{"id":514,"date":"2015-01-22T06:30:17","date_gmt":"2015-01-22T06:30:17","guid":{"rendered":"http:\/\/biomedpharmajournal.org\/?p=514"},"modified":"2020-04-25T06:31:08","modified_gmt":"2020-04-25T06:31:08","slug":"effect-of-organic-additives-on-activity-of-urease","status":"publish","type":"post","link":"https:\/\/biomedpharmajournal.org\/staging\/vol1no2\/effect-of-organic-additives-on-activity-of-urease\/","title":{"rendered":"Effect of Organic Additives on Activity of Urease"},"content":{"rendered":"

Introduction<\/strong><\/p>\n

In 1926 James Sumner showed that urease is a protein. Urease is found in bacteria,\u00a0\u00a0\u00a0 yeast and several higher plants.urease is significant in the history of enzymology as the first\u00a0\u00a0 enzyme to be purified and crystallized.\u00a0 <\/strong>James B. Sumner of Cornell University in (1946) received the Nobel Prize for his work with the enzyme urease, extracted from the jack bean. Urease is an enzyme that catalyzes the conversion of urea to ammonia and carbon dioxide. Certain bacteria that convert urea to ammonia as part of the nitrogen cycle contain this enzyme.Nomenclature Committee of the\u00a0\u00a0 International Union of Biochemistry and Molecular Biology (NC-IUBMB)\u00a0 <\/strong>has given nomenclature to the urease as E.C (3.5.1.5).Enzyme commission 3.5 of enzyme urease denotes its action that it is a hydrolase\u00a0 which breaks the bond by addition of water molecule(no:3) and specifically breaks carbon-nitrogen non peptide bond.(no:5).\u00a0 An unusual feature of urease is its dependence on nickel to grab onto and break up urea in the enzyme’s active site. In 1982, Australian researchers Barry Marshall and Robin Warren discovered spiral-\u00a0\u00a0 shaped bacteria in the stomach, later named Helicobacter pylori (H. pylori). After closely studying H. pylori’s effect on the stomach, they proposed that the bacteria were the underlying cause of gastritis and peptic ulcers by using enzyme urease.<\/p>\n

Materials and Methods<\/strong><\/p>\n

Beans of Dolichos lab lab,<\/em> Nesslers reagent,. Tris acetate buffer (10%TCA), 4. Sulphuric Acid (1N H2SO4), phosphate buffer (0.2M), ammonium sulphate solution, pure enzyme urease (0.1%)<\/p>\n

Experimental<\/strong><\/p>\n

Urease enzyme was isolated from seeds of\u00a0 beans using mortar pestle and phosphate buffer and then stored at 4 C .Standardization of\u00a0 ammonium sulphate using Nessler\u2019s reagent will be done by drawing standard graph.Then determination of urease activity under various modulators with substrate (urea) by using the standard curve. Then determination of pure urease activity under the same modulators. Then comparison of\u00a0 the results obtained.<\/p>\n

Determination of urease activity<\/strong><\/p>\n

3 clean test tubes are taken and marked as control, test, blank. To the tubes control and test 2 ml of urea substrate were added for the blank 4.5 ml of phosphate buffer was added. The tubes were incubated for 10 min and add 0.5ml of enzyme to the test.. After 15 min 0.5 ml of enzyme was added to the tube control and immediately to all the tubes 0.5 ml of 10% trichloro acetic acid and 0.5 ml of 1N H2SO4 were added. All the tubes were centrifuged for 10 min at 2000rpm. After centrifugation 0.5 ml of supernatant was taken in a tube separately from all the test tubes. To the supernatant 0.5 ml of Nesslers reagent was added and the yellow colour\u00a0\u00a0 developed was measured at 540 nm.<\/p>\n

Results and Discussion<\/strong><\/p>\n

After performing the said experiments, to the enzyme sample-varying conceentration of the inhibitor glycerol was taken and the activity of the enzyme was studied under specified and defined conditions. Enzyme activity is measured at various time intervals like 15min, 30min, 45min and maximum enzyme activity is shown at 30min, decrease for 45minutes. The results of analysis are presented in Table -1<\/p>\n\n\n\n
\"Table<\/a><\/td>\nTable 1:\u00a0<\/strong>Effect of Glycerol on activity of urease.<\/strong><\/p>\n

 <\/p>\n

Click here to View figure<\/a><\/td>\n<\/tr>\n<\/tbody>\n<\/table>\n

 <\/p>\n

Conclusion<\/strong><\/p>\n

Temperature fluctuations have significant effect on enzyme activity. Following conclusions are drawn for the enzyme activity and effect of inhibitors on enzyme activity for crude enzyme source and pure enzyme. Glycerol showed maximum inhibition at 20% concentration for crude enzyme source, and for pure enzyme maximum inhibition is shown at 20%.<\/p>\n

Acknowledgements<\/strong><\/p>\n

The authors are grateful to Management of Koneru Lakshmaiah College of Engineering, Vaddeswaram, Guntur Dist, for their continuous support and encouragement and for providing the necessary facilities for carrying out the project.<\/p>\n

References<\/strong><\/p>\n

    \n
  1. \u00a0<\/strong>BURNE, R.A. AND CHEN, Y.Y.\u00a0\u00a0 Bacterial ureases in infectious diseases.\u00a0MICROBES INFECT<\/em>. 2 533-542 (2000).<\/li>\n
  2. \u00a0MOBLEY, H.L., ISLAND, M.D. AND HAUSINGER, R.P.\u00a0\u00a0 Molecular biology of microbial, ureases. MICROBIOL.REV<\/em>. 59 451-480 (1995).<\/li>\n
  3. \u00a0MOBLEY, H.L., ISLAND, M.D. AND MASSAD, G.\u00a0\u00a0 Virulence determinants of \u00a0uropathogenic, Escherichia coli and Proteus mirabilis. KIDNEY INT.SUPPL<\/em>. 47 \u00a0S129-136 (1994).<\/li>\n
  4. \u00a0MCGEE, D.J. AND MOBLEY, H.L. Mechanisms of Helicobacter pylori infection: bacterial, factors. R.TOP.MICROBIOL.IMMUNOL<\/em>. 241 155-180 (1999).<\/li>\n
  5. HA, N.C., OH, S.T., SUNG, J.Y., CHA, K.A., LEE, M.H. AND OH, B.H. Supramolecular assembly and acid resistance of Helicobacter pylori urease.\u00a0NAT.STRUCT.BIOL<\/em>. 8 505-509 , (2001) .<\/li>\n
  6. P Bauerfeind, R Garner, B E Dunn, and H L Mobley, Synthesis and activity of Helicobacter pylori urease and catalase at low pH Gut<\/em>. 1997 January; 40(1):25\u201330.<\/li>\n
  7. Susanne Klose, M.A. Tabatabai, Soil Biology and Biochemistry<\/em>, Volume , 31, issue 2, February 99, Pages 205-211.<\/li>\n
  8. A. Rivadeneyra, A. Gutierrez-Calder\u00f3n, A.M. Rivadeneyra, A. Ramos-Cormenzana, A Study of Struvite Precipitation and Urease Activity in Bacteria Isolated from Patients with Urinary Infections and Their Possible Involvement in the Formation of Renal Calculi,\u00a0 Urologia Internationalis<\/em>, Vol-63, No.3 1999.<\/li>\n
  9. K.\u00a0Sanyal, B.\u00a0Banerjee, Urease activity in Trichophyton\u00a0<\/em>Mentagrophytes<\/em>, folia microbiologica<\/em>, Volume 28, number 2 , march 1983.<\/li>\n<\/ol>\n","protected":false},"excerpt":{"rendered":"

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