Effect of Calcium on Immobilization of Rice (Oryza sativa L.) Peroxidase for Bioassays in Sodium Alginate and Agarose Gel

다국어 초록 (Multilingual Abstract)

The direct immobilization of soluble peroxidase isolated and partially purified from shoots of rice seedlings in calcium algi-nate beads and in calcium agarose gel was carried out. Peroxidase was assayed for guaiacol oxidation products in pres-ence of hydrogen peroxide. The maximum specific activity and immobilization yield of the calcium agarose immobilized per-oxidase reached 2,200 U mg-¹ protein (540 mU cm-³ gel) and 82%, respectively. In calcium alginate the maximum activity of peroxidase upon immobilization was 210 mU g-¹bead with 46% yield. The optimal pH for agarose immobilized peroxidase was 7.0 which differed from the pH 6.0 for soluble peroxidase. The optimum temperature for the agarose immobilized per-oxidase however was 30°C, which was similar to that of soluble peroxidase. The thermal stability of calcium agarose immo-bilized peroxidase significantly enhanced over a temperature range of 30~60°C upon immobilization. The operational stabil-ity of peroxidase was examined with repeated hydrogen peroxide oxidation at varying time intervals. Based on 50% conver-sion of hydrogen peroxide and four times reuse of immobilized gel, the specific degradation of guaiacol for the agarose im-mobilized peroxidase increased three folds compared to that of soluble peroxidase. Nearly 165% increase in the enzyme protein binding to agarose in presence of calcium was noted. The results suggest that the presence of calcium, ions help in the immobilization process of peroxidase from rice shoots and mediates the direct binding of the enzyme to the agarose gel and that agarose seems to be a better immobilization matrix for peroxidase compared to sodium alginate.

참고문헌 (Reference)

1 Dulik, D.M., "Use of Immobilized enzymes in drug metabolism studies" 2 : 2235-2240, 1988

2 Weetall, H.H., "Urease covalently coupled to porous glass" 185 : 464-465, 1969

3 Moynihan, H. J., "Urea hydrolysis by immobilized urease in a fixed-bed reactor: Analysis and kinetic parameter estimation" 34 : 951-963, 1989

4 Haogland, D.R., "The water culture method for growing plants without soil" 3 : 346-347, 1938

5 Ginalska, G., "The mode of solidification (immobilization) of plant peroxidases in salt stress as possible recognizing of the changes in their active sites" 17 (17): 1999

6 Brzyska, M., "The effect of simultaneous operation of two metal ions on soluble and immobilized peroxidase" 68 : 101-109, 1997

7 Khan, A. A., "Simultaneous purification and immobilization of mushroom tyrosinase on an immunoaffinity support" 40 : 2379-2386, 2005

8 Akhtar, S., "Simultaneous purification and immobilization of bitter gourd (Momordica charantia) peroxidases on bioaffinity support" 80 : 198-205, 2005

9 Egley, G. H., "Role of peroxidase in the development of water-impermeable seed coats in Sida spinosa L" 157 : 224-232, 1983

10 Fernandez-Lafuente, R., "Reducing enzyme conformational flexibility by multipoint covalent immobilization" 9 : 1-6, 1995

11 Lowry, O. H., "Protein measurement with the Folin phenol reagent" 193 : 265-275, 1951

12 Torres, E., "Potential use of oxidative enzymes for the detoxification of organic pollutants" 46 : 1-15, 2003

13 Shah, K., "Plant peroxidases – a brief note on response to metal pollutants ; In Advances in Plant Physiology(Molecular Plant Physiology and Biology)" Scientific Publishers 113-122, 2005

14 Nam-Soon Oh, "Phytate Degradation by ImmobilizedSaccharomyces cerevisiae Phytase inSoybean-curd Whey" 한국생물공학회 12 (12): 348-353, 2007

15 Prakash, O., "Physicochemical characterization of watermelon urease upon immobilization in alginate beads" 14 : 209-213, 2005

16 Yoshida, K., "Molecular biology and application of plant peroxidase genes" 60 : 665-670, 2003

17 Bickerstaff, G.F., "Impact of genetic technology on enzyme technology" 15 : 13-30, 1995

18 Tischer, W., "Immobilized enzymes: crystals or carriers" 17 : 326-335, 1999

19 Torchilin, V. P., "Immobilized Enzymes in Medicine" Springer-Verlag 1991

20 Reddy, K. R. C., "Immobilization of pigeonpea (Cajanus cajan) urease on DEAE-cellulose paper strips for urea estimation" 39 : 323-327, 2004

21 Fernandes, K. F., "Immobilization of horseradish peroxidase onto polyaniline polymers" 38 : 1379-1384, 2003

22 Om Prakash, "Immobilization of Watermelon (Citrullus vulgaris) Urease in Agarose Gel for Urea Estimation" 한국생물공학회 12 (12): 131-135, 2007

23 Quan, C. S., "Immobilization of Candida krusei cells producing phytase in alginate gel beads: an application of the preparation of myoinositol phosphatases" 62 : 41-47, 2003

24 Bhunia, A., "Horseradish peroxidase catalyzed degradation of industrially important dyes" 72 : 562-567, 2001

25 Srere, P.A., "Functional groups on enzymes suitable for binding of matrices" 44 : 11-19, 1976

26 Fraser, J.E., "Entrapment in calcium alginate ; In Immobilization of Enzymes and Cells" Humana Press 61-66, 1997

27 Shah, K., "Effect of cadmium on lipid peroxidation, superoxide anion generation and activities of antioxidant enzymes in growing rice seedlings" 161 : 1135-1144, 2001

28 Kulshrestha, Y., "Direct immobilization of peroxidase on DEAE cellulose from ammonium sulphate fractionated proteins of bitter gourd (Momordica charantia)" 38 : 470-477, 2006

29 Husain, Q., "Detoxification of phenols and aromatic amines from polluted wastewater by using phenol oxidases" 59 : 286-293, 2000

30 Taylor, R.F., "Commercially available supports for protein immobilization ; In Protein Immobilization" Marcel Dekker 139-160, 1991

31 Liang, J. F., "Biomedical application of immobilized enzymes" 89 : 979-990, 2000

32 Kulshrestha, Y., "Bioaffinity-based an inexpensive and high yield procedure for the immobilization of turnip (Brassica rapa) peroxidase" 38 : 470-477, 2003

33 Duran, N., "Applications of laccases and tyrosinases (phenoloxidases) immobilized on different supports: a review" 31 : 907-931, 2002