Immobilization of Vibrio cholerae S1 (NAG) L-Glutaminase on Different Supports
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Abstract
Sixteen Vibrio isolates producing L-glutaminase were obtained from clinical and water samples , one isolate was selected according to its’ highest enzyme productivity , it was identified as Vibrio cholerae (NAG) and coded as V. cholerae S1.
The bacteria was cultured in a liquid medium (containing L-glutamine) , L-glutaminase was extracted from the cells by ultrasonication , the enzyme was precipitated by 30 % saturation of ammonium sulphate , dialyzed and immobilized by adsorption on different supports including Sephadex G-100 , cellulose powder , starch , silica gel , glass beads and charcoal.
Sephadex G-100 retained most of enzyme activity (90 %) followed by starch (78 %) , then silica gel and cellulose powder (71 %) while glass beads and charcoal retained 58 % only.
The immobilized enzyme was subjected to different temperatures and pHs. The results showed that the immobilized enzyme is more stable than the free enzyme in different temperatures and pHs. Silica gel was the best matrix for protecting L-glutaminase against heat , it retained 52 and 22 % of the original activity after 2 hrs of incubation at 50 and 60 ۫c respectively while the free enzyme retained 30 and 10 % at the same conditions.
The immobilized enzyme was more stable at pH 7 than at pH 4 or 10 . the enzyme adsorbed on Sephadex G-100 retained the maximum activity (98 %) at pH 7 for 2 hrs , while it was 73 % for the free enzyme.
The immobilized L-glutaminase of V. cholerae S1 (with Sephadex G-100) was stored at 4 ۫c for 30 days , the remaining activity was 35 % , while it was 18 % for the free enzyme.
It can be concluded from these results that V. cholerae S1 L-glutaminase can be immobilized on different inert materials, Sephadex G-100 is more suitable in this project , Silica gel can protect the enzyme against heat. In general the immobilized enzyme is more stable at different temperatures , pH and time than the free enzyme.
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Bhattcharya, P. and Matty, P. 2001. Localization of phosphate dependent glutaminase in ascites fluid of ovarian cancer patient. Path. Ecol. Res. 6: 217 – 223.
Matsuno, T. and Goto, I. 2004. Glutaminase and glutamine synthetase activities in human cirrhotic liver and heptocellular carcinoma. Am. J. Physiol. Gastrointes. Liverphysiol. 286 (3) : 467 – 478.
Chandrasekaran, M. 1997. Industrial enzymes from marine microorganisms. J. Marine Biotechnol. 5 : 1432 – 1438.
Asabu, S. 2003. Sources properties and application of therapeutic enzymes. Indian Journal of Biotechnology. 2 : 334 – 341.
Roediger, W. E. ; Millard, S. H. and Bird, A. R. 2001. Focused gut mucosal nutrition for diarrheoeal disease : imported nutrient therapy. Asia Pasific. J. Clin. Nut.10(1) : 67 – 68.
Smith,J.E.1996.Biotechnology.Cambridge University Press.pp 83-90.
Collee, J. G.; Fraser, A. G.; Marmion, B. P. and Simmons, A. 1996. Practical and Medical Microbiology 14th ed. Churchill Livingstone.
Holt, J. G., Krieg, N. R.; Sneath, P. H. A. ; Staley, J. T. and Williams, S. T. 1994. Bergey’s Manual of Determination Bacteriology. 9th ed. Williams and Wilkins
Beck, J. V. 1971. Enrichment culture and isolation techniques particularly for aerobic bacteria. In-Methods In Enzymology (ed. Jakoby, W. B.) Vol. 22. pp. 46 – 61. Academic Press. New york.
Novak, E. K. and Philips, A. W. 1974. L-glutamine as a substrate for L-asparaginase from Serratia marcescens. J. Bacteriol. 117 (2) : 593 – 600.
Scopes, R. k. (1987). Protein Purification )2nd ed.) Springer. Velag. New York.
Imada, A.; Igarasi, S.; Nakahama, K. and Isono, M. 1973. Asparaginase and glutaminase activities of microorganisms. J. Gen. Microbiol. 76 : 85 – 99.
Sarquiz, M. I. M. ; Oliviera, E. M. ; Santos, A. S. and Costa, G. L.2004. Production of L-glutaminase by filamentous fungi. Mem. Inst. Oswaido Gruz. 99 (5) : 489 – 492.
Prusiner, S. 1975. Regulation of glutaminase levels in Escherichia coli. J. Bacteriol. 123 (3) : 992 – 999.
Morehouse, R. F. and Curthoys, N. P. 1981. Properties of rat renal phosphate – dependent glutaminase couples to Sepharose. Biochem. J. 193 : 709 – 716.
Truong, V. Clare, D. A. ; Catignan, G. L. and Swaisgood, H. E.2004. Cross linking and rheological changes of whey proteins treated with microbial transglutaminase. J. Agr. Food. Chem. 52 : 1170 – 1176.
Meibom, K. L.; Li, X. B. ; Nielson, A. T. ; Wu, C. ; Roseman, S. and Schoolnik, G. K. 2004. The Vibrio cholerae chitin utilized program. Natt. Acad. Sci. U. S. A. 101 : 2524 – 2529.
Gancz, H.; Niderman, O.; Broza, M. ; Kashi, Y. and Shimoni, E. 2005. Adhesion of Vibrio cholerae to granular starches. J. Trop. Perdiatr. 139: 157 – 163.
Sabu, A. ; Kumar, S. R. and Chandrasekaran, M. 2002. Continuous production of extracellular glutaminase by Ca-alginate immobilized marine Beauveria bassiana BTMFS-10 in packed – bed reactor. Appl. Biochem.Biotechnol.102–103(1-6).
Belgoudi, J.1999. Polyethylene glycol – bovine serum hydrogel as a matrix for enzyme immobilization. In vitro biochemical characterization. J. Bioactive & Compatible Polymer. 14 (1) : 31 – 53.
Toren, A. ; Landau, l. ; Kushmaro, A. ; Loya, Y. and Rosenberg, G. 1998. Effect of temperature on adhesion of Vibrio strain Ak-1 to Oculina patagonica and on coral bleaching. Appl. Environ. Microbiol. 64 (4) : 1379 – 1384.
Cortassa, S.; Sun, H.; Kernevez, P. and Thomas, D. 1990. Pattern formation in an immobilized bienzyme system. J. Biochem. 269: 115 – 122.
Madara, M. B.; Spokane, R. B.; Johnson, J. M. and Woolward, J. R. 1997. Glutamine biosensors for biotechnology applications with suppression signal. Anal. Chem. 69 (18): 3674 – 3678.