Immobilization Imparts Stability to Watermelon Urease to Work in Water Miscible Organic Media

  • Prakash, Om (Department of Biochemistry Faculty of Science, Banaras Hindu University) ;
  • Upadhyay, Lata Sheo Bachan (Department of Biochemistry Faculty of Science, Banaras Hindu University)
  • Published : 2006.04.30

Abstract

The behaviour of alginate immobilized and soluble watermelon (Citrullus vulgaris) urease in water miscible organic solvents like, acetonitrile, dimethylformamide (DMF), ethanol, methanol, and propanol is described. The organic solvents exhibited a concentration dependent inhibitory effect on both the immobilized and the soluble urease in the presence of urea. Pretreatment of soluble enzyme preparations with organic solvents in the absence of substrate for 10 min at $30^{\circ}C$ led to rapid loss in the activity, while similar pretreatment of immobilized urease with 50% (v/v) of ethanol, propanol, and acetonitrile was ineffective. Time-dependent inactivation of immobilized urease, both in the presence and in the absence of urea, revealed stability for longer duration of time even at very high concentration of organic solvents. The soluble enzyme, on the other hand, was rapidly inactivated even at fairly lower concentrations. The results suggest that the immobilization of watermelon urease in calcium alginate make it suitable for its application in organic media. The observations are discussed.

Keywords

References

  1. Zaks, A. and A. M. Klibanov (1984) Enzymatic catalysis in organic media at 100$^{\circ}$C. Science 224: 1249-1251 https://doi.org/10.1126/science.6729453
  2. Griebenow, K. and A. M. Klibanov (1996) On protein denaturation in aqueous-organic mixtures but not in pure organic solvents. J. Am. Chem. Soc. 118: 11695-11700 https://doi.org/10.1021/ja961869d
  3. Schmitke, J. L., L. J. Stern, and A. M. Klibanov (1997) The crystal structure of subtilisin Carlsberg in anhydrous dioxane and its comparison with those in water and acetonitrile. Proc. Natl. Acad. Sci. USA 94: 4250-4255
  4. Zhu, G., Q. Huang, Z. Wang, M. Qian, Y. Jia, and Y. Tang (1998) X-ray studies on two forms of bovine $\beta$-trypsin crystals in neat cyclohexane. Biochim. Biophys. Acta 1429: 142-150 https://doi.org/10.1016/S0167-4838(98)00226-X
  5. Zaks, A. and D. R. Dodds (1997) Application of biocatalysis and biotransformations to the synthesis of pharmaceuticals. Drug Discov. Today 2: 513-531 https://doi.org/10.1016/S1359-6446(97)01078-7
  6. Luisi, P. L. and C. Laane (1986) Solubilization of enzymes in apolar solvents via reverse micelles. Trends Biotechnol. 4: 153-161 https://doi.org/10.1016/0167-7799(86)90166-6
  7. Klibanov, A. M. (1989) Enzymatic catalysis in anhydrous organic solvents. Trends Biochem. Sci. 14: 141-144 https://doi.org/10.1016/0968-0004(89)90146-1
  8. Zaks, A. and A. M. Klibanov (1988) Enzymatic catalysis in nonaqueous solvents. J. Biol. Chem. 263: 3194-3201
  9. Carrea, G., G. Ottolina, and S. Riva (1995) Role of solvents in the control of enzyme selectivity in organic media. Trends Biotechnol. 13: 63-70 https://doi.org/10.1016/S0167-7799(00)88907-6
  10. Okahata, Y., M. Yamaguchi, F. Tanaka, and I. Fujii (1995) A lipid-coated catalytic antibody in water-miscible organic solvents. Tetrahedron 51: 7673-7680 https://doi.org/10.1016/0040-4020(95)00407-Y
  11. Tanaka, K. and Y. Okahata (1996) A DNA-lipid complex in organic media and formation of an aligned cast film. J. Am. Chem. Soc. 118: 10679-10683 https://doi.org/10.1021/ja9617855
  12. Carrea, G. and S. Riva (2000) Properties and synthetic applications of enzymes in organic solvents. Angew. Chem. Int. Ed. Engl. 39: 2226-2254 https://doi.org/10.1002/1521-3773(20000703)39:13<2226::AID-ANIE2226>3.0.CO;2-L
  13. Prakash, O. and G. Bhushan (1997) Isolation, purification, and partial characterization of urease from seeds of water melon (Citrullus vulgaris). J. Plant Biochem. Biotechnol. 6: 45-47 https://doi.org/10.1007/BF03263009
  14. Prakash, O. and G. Bhushan (1998) A study of inhibition of urease from seeds of the water melon (Citrullus vulgaris). J. Enzym. Inhib. 13: 69-77 https://doi.org/10.3109/14756369809035828
  15. Prakash, O. and G. Bhushan (1998) Molecular asymmetry in urease of water melon (Citrullus vulgaris). J. Plant Biochem. Biotechnol. 7: 53-55 https://doi.org/10.1007/BF03263035
  16. Blakeley, R. L. and B. Zerner (1984) Jack bean urease: the first nickel enzyme. J. Mol. Catal. 23: 263-292 https://doi.org/10.1016/0304-5102(84)80014-0
  17. Prakash, O. and L. S. B. Upadhyay (2003) Effect of thiols on the activity of urease from dehusked seeds of watermelon (Citrullus vulgaris). Plant Sci. 164: 189-194 https://doi.org/10.1016/S0168-9452(02)00382-5
  18. Prakash, O. and L. S. B. Upadhyay (2005) Physicochemical characterization of watermelon urease upon immobilization in alginate beads. J. Plant Biochem. Biotechnol. 14: 209-213 https://doi.org/10.1007/BF03263249
  19. Lowry, O. H., N. J. Rosebrough, A. L. Farr, and R. J. Randall (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193: 265-275
  20. Janda, K. D., J. A. Ashley, T. M. Jones, D. A. McLeod, D. M. Schloeder, and M. I. Weinhouse (1990) Immobilized catalytic antibodies in aqueous and organic solvents. J. Am. Chem. Soc. 112: 8886-8888 https://doi.org/10.1021/ja00180a035
  21. Anupma (1997) Immobilization and Chemical Modification of Proteases. Ph.D. Thesis. Banaras Hindu University, Varanasi, India
  22. Adlercreutz, P. (1991) On the importance of the support material for enzymatic synthesis in organic media. Support effects at controlled water activity. Eur. J. Biochem. 199: 609-614 https://doi.org/10.1111/j.1432-1033.1991.tb16161.x
  23. Khmelnitsky, Y. L., S. H. Welch, D. S. Clark, and J. S. Dordick (1994) Salts dramatically enhance activity of enzymes suspended in organic solvents. J. Am. Chem. Soc. 116: 2647-2648 https://doi.org/10.1021/ja00085a066
  24. Clark, D. S. and J. E. Bailey (1983) Structure-function relationships in immobilized chymotrypsin catalysis. Biotechnol. Bioeng. 25: 1027-1047 https://doi.org/10.1002/bit.260250412
  25. Clark, D. S. and J. E. Bailey (1984) Characterization of heterogeneous immobilized enzyme subpopulations using EPR spectroscopy. Biotechnol. Bioeng. 26: 231-238 https://doi.org/10.1002/bit.260260306
  26. Skerker, P. S. and D. S. Clark (1988) Catalytic properties and active-site structural features of immobilized horse liver alcohol dehydrogenase. Biotechnol. Bioeng. 32: 148- 158 https://doi.org/10.1002/bit.260320205