International Journal of Science and Advanced Technology (ISSN 2221-8386)

http://www.ijsat.com

Volume 1 No 6 August 2011

Kinetics of Urea decomposition by commercial grade

Urease
Sayantan Chowdhuri

Joydeep Chakraborty

Department of Biotechnology
Heritage Institute of Technology
Kolkata, India
sayan.chowdhuri@gmail.com

Department of Biotechnology
Heritage Institute of Technology
Kolkata, India
joydeep.c87@gmail.com

Tapan Kumar Ghosh

Binoy Ranjan Maiti

Department of Biotechnology
Heritage Institute of Technology
Kolkata, India
tapanswatighosh@gmail.com
Abstract A commercial grade Urease has been used to study
the kinetics of urea decomposition in a batch reactor at a room
temperature of 280C. A kinetic model has been developed in the
form of Michaelis Menten Equation and the evaluated kinetic
parameters; Km and Vmax are comparable with the reported
literature data, though the lumped kinetic parameter, Vmax
widely varied with a reduced value compared to the earlier
reported value. This anomaly has been attributed to the presence
of impurity in the commercial sample due to strong inhibition
effect of a likely impurity like Thiophene. A simple pseudo-first
kinetics have also been fitted to the batch data for the system.
Keywords-decomposition; kinetic; parameter; inhibition

INTRODUCTION
Very few studies have been reported for Urea decomposition
by Urease. Levine and Lacourse (1967) reported Urea
decomposition during their investigation on artificial kidney.
In the microencapsulation scheme proposed by them, the
enzyme Urease could be used in the removal of Urea from the
blood stream by the catalytic decomposition of Urea to
Ammonia and Carbon di oxide. This reaction has been
extensively used in the development of biosensors for the
detection of Urea in blood (Hall, 1990).
The objective of the present investigation is to evaluate the
kinetics of the process on the basis of some sequential steps of
the reaction.

MECHANISM AND KINETIC MODEL OF REACTION

Urease is a Nickel metallo-enzyme that catalyzes the
degradation of the Urea to Ammonia and Carbamine Acitd.
The latter compound further decomposes to generate a second
molecule of Ammonia and Carbon dioxide. The aqueous
solution turns phenolphthalein red due to the presence of
hydroxide ions. The following sequence of reactions may be
present as follows:

Department of Biotechnology
Heritage Institute of Technology
Kolkata, India

CO(NH2)2 + H2O

K1 Urease

H2N-COOH

K2 Urease

2 NH3 + 2 H2O + CO2

NH3 + NH2-COOH (Carbaminic Acid) - (1)

NH3 +CO2 ------ (2)
fast

2 NH4- + OH- + HCO3- ---- (3)

The crystal structure of the active center of Urease contains

probably two simple co-ordinated water molecules and a
bridging OH group. The specificity of the enzyme is closely
related to the shape of its active center. One Ni-ion acts as a
Lewis acid and the metal ion polarizes the carbonyl group of
Urea and activates it towards nucleophillic attack. In the
process 2 water molecules are replaced by Urea. The other Ni
binds a hydroxide ion. The OH- ion attacks the partially
positive Carbonyl carbon of the Urea molecule. One of the
2NH2 groups is postulated Histadine is postulated to be the
acid.
Urease is absolutely specific for its substrate. Urea and the
structural analogues e.g. Thio Urea are not degraded by
Urease.
Based on the first two reactions and assuming water is in
excess, the rate of Urea(H) decomposition can be given as a
pseudo-first order reaction rate
-dCA/dt = K1 CA,
where K = K1 Cw, where K1 is the second order rate constant
and Cw is the concentration of water which is in excess.
The second approach to the kinetic evaluation of Urea
decomposition is the enzymatic reaction mechanism involving
the formation of enzyme-substrate (E-S) complex.
The following reaticon steps can be written using the
following abbreviations:

101

International Journal of Science and Advanced Technology (ISSN 2221-8386)

http://www.ijsat.com
E (Urease), S(Substrate), W(Water), E-S (Enzyme-Substrate
complex), and P(Products 2NH3 + CO2)
E+S

E.S

------ (4)

E.S

E+S

------ (5)

E.S

P+E

------ (6)

[(So )/ln{1/(1- )}] = -Km + Vmax[t/{ln (1- )-1}]

------ (17)

From the linear plot of [(So )/ln{1/(1- )}] against [t/{ln (1)-1}], the intercept will give Km and the slope is equal to Vmax
MATERIALS AND METHOD

The rate of Urea decomposition, rs can be given as:

-rs = K1 (E)(S) K2 (E.S) ------- (7)
Assuming pseudostate assumptions for E.S i.e. d(E.S)/dt = 0
and the total enzyme is constant:
d(E.S)/dt = 0 = K1 [(E0) (E.S)] S - K2(E.S) K3(E.S)(W)
------- (8)
(E) = (Eo) (E.S) ------- (9)
Substituting Eq (9) into Eq (7), we have,
-rs = K1 [(Eo) (E.S)] K2 (E.S) ----- (10)
Solving for (E.S) from equation (8)
(E.S) = [K1 (Eo) (S)]/[K1(S) + K2 + K3(W)]

Volume 1 No 6 August 2011

------- (11)

In a batch reactor of 250 ml conical flask, 200 ml of 0.7%

solution of pure Urea (7gm/L) and 0.1% of commercial
Urease were taken and stirred with a magnetic stirrer. The
reaction was found to be very slow. Three samples of 5ml
were collected at intervals of 4hrsm 14hrs and 19hrs. The
sample solution turned red with 1% phenolphthalein indicator
and titrated with standard Oxalic Acid to find out the
concentration of ammonia from which the unreacted Urea was
calculated.
RESULTS AND DISCUSSIONS
A set of batch data of Urea decomposition has been obtained
at room temperature (280C) and pH = 7.0 and presented in
Table 1.
Calculations for evaluations of Kinetic Parameters with batch
data :
So = 0.115 gm mole/L (7gm/L) and Eo = 1.0 gm/L
S
l.

Time
(hrs)

S
(mole/L)

= [(So)
(S)]/
(So)

0.112

0.0027

14

0.1044

-rs = [K1K3 (W)(Eo)(S)]/[K1(S) + K2 + K3(W)] --------- (13)

19

0.100

Since the reaction of Urea and Urease is carried out in aqueous

solution, water is in excess and the concentration of water (W)
is assumed constant. Thus,

Table 1 Kinetic Parameters with batch data

Subtracting Eq (8) from Eq (10), we get

-rs = K3 (W) (E.S) -------- (12)
Now substituting (E.S) from Eq (11) into Eq (12),

K3 = K3 (W) and Km = (K3 + K2) ------ (14)

and Vmax = K3 (Eo)
Introducing above Eq(14) with Eq(13), we obtain an equation
of Michaelis-Menten type:
-rs = -d(S)/dt = {Vmax(S)}/ {Km + S} --------- (15)

So

ln{1/(1)}

[(So
)/ln{1/
(1- )}]

[t/{ln
(1- )1
}]

0.00313

0.0273

0.1146

146.5

0.100

0.0116

0.1053

0.1104

133.0

0.138

0.0160

0.1487

0.1078

128.0

A pseudo- first order plot of - ln{1/{1- )} vs t yields a pseudo

first order rate constant, K1 = 0.0008 hr-1. The excellent
linearity of the plot nicely corroborated the pseudo-first order
kinetics of the process. In the second model testing, the
linearized integrated plot of Michaelis-Menten equation has
been illustrated in Figure 2 where [(So )/ln{1/(1- )}] has
been plotted against [t/{ln (1- )-1}] and the kinetic
parameters, Km and Vmax have been evaluated from the
intercept and the slope respectively.

where Vmax and Km are the kinetic parameters of the

equation.
To obtain the kinetic parameters from a batch reactor data, the
Eq(15) is integrated and linearized and expressed in terms of
fractional conversion,
= [(So) (S)]/ (So)

------- (16)

102

International Journal of Science and Advanced Technology (ISSN 2221-8386)

http://www.ijsat.com

Volume 1 No 6 August 2011

The values are tabulated with the reported literature data:

Present Study

Literature Reported
(Fogler, 1995)

Km = 0.025 gm mole/L

Km = 0.266 gm mole/L

Vmax = 1.022 x 10-3 gm mole L-1

hr-1

Vmax = 4.788 x 10-3 gm mole

L-1 hr-1

K1 (Pseudo-first order rate

constant) = 8.0 x 10-3 hr-1

--

Table 2 Comparative Kinetic parameters

From the Table 2 it has been observed that Km values in both
cases have been found to be of identical magnitude, whereas
Vmax varied widely. In the present study, Vmax is very small
indicating a very slow decomposition from (E-S) complex
with the given commercial enzyme. It is suspected that some
structural analogue of Urea like Thiophene may be present in
the system. Like Urea, Thiophene may be bound to the active
center of the enzyme without producing ammonia.

Fig 3: Simulated curve for Urea decomposition based on

Experimental data
CONCLUSION
Since the data are scanty, it is difficult to predict the
accurate kinetic behavior of the Urea decomposition, using
other types of models.
A simulation study for the system has been carried out with
the evaluated parameters which adequately match the
experimental data as shown in Fig 3.
It is desired that more experimental data are to be obtained
at different process conditions like pH, substrate
concentration, temperature etc for sophisticated kinetic
modeling.
ACKNOWLEDGEMENT
Experimental works were carried out by Sayantan
Chowdhuri. Data analysis, representations and reference
collection, done by Joydeep Chakraborty.

Fig 1: Pseudo- first order plot for Urea decomposition

REFERENCES
Slope = (0.124 0.060)/(16-8) = 0.008 hr-1
1.
2.
3.
4.

5.

6.
Fig 2: Linearized integrated Michaelis Menten Equation

7.

Levine N and W.C. Lacourse, J. Biomed. Mater,

Res. 1, 275 (1967)
Fogler, S.H., Elements of Chem. Reaction Engg,
PHI, 1995
P.F. Hall, Biosensors, Milton-Kaynes, Open Univ.
Press, 1990
Lucia Andrich, Marco Esti, Mauro Moresi, Enzyme
and Microbial Technology, Vol 46, Issue 5, April
2010, Pg 397-406
A. Anita, C.A. Sastry and M.A. Hashim, Bioprocess
and Biosystems Engineering Vol 17, Number 4, 241245, DOI: 10.1007/s004490050381, April 1997
M. Fidaleo and R. Lavecchia, Chem. Biochem. Eng.
Q. 17 (4) 311318 (2003)
William N. Fishbein, Annals of the New York
Academy of Sciences Volume 147, pages 857
881, November 1969.

103