J. Plant Biochemistry & Biotechnology Vol.

19(1), 73-77, January 2010

Urease Immobilization on Arylamine Glass Beads and its

Characterization

Keyurkumar S Mangaldas1, Yudhishthir S Rajput2* and Rajan Sharma1

1
Department of Dairy Chemistry, 2Department of Animal Biochemistry, National Dairy Research Institute, Karnal 132 001, India

Jack bean urease has been immobilized on arylamine glass beads (200-400 mesh size, 75-100 Å pore size) and its properties
compared with soluble enzyme. The binding of urease was 13.71 mg per gram beads. The K m for soluble and immobilized
urease for urea was 4.20 mM and 8.81 mM, respectively. Vmax values of urease decreased from 200 to 43.48 µmol of ammonia
formed per min per mg protein at 37°C on immobilization. Both pH and buffer ions influenced the activities of soluble as well
as immobilized urease. Soluble urease exhibited pH optima at 5.5 and 8.0. However, immobilized urease showed one
additional pH optimum at 6.5. In comparison to phosphate buffer, citrate buffer was inhibitory to urease activity. Immobilization
of urease on arylamine glass beads resulted in improved thermal, storage and operational stability. Because of inertness of
support and stability of immobilized urease, the preparation can find applications in ‘artificial kidney’ and urea estimation in
biological fluids viz., blood, milk etc.

Key words: Urease, immobilization, arylamine glass beads.

Urease (E.C. 3.5.1.5) catalyses hydrolysis of urea into
ammonia and carbamates, latter being highly unstable
decomposes spontaneously into ammonia and carbonic
acid. Urease is widely distributed in nature and is found in
bacteria, fungi, algae, some invertebrates, higher plants,
and in soil (1, 2). Urease from plants including jack bean
( Canavalia ensiformis) urease is 540 kDa enzyme
comprising of six identical α-subunits. Fungal ureases also
have similar structure. In contrast, bacterial urease are, in
general, heterotrimers (αβγ)3, whose αβγ units exhibits high
homology of amino-acid sequences with α subunit of jack
bean urease (3). However, urease from Helicobacter pylori
consists of 2 subunits (αβ) but present in dodecameric
complex [(αβ)3]4 (4). The active site is located in α-subunit
(3) which is present in urease from different sources.
Urease from jack bean was the first enzyme ever
crystallized (5) and the first nickel containing enzyme
identified (6). Recent work suggested more complex pH-
dependence of urease action involving three groups (7).
Urease in soil allows urea utilization by plants.
Urease has found its applications in medical and
analytical sciences. Elevated blood urea levels can be
controlled by use of urease in artificial kidney (1). Urease
based methods for estimation of urea are accurate and
are increasingly used (8-11). Urea estimation in blood of
*Corresponding author. E-mail: ys_rajput@rediffmail.com
patients suffering renal insufficiency by urea biosensor
will require immobilized urease (12, 13). Immobilization of
urease allows its use repetitively and thus enables its use
on economic lines. Jack bean and pigeon pea are good
sources for urease and therefore, immobilization of urease
from these plant sources is widely studied. These include
immobilization of pigeon pea urease on flannel cloth (14),
DEAE-cellulose (15), polyacrylamide and calcium alginate
(16), gelatin (17), chitosan (18), reverse micelles (19),
microspheres (20), agar tablets (21) and on various types
of glass surfaces (22). Examples of immobilization supports
for jack bean urease include hydroxyapatite (23), clay
minerals (24), polyvinyl alcohol para formaldehyde (25),
nanoporous alumina membrane (26) and non-porous
hydroxy ethylene methacrylate incorporated microbeads
(27). In this study, immobilization of jack bean urease on
arylamine glass beads and its characterization are
reported.
Materials and Methods
Urease immobilization — Jack bean urease (type III,
Sigma) was immobilized on arylamine glass beads
(controlled pore, 75-100 Å, 200-400 mesh, Sigma). The
beads were activated by diazotization as per method
described by Weetall (28). Four ml of 2N HCl (chilled) were
added to 400 mg glass beads in glass vial (30 ml).
Immediately, 100 µl aqueous NaNO2 (1 g ml-1, chilled) were
74 J Plant Biochem Biotech
added. The vial was kept on ice for 30 min and contents
were intermittently swirled. Supernatant containing nitrous
acid was removed. The beads were washed several times
with phosphate buffer (0.1 M, pH 7.0, chilled) till pH of
washings became neutral. At this stage the colour of beads
changed from orange to magenta. Then, 2 ml urease (20
mg ml-1) prepared in Tris-acetate buffer (50 mM, pH 7.3)
was added to activated beads. The contents were swirled
and vial was kept on ice for 24 h with intermittent mixing.
Supernatant was collected. Beads were washed several
times with 5 ml phosphate buffer (0.1 M, pH 7.0) till
washings became negative for urease activity. Supernatant
and washings were pooled for assaying protein and urease
activity. Beads were then stored in 50 mM Tris-acetate
buffer, pH 7.3 at 4°C.
Protein estimation — Protein was estimated by dye
binding method described by Bradford [29] using BSA as
standard. The amount of protein immobilized was
determined by subtracting the amount of protein
determined in the supernatant following immobilization
from the total protein used for immobilization.
Urease activity — Urease was assayed as per the method
described by Das et al (16). The reaction mixture (2 ml)
contained 100 mM urea in 50 mM Tris-acetate, pH 7.3 and
0.1 ml urease solution. The reaction mixture in test tube
was incubated for 15 min at 37°C and reaction was stopped
by addition of 1 ml of 10 % TCA. The test tubes were sealed
with parafilm and placed in ice bath. Released ammonia
(ammonium ion) was measured through Nesslerization
reaction. An aliquot (50 µl to 1.0 ml) of reaction mixture
was added to 20 ml distilled water in 50 ml graduated
glass tube. While vortexing the contents, 1 ml Nessler
reagent was added. The contents were then diluted to 50
ml with distilled water and mixed. After 15 min, absorbance
was recorded at 405 nm. Amount of ammonia was
calculated from standard curve of ammonia (1 to 8 µmol)
which was prepared by Nesslerization of ammonium
sulphate. One unit of urease is defined as amount of
enzyme required to liberate 1 µmol of ammonia in 1 min at
pH 7.3 and 37°C.
Activity of immobilized urease on glass beads was
measured similarly. For each assay, 0.1 ml of suspended
beads (4 mg) was used. After 15 min of enzymatic reaction,
1.0 ml of supernatant was transferred to another glass
tube containing 0.5 ml 10% TCA. Known aliquots were
used for ammonia assay. Beads were recovered, washed
several times with 50 mM Tris-acetate buffer, pH 7.3 and
stored in this buffer at 4°C for future use.
Measurement of Km, Vmax and pH-optima — The Km and
Vmax were determined by measuring reaction velocity at
different concentrations of urea (0.5 mM to 30 mM) in 50
mM Tris-acetate buffer, pH 7.3 at 37°C. Reciprocals of urea
concentration and velocity were plotted to calculate Km
and Vmax. For determining optimum pH, urease activity was
measured at pH 5.0, 5.5 and 6.0 in 50 mM citrate buffer, at
pH 6.0, 6.5, 7.0 and 7.5 in 50 mM phosphate buffer, and at
pH 7.5, 8.0, 8.5 and 9.0 in 50 mM Tris-acetate buffer.
Thermal inactivation — Soluble or immobilized urease in
50 mM Tris-acetate buffer, pH 7.3 was treated at different
temperatures ranging from 37°C to 75°C for 10 min, and
immediately cooled. Remaining activity was measured at
37°C.
Operational stability — Urease activity of immobilized
enzyme was measured each time after its successive use.
After each use, the preparation was washed at least 5
times with 50 mM Tris-acetate buffer, pH 7.3.
Storage stability — Immobilized urease glass beads were
stored in 50 mM Tris-acetate buffer, pH 7.3 at 4°C. The
activity of immobilized urease was measured on different
days, and for each assay fresh beads were used.
Results and Discussion
Immobilization — The immobilized enzyme exhibited 752
urease units activity per gram of bead containing 13.73
mg urease protein. The K m value (calculated from
Lineweaver-Burk plot) for soluble and immobilized urease
was found to be 4.2 mM and 8.91 mM, respectively. On
immobilization, Vmax of urease decreased from 200 units to
43.38 units per mg protein resulting 21.69% immobilization
yield. The increase in Km is perhaps related to hindrances
caused by the groups for diffusion of substrates (urea) to
the active site and also constraints associated in diffusion
of the reaction product. The reactant (urea) and the reaction
product (NH3 and CO2) are small in size and therefore,
diffusion constraints are expected to be of low order but it
resulted in nearly 2 fold increase in Km value. Further, there
is considerable decrease in reaction velocity when urease
is immobilized on arylamine glass. This decrease can be
attributed to stearic effects resulting from limitations of the

accessibility of substrate to active site, conformational
changes in the structure of active site of the enzyme upon
immobilization or denaturation of enzyme protein under
the conditions of immobilization. The immobilization method
uses coupling of diazo group on support to primarily
tyrosine side chain of enzyme. The diazo coupling is not
very specific and it can covalently couple to imidazole side
chain. Further, two of the imidazole groups from histidine
are coordinated to two nickel ions (3, 7) which are essential
for functional urease active site. Perhaps, imidazole
coupling can alter intactness of urease active site and this
may account for decreased urease activity. Km value of free
urease from jack bean reported in literature is variable
and is perhaps dependent on assay conditions. A Km value
of as low as 0.94 mM or as high as 521 mM for jack bean
urease has been reported (30). In most of the covalent
immobilized methods for jack bean urease, the Km value
enhanced by 1.5 to 6 folds (30). In the present study, the
increase in Km was 2.1 fold. However, pigeonpea urease
immobilized on arylamine or alkylamine glass beads
exhibited no virtual increase in Km values (22). There are
only a few studies where a Km value of urease is not
increased on immobilization.
Effect of pH on urease activity — The effect of pH on
soluble and immobilized urease activity (% of maximum
activity) is shown in Fig. 1. There is no sharp single pH
optimum either for soluble or immobilized urease. Soluble
Fig. 1. Effect of pH on soluble and immobilized urease activity.
Activities of soluble ( , , ) and immobilized urease ( , , )
urease was measured at pH 5.0, 5.5 and 6.0 in 50 mM citrate
buffer ( , ), at pH 6.0, 6.5, 7.0 and 7.5 in 50 mM phosphate
buffer ( , ) and at pH 7.5, 8.0, 8.5 and 9.0 in 50 mM Tris-acetate
buffer ( , ).
Urease Immobilization on Glass 75
urease had two pH-maxima at pH 5.5 and 8.0. Immobilized
urease had one more additional maximum at pH 6.5.
Further, urease activity at pH 6.0 in citrate buffer (50 mM)
was about 18% lower than in 50 mM phosphate buffer
indicating that citrate buffer is inhibitory. A detailed study is
needed to understand how citrate buffer inhibit the urease
activity. The soluble urease activity at pH 7.5 is identical in
50 mM phosphate buffer and 50 mM Tris-acetate buffer
indicating that these buffer components were not inhibitory
to urease activity at pH 7.5.
Numerous studies indicated that urease exhibited
single pH optimum. However, more than one pH optima
ranging from 5.8 to 8.0 have also been noted (17, 23, 25,
31-33]. Both the pH-optima (pH 5.5 and 8.0) for soluble
urease observed in present studies lie in this range. H2PO4-
and borate are competitive inhibitors of urease (7). The
concentration of H2PO4- in phosphate buffer is dependent
on pH and molarity of buffer and therefore, effect of pH on
urease activity in phosphate buffer will be sum total of
effects arising out from change in pH and H 2 PO 4 -
concentration. However, H2PO4- ion is inhibitory to urease
action below pH 7.5 (7). Krajewska et al (1) studied effect
of pH by using 20 mM MES and 20 mM HEPES buffers and
obtained two pH optima corresponding to pH values 6.5
and 7.2. These indicated importance of at least two
ionizable groups in urease catalysis. Studies also
suggested that involvement of three groups with pKa 5.3,
6.6 and 9.1 (7), indicating urea catalysis mechanism by
urease is more complicated than what was earlier thought.
The pKa of 5.3 and 6.6 were related to carboxyl group and
imidazole groups, respectively while pKa of 9.1 could be
related to a nickel-bridging hydroxide (7).
Thermal stability of soluble and immobilized urease —
Immobilization invariably results in enhanced thermal
stability which is a useful property of immobilized enzyme
for its efficient use. For assessing thermal stability of
immobilized urease vis-a-vis soluble urease, ureases were
subjected to heat treatment for 10 min at different
temperatures ranging from 37°C to 75°C, and remaining
urease activities were measured. Immobilized urease on
arylamine glass beads was thermally more stable as
compared to soluble urease (Fig. 2). As temperature of
heat treatment was enhanced, there was small but gradual
decrease in urease activity. However, in case of soluble
urease, the loss in activity was significant and more
pronounced above 50°C. Heat treatment at 75°C for 10
min resulted in 20% loss in immobilized urease activity
76 J Plant Biochem Biotech
Fig. 2. Effect of heat treatment (10 min) at indicated temperature
on urease activity of soluble ( ) and immobilized urease ( )
while, under similar conditions, soluble enzyme lost 82.5%
of initial activity (Fig. 2). The results are in conformity with
immobilization of pigeon pea urease on glass beads (22)
and poly-acrylonitrile/chitosan composite membranes (32).
Enhanced thermal stability of urease on immobilization on
glass support may be attributed to limited flexibility of the
molecule due to covalent coupling which may protect
unfolding of urease due to heat treatment.
Operational stability — Operational stability of urease
immobilized on arylamine glass beads was checked by
measuring urease activity after each of five successive
operations. The remaining activities are plotted as
percentage of activity calculated from first operation (Fig.
3). Only 6% activity is lost in four operations. Covalent
coupling minimizes leaching of urease during use and
Fig. 3. Effect of repeated use of immobilized urease on its activity.
provides more operational stability. Ar ylamine (22),
nanoporous alumina (26) and poly(acrylamide co-acrylic
acid)/k-carrageenan (34) appear to be excellent support
for providing operational stability to immobilized urease.
The other methods of immobilization have not provided
desirable operational stability. These include urease
immobilization on Vermiculite using glutaraldehyde (39%
activity loss after four repeated uses) (35), urease
immobilization on aminated butyl acrylate ethylene
dimethacrylate copolymer (50% activity loss after seven
repeated uses) (36), and urease immobilization on chitosan
membrane (complete loss after nine repeated uses) (37).
Effect of storage on stability of soluble and immobilized
urease — Soluble urease and immobilized urease were
stored in 50 mM Tris-acetate buffer pH 7.3 at 4°C up to 60
days (Fig. 4). The immobilized urease on arylamine glass
beads retained 89% of initial activity after 60 days. However,
soluble urease lost its activity very fast and became
completely inactive at 15 days of storage. The loss in activity
of soluble urease was unusually very fast. This clearly
signifies that coupling of enzyme to glass matrix had
tremendously improved the storage stability of urease. High
storage stability after immobilization has also been noted
in case of pigeon pea urease immobilized on glass beads
with 15-17% loss in activity after 70 days of storage at 4°C
(22); aminated butyl acrylate ethylene dimethacrylate
Fig. 4. Effect of storage on activity of soluble ( ) and immobilized
urease ( ) at 4°C in 50 mM Tris-acetate buffer, pH 7.3.
 Urease Immobilization on Glass 77

copolymer with almost no loss of activity for 85 days (36);
38% and 27% loss in case of Amberlite MB-1 (38) and
Chitosan-poly (GMA) Copolymer (39) at 4°C after 60 days,
respectively. Urease immobilized on modififed
polysulphone membrane and on nanoporous alumina
membranes have 70-73% residual activity after 30 days of
storage at 4°C (26, 31). The urease immobilized on glass
showed better shelf-life than many matrices used so far.
Thus, urease immobilization on arylamine glass beads
results in improved thermal, operational and storage
stability. Since there is an increase in Km and decrease in
Vmax, more amount of enzyme may be required to be
immobilized to achieve same rate of reaction. However, as
15 Reddy KRC, Srivastava PK, Dey PM & Kayastha AM,
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there is tremendous improvement in thermal, operational 22
and storage stability, immobilized preparation can be used
economically in medical and analytical applications. 23
Received 2 February, 2009; accepted 14 September, 2009 24
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