Deactivation Kinetics and Response Surface Analysis of the Stability of α-l-Rhamnosidase from Penicillium decumbens
- 173 Downloads
The stability of the mixed enzyme preparation Naringinase from Penicillium decumbens was studied in dependence of the temperature, the pH value, and the enzyme concentration by means of response surface methodology. Deactivation kinetics by formation of an intermediate state was proposed for fitting deactivation data. Empirical models could then be constructed for prediction of deactivation rate constants, specific activity of intermediate state, and half-life values under different incubation conditions. From this study, it can be concluded that (1) Naringinase is most stable in the pH range of 4.5–5.0, being quite sensitive to lower pHs (<3.5) and (2) the glyco-enzyme is a rather thermo-stable enzyme preserving its initial activity for long times when incubated at its optimal pH up to temperatures of 65 °C. Enriched α-l-rhamnosidase after column treatment and ultrafiltration presented similar deactivation kinetics pattern and half-life values as the unpurified enzyme. Thus, any influence of low molecular weight substances on its deactivation is most probably negligible. The intermediate state of the enzyme may correspond to unfolding and self-digestion of its carbohydrate portion, lowering its activity relative to the initial state. The digestion- and unfolding-grade of this intermediate state may also be controlled by the pH and temperature of incubation.
KeywordsNaringinase Doehlert array Response surface methodology Series-type deactivation kinetics α-Rhamnosidase Enzyme stability
The authors would like to thank the financial support of this project, carried out in the framework of a EU-CRAFT project (1999-72243) entitled “Integrated process for bio-surfactant synthesis at competitive cost allowing for their application in household cleaning and bio-remediation” (InBioSynAp). We also thank Rebecca Lorenz for her helpful practical input during the research project.
- 1.Dunlap, W. J., Hagen, R. E., & Wender, S. H. (1962). Journal of Food Science, 27(6), 597.Google Scholar
- 10.Meiwess, J., Wullbrant, D., & Giani, C. (1994) EP0599159.Google Scholar
- 13.Manzanares, P., de Graaff, L. H., & Visser, J. (1997). FEMS Microbiology Letters, 157(2), 279–283.Google Scholar
- 17.Gabor, F., & Pittner, F. (1984). Hoppe-Seylers Zeitschrift Fur Physiologische Chemie, 365(9), 914–914.Google Scholar
- 22.Biselli, M., Krugl, U., & Wandrey, C. (1995). In K. Drauz, & H. Waldman (Eds.), Enzyme catalysis in organic synthesis—a comprehensive handbook, Vol. 1 (pp. 89–155). Weinheim: VCH.Google Scholar
- 24.Bisswanger, H. (1999). Enzymkinetik: Theorie und methoden (3rd ed.). Weinheim: Wiley-VCH.Google Scholar
- 25.Sadana, A. (1991). Biocatalysis: Fundamentals of enzyme deactivation kinetics. New Jersey: Prentice Hall.Google Scholar
- 27.Aktinson, C. (1992). Optimum experimental designs. Oxford: Clarendon.Google Scholar
- 28.Box, G. E. P., Hunter, W. G., & Hunter, J. S. (1978). Statistic for experimenters: An introduction to design, data analysis and model building. New York: Wiley.Google Scholar
- 29.Khuri, A. I., & Cornell, J. A. (1987). Response surfaces, design and analyses. New York: Marcel Dekker.Google Scholar
- 30.Rasch, D., Verdooren, L. R., & Gowers, J. I. (1999). Grundlagen der Planung und Auswertung von Versuchen und Erhebungen, R. Oldenbourg Verlag, München, WienGoogle Scholar
- 32.Oliveros, E., et al. (2000). In Proceedings of the third Asia pacific conference (pp. 577–581). Singapore, Work Scientific.Google Scholar
- 34.NEMRODW LPRAI, B.P. no. 7, Marseille - Le Merlan, 13311 Marseille Cedex 14, France. Retrieved from www.nemrodw.com.
- 35.Scopes, R. (1994). Protein purification, principles and practice (3rd ed.). New York: Springer.Google Scholar
- 37.Greco, G., et al. (1992). In Stability and stabilization of enzymes (Proceedings of an International Symposium) (pp. 429–435). Maastricht: Elsevier Science.Google Scholar
- 38.Prazeres, D. M. F., Garcia, F. A. P., & Cabral, J.M. S. (1992). In Stability and stabilization of enzymes (Proceedings of an International Symposium) (pp. 445–450). Maastricht: Elsevier Science.Google Scholar