks are assigned to the introduction of urease to the HANCD
crystalline phase (Fig. 3).
Fig. 3 X-ray diffraction patterns of HANCD and urease immobilized Fig. 4 FESEM of MC (a), NC (b), HANCD (c), and immobilized urease
on HANCD.
on HANCD (d).
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Fig. 5 Thermal gravimetric analysis of HANCD and HANCD@urease.
determination by the biosensor HANCD@urease was conrmed
by repeating the experiments ve times under similar condi-
tions, with a relative standard deviation (SD) of 2.8% derived by
statistic analysis (Fig. 6a and b).
Based on the results in Fig. 6a, the optimum pH for
maximum activity of the free and HANCD-immobilized urease
is neutral pH 7, which means the same diffusion of the urea
substrate into both the free and immobilized enzymes. The
maximum activities of free and immobilized urease were at
60 ꢁC and 70 ꢁC, respectively, while the residual activities for the
HANCD@urease are higher than free urease at all temperatures.
With a milder slope for HANCD@urease, the decrease in
ꢁ
enzyme activity at higher than 80 C is attributed to the dena-
turation of urease (Fig. 6b). To determine the stability of the
HANCD covalent-bonded urease and conrm that the residual
activity is from the HANCD@urease and not due to the free
urease released by breaking the Schiff-base bonded enzyme, the
aldehyde content for the HANCD@urease was evaluated under
various conditions. The aldehyde content was determined for
the immobilized urease at certainꢁbufferꢁpHs ofꢁ2, 3, 4, 7, and 9
ꢁ
and screened temperatures of 25 C, 60 C, 70 C and 80 C in
controlled pH 7 (Fig. 6c). Based on the results, the aldehyde
ꢁ
content of HANCD@urease did not change between 20–60 C,
while the mere 2% increase in aldehyde content at 80 ꢁC
revealed 2% urease releasing at this temperature (Fig. 6c).
Similarly, urease Schiff-base covalently bonded to HANCD is
stable at about pH 7 and relatively stable at alkaline pH, with
release of only 1% urease at pH 9. The 42% and 20% aldehyde
contents for HANCD@urease at acidic pHs 2 and 3 were
recorded and illustrate the breaking of C]N bonds and release
of a similar percent of free urease at these acidic pHs. No
change in aldehyde content at optimum pH ꢃ7 for urease
enzyme displayed the performance of HANCD@urease (Fig. 6c).
Changes in the absolute activities for the free and HANCD
anchored urease were also evaluated at various pHs from 4 to 9
(Fig. 6d) and temperatures from 20 C to 80 C (Fig. 6e). The
maximum specic activity for both free and HANCD-
immobilized urease was at pH ¼ 7, while increasing or
decreasing pH reduces the enzyme activity due to the denatur-
ation of the peptide part of enzyme in acidic or basic conditions.
Because of the importance of the thermal stability, reus-
ability, and storage of enzymes in practical uses,50 the activity-
loss of the incubated free and HANCD-immobilized ureases at
Fig. 6 The relative residual activities of the free and immobilized
urease at adjusted pHs at 35 ꢁC (a) and at temperatures from 20–80 ꢁC
at pH ¼ 7 (b); the aldehyde content of HANCD@urease at various
temperatures and pHs (c); change in the absolute activities for free and
HANCD anchored urease at various pHs (d) and temperatures (e). Each
experimental point represents the mean of five determinations with
2.8% SD.
ꢁ
ꢁ
70 ꢁC were evaluated at numerous time intervals, with assays for
urea detection repeated for ve runs and the results analyzed to
estimate the experimental error using Statistic soware version
6 (Fig. 7).
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recycled immobilized urease was washed with deionized water
and reused, and 90% of the residual enzyme activity was
retained aer the sixth run. Tracking the aldehyde content for
the reused HANCD@urease aer enzymatic reaction runs
showed 5% aldehyde content for the recovered immobilized
enzyme aer the sixth run (Fig. 8).
To check the HANCD@urease for the estimation of blood
urea, three human blood serum samples with 82, 40, and 20 mg
mLꢀ1 urea (from a clinical auto-analyzer in Yazd Medical Lab)
were assayed with the biosensor HANCD@urease. The selection
of these samples was due to the available experimental data for
immobilized urease on alginate@urease and chitosan@urease
urea biosensors, as prepared and used by Kumar et al.24 We
repeated their procedure ve times and the results were
expressed as means ꢄ deviation standard (DS) for logical
comparison (Table 2). Nessler's reagent was added every 5
minutes to the lab serum samples treated with HANCD@ur-
ease, the absorbance of the color complex was measured at
wavelength 460 nm, and the urea concentration was calculated
by a calibration curve.
Fig. 7 Comparative residual activity of the free- and immobilized-
urease versus time.
Table 3 Optimization of reaction conditions
Fig. 8 Operating reusability of the HANCD@urease evaluation of
serum urea by HANCD@urease.
NANCD@urease
(mg)
Entry
Solvent/ꢁC
Time (min)
Yield (%)
1
2
3
4
5
6
7
8
0.005
0.005
0.005
0.01
0.02
0.01
0.01
0.01
0.01
0.01
0.01a
—
EtOH/60
CH2Cl2/60
H2O/60
H2O/60
H2O/60
H2O/60
H2O/60
H2O/60
H2O/70
H2O/80
H2O/60
H2O/60
50
50
50
50
50
60
70
80
70
70
70
70
45
15
65
80
76
85
96
95
75
60
92
0
The results supported the higher residual activity and lower
activity-loss rate for HANCD@urease than for free urease at all
incubation times. This shows the efficacy of urease immobili-
zation on HANCD. Moreover, the month-long control experi-
ment for the biosensor HANCD@urease in 1 mM urea solution
showed 100% preservation of the enzyme activity aer 15 days
and 10% activity-loss aer 35 days.
9
10
11
12
Alternatively, to overcome the recovery issues for free
urease,10 the operating stability of the HANCD@urease was
examined by reusability tests. Aer each experiment, the
a
Reaction with free urease.
Table 2 Performance of HANCD@urease for determination of the serum ureaa
Auto-analyzer
HANCD@urease urea
(mg dLꢀ1) (this work)
Clinical serum sample
urea (mg dLꢀ1
)
Alginate@urease urea24 (mg dLꢀ1
)
1
2
3
40 ꢄ 0.2
82 ꢄ 0.2
20 ꢄ 0.2
38 ꢄ 0.3
81 ꢄ 0.2
19 ꢄ 0.5
40 ꢄ 0.3
82 ꢄ 0.2
20 ꢄ 0.2
a
The urea amount was determined based on Experimental section “The performance assays for urea detection by HANCD@urease” (n ¼ 3).
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The results from HANCD@urease were similar to the clinical and reused. The results showed that aer the sixth run, the yield
auto-analyzer amounts and compatible with the previous data of product (94%) was reduced only 2%.
reported by Kumar et al.24 with respect to both precision and
Next, we extended the substrate and catalytic activity of
accuracy (Table 2). However, HANCD@urease can also be used HANCD@urease for bio-production of ammonia from urea by
as a biosensor to determine the urea in the other real biological employing 0.01 g of this catalyst with 0.18 g urea per 1 mmol of
samples.
aldehyde in a pseudo-six-component reaction of a number of
alkyl acetoacetates and aldehydes with hydrazine hydrate in
a molar ratio of 2 : 1 : 2 in water at 70 C (Table 4).
ꢁ
Catalytic evaluation of HANCD@urease for bio-production of
NH3 in THPPs synthesis
Given that THPPs are synthesized by MCRs with excess hygro-
scopic ammonium salts, organic solvents, and bio-
incompatible catalysts,41–45 we attempted the in situ bio-
production of ammonia by HANCD@urease-catalyzed hydro-
lysis of urea in water for the synthesis of THPPs. To optimize the
reaction settings, we chose hydrazine hydrate, ethyl acetoace-
tate, and benzaldehyde (2 : 2 : 1) as model substrates and
screened their reactions in water with the urea/HANCD@urease
couple for the synthesis of 3,5-dimethyl-4-phenyl-1,4,7,8-tetra-
hydrodipyrazolo[3,4-b:40,30-e]pyridine under various conditions
(Table 3).
As the results show, carrying out the model reaction with
0.01 g HANCD@urease in water at 70 ꢁC gives a maximum 96%
yield of the precipitated product within 70 min (entry 8), while
the reaction with free urease at the same time resulted in 92%
yield of product (entry 11). To isolate the catalyst, the precipi-
tated product was dissolved in EtOAc; the catalyst HANC-
D@urease was ltered, washed with EtOAc and then EtOH/H2O;
and the product was isolated aer evaporation of the solvent
and ltration aer addition of water. Aer structural conr-
mation of the product, further experiments were followed with
the HANCD@urease because of the higher efficiency and more
feasible reusability of HANCD-loaded urease than free-urease.
Also, the reusability of the biocatalyst HANCD@urease was
examined, so aer each model experiment, the recycled
HANCD@urease was washed with phosphate buffer solution
Scheme 2 Proposed mechanism for the synthesis of THPPs using
HANCD@urease.
Table 4 HANCD@urease catalyzed synthesis of THPPs
Entry
R
R11
Time (min)
Yield (%)
Mp (ꢁC)43–45, found (reported)
1
2
3
4
5
6
7
8
C6H5
OEt
OEt
OMe
OEt
OEt
OEt
OEt
OMe
OEt
OEt
OMe
80
110
90
60
70
70
110
80
90
70
55
96
89
90
98
95
97
90
91
92
89
90
240–242 (240–242)
215–217 (217–218)
215–217 (217–218)
<300 (<300)
251–253 (252–254)
284–286 (286–288)
216–219 (217–218)
216–219 (217–218)
219–221 (220–221)
185–187 (187–188)
185–187 (187–188)
4-Me2NC6H4
4-Me2NC6H4
4-O2NC6H4
4-ClC6H4
3-O2NC6H4
4-OH
4-OH
9
10
11
2-ClC6H4
2-O2NC6H4
2-O2NC6H4
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According to the results, HANCD-catalyzed synthesis of
RSC Advances
4 J. Zdarta, A. S. Meyer, T. Jesionowski and M. Pinelo,
THPPs in water provides access to a green high yield synthesis
of THPPs from the ammonia produced in situ by urea hydro-
lysis. The performance of HANCD@urease with respect to the
reaction yield and time is better than those of previously re-
ported syntheses of THPPs using ammonium acetate.
Catalysts, 2018, 8, 92.
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104692.
´
The possible reaction mechanism for HANCD@urease-
catalyzed synthesis of THPPs is proposed in Scheme 2. It was
presumed that the reaction proceeds by activation of ethyl
acetoacetate with HANCD@urease, bio-production of ammonia
from urea in the presence of HANCD@urease, and the cooper-
ative role of immobilized urease in closing the reaction
components by hydrogen bonding and cyclization of interme-
diates to nal products (Scheme 2).
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Conflicts of interest
The authors declare no conicts of interest.
Acknowledgements
The authors gratefully acknowledge the Yazd University
Research Council.
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