Extraction,
purification, kinetic and thermodynamic properties of urease from
germinating Pisum Sativum L.seeds
- *Corresponding
author: Mohamed E EL-Hefnawymalhefnawy@kau.edu.sa
1Department of Chemistry, Rabigh College of
Science and Arts, King Abdulaziz University, P.O. Box 344, Rabigh 21911, Saudi
Arabia
2Department of Biochemistry, Faculty of Science,
Tabuk University, Tabuk, Saudi Arabia
3Department of Chemistry, Faculty of Science,
Tanta University, Tanta 31527, Egypt
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BMC Biochemistry 2014, 15:15 doi:10.1186/1471-2091-15-15 The electronic version of this article is the
complete one and can be found online at:http://www.biomedcentral.com/1471-2091/15/15
This is an Open Access article distributed under the terms
of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0),
which permits unrestricted use, distribution, and reproduction in any medium,
provided the original work is properly credited. The Creative Commons Public
Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/)
applies to the data made available in this article, unless otherwise stated.
Background
Urease, one of the highly efficient known enzymes, catalyzes
the hydrolysis of urea into ammonia and carbon dioxide. The present study aimed
to extract urease from pea seeds (Pisum Sativum L).The enzyme was then
purified in three consequence steps: acetone precipitation, DEAE-cellulose
ion-exchange chromatography, and gel filtration chromatography (Sephacryl S-200
column).
Results
The purification fold was 12.85 with a yield of 40%. The
molecular weight of the isolated urease was estimated by chromatography to be
269,000 Daltons. Maximum urease activity (190 U/g) was achieved at the optimum
conditions of 40°C and pH of 7.5 after 5 min of incubation. The kinetic
parameters, Km and Vmax, were estimated by
Lineweaver-Burk fits and found to be 500 mM and 333.3 U/g, respectively.
The thermodynamic constants of activation, ΔH, Ea, and ΔS, were
determined using Arrhenius plot and found to be 21.20 kJ/mol,
23.7 kJ/mol, and 1.18 kJ/mol/K, respectively.
Conclusions
Urease was purified from germinating Pisum Sativum L. seeds.
The purification fold, yield, and molecular weight were determined. The effects
of pH, concentration of enzyme, temperature, concentration of substrate, and
storage period on urease activity were examined. This may provide an insight on
the various aspects of the property of the enzyme. The significance of extracting
urease from different sources could play a good role in understanding the
metabolism of urea in plants.
Keywords:
Urease; Enzyme activity; Enzyme purification; Pisum
Sativum L; Pea seeds - ver mas en
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