Temperature and pH Dependent Deactivation of Cutinases from Thermobifida fusca : A Comparative Study of Homologous Enzymes


Thermostability of two homologous cutinases, Cut1 and Cut2 from Thermobifida fusca NRRL B-8184 was inves-tigated at combination of different pH and temperature in the range of pH 6 - 9 and temperature 45 - 80, re-spectively. The deactivation rate constants, the half-life and thermodynamic parameters, viz., H*, S*, G* and activation energy kinetics of inactivation of the cutinases were assessed at different combinations of pH and temperature and compared. The optimal pH and temperature for the least degree of deactivation for Cut1 and Cut2 were found to be 8℃ and 45℃, respectively. The deactivation process was found to be faster at pH 6 and 9, with minimum deactivation at pH 8 for both the cutinases. It was found that S* values are negative for both the enzymes and △H* value of Cut2 was 1.5 fold higher than that of Cut1 in the range of pH studied. Cut2 was found to be thermodynamically more stable with 1.7 fold higher deactivation energy at pH 6 and 7 and 1.4 fold higher deactivation energy at pH 8 and 9 in comparison to Cut1.

Share and Cite:

Hegde, K. and Veeranki, V. (2014) Temperature and pH Dependent Deactivation of Cutinases from Thermobifida fusca : A Comparative Study of Homologous Enzymes. Modern Research in Catalysis, 3, 128-135. doi: 10.4236/mrc.2014.34016.

Conflicts of Interest

The authors declare no conflicts of interest.


[1] Chitnis, A. and Sadana, A. (1989) pH-Dependent Enzyme Deactivation Models. Biotechnology and Bioengineering, 34, 804-818. http://dx.doi.org/10.1002/bit.260340610
[2] Sadana, A. (1995) Biocatalysis: Fundamentals of Deactivation Kinetics. Prentice-Hall, Englewood Cliffs.
[3] Joly, M (1965) Physico-Chemical Approach to the Denaturation of Proteins. Academic Press, New York.
[4] Purdy, R.E. and Kolattukudy, P.E. (1975) Hydrolysis of Plant Cuticle by Plant Pathogens—Purification, Amino-Acid Composition, and Molecular-Weight of 2 Isoenzymes of Cutinase and a Nonspecific Esterase from Fusarium solani f. pisi. Biochemistry, 14, 2824-2831. http://dx.doi.org/10.1021/bi00684a006
[5] Araujo, R., Silva, C., O’Neill, A., Micaelo, N., Guebitz, G., Soares, C.M., Casal, M. and Cavaco-Paulo, A. (2007) Tailoring Cutinase Activity towards Polyethylene Terephthalate and Polyamide 6.6 Fibers. Journal of Biotechnology, 128, 849-857. http://dx.doi.org/10.1016/j.jbiotec.2006.12.028
[6] Ribitsch, D., Yebra, A.O., Zitzenbacher, S., Wu, J., Nowitsch, S., Steinkellner, G., Greimel, K., Doliska, A., Oberdorfer, G., Gruber, C.C., Gruber, K., Schwab, H., Stana-Kleinschek, K., Acero, E.H. and Guebitz, G.M. (2013) Fusion of Binding Domains to Thermobifida cellulosilytica Cutinase to Tune Sorption Characteristics and Enhancing PET Hydrolysis. Biomacromolecules, 14, 1769-1776. http://dx.doi.org/10.1021/bm400140u
[7] Kim, Y.H., Ahn, J.Y., Moon, S.H. and Lee, J. (2005) Biodegradation and Detoxification of Organophosphate Insecticide, Malathion by Fusarium oxysporum f. sp. pisi Cutinase. Chemosphere, 60, 1349-1355.
[8] Dutta, K. and Dasu, V.V. (2011) Synthesis of Short Chain Alkyl Esters Using Cutinase from Burkholderia cepacia NRRL B 2320. Journal of Molecular Catalysis B: Enzymatic, 72, 150-156.
[9] Dutta, K., Sen, S. and Veeranki, V.D. (2009) Production, Characterization and Applications of Microbial Cutinases. Process Biochemistry, 44, 127-134. http://dx.doi.org/10.1016/j.procbio.2008.09.008
[10] Egmond, M.R. and Vlieg, J.D. (2000) Fusarium solani pisi Cutinase. Biochimie, 82, 1015-1021.
[11] Fett, W.F., Gerard, H.C., Moreau, R.A., Osman, S.F. and Jones, L.E. (1992) Screening of Nonfilamentous Bacteria for Production of Cutin-Degrading Enzymes. Applied and Environmental Microbiology, 58, 2123-2130.
[12] Hegde, K. and Veeranki, V.D. (2013) Production Optimization and Characterization of Recombinant Cutinases from Thermobifida fusca sp. NRRL B-8184. Applied Biochemistry and Biotechnology, 170, 654-675.
[13] Dutta, K., Krishnamoorthy, H. and Dasu, V.V. (2013) Novel Cutinase from Pseudomonas cepacia NRRL B 2320: Purification, Characterization and Identification of Cutinase Encoding Genes. The Journal of General and Applied Microbiology, 59, 171-184.
[14] Maeda, H., Yamagata, Y., Abe, K., Hasegawa, F., Machida, M., Ishioka, R., Gomi, K. and Nakajima, T. (2005) Purification and Characterization of a Biodegradable Plastic-Degrading Enzyme from Aspergillus oryzae. Applied Microbiology and Biotechnology, 67, 778-788. http://dx.doi.org/10.1007/s00253-004-1853-6
[15] Skamnioti, P., Furlong, R.F. and Gurr, S.J. (2008) Evolutionary History of the Ancient Cutinase Family in Five Filamentous Ascomycetes Reveals Differential Gene Duplications and Losses and in Magnaporthe grisea Shows Evidence of Suband Neo-Functionalization. New Phytologist, 180, 711-721.
[16] Bellamy, W.D. (1977) Cellulose and Lignocellulose Digestion by Thermophilic Actinomycetes for Single Cell Protein Production. Developments in Industrial Microbiology, 18, 249-254.
[17] Degani, O., Gepstein, S. and Dosoretz, C.G. (2002) Potential Use of Cutinase in Enzymatic Scouring of Cotton Fiber Cuticle. Applied Biochemistry and Biotechnology, 102, 277-289.
[18] Hegde, K. and Veeranki, V.D. (2014) Structural Stability and Unfolding Properties of Cutinases from Thermobifida fusca. Applied Biochemistry and Biotechnology, 174, 803-819.
[19] Eyring, H. and Stearn, A.E. (1939) The Application of the Theory of Absolute Reaction Rates to Proteins. Chemical Review, 24, 253-270. http://dx.doi.org/10.1155/S1110724301000249
[20] Kapat, A. and Panda, T. (1997) pH and Thermal Stability Studies of Chitinase from Trichoderma harzianum: A Thermodynamic Consideration. Bioprocess Engineering, 16, 269-272. http://dx.doi.org/10.1007/s004490050321
[21] Petersen, S.B., Fojan, P., Petersen, E.I. and Petersen, M. (2001) The Thermal Stability of the Fusarium solani pisi Cutinase as a Function of pH. Journal of Biomedicine and Biotechnology, 1, 62-69.
[22] Relkin, P. (1996) Thermal Unfolding of β-Lactoglobulin, α-Lactalbumin, and Bovine Serum Albumin: A Thermodynamic Approach. International Journal of Food Sciences and Nutrition, 36, 556-601.
[23] Daniel, R.M. (1996) The Upper Limits of Enzyme Thermal Stability. Enzyme and Microbial Technology, 19, 74-79.
[24] Eisenberg, H., Mevarech, M. and Zaccai, G. (1992) Biochemical, Structural, and Molecular Genetic Aspects of Halophilism. Advances in Protein Chemistry, 43, 1-62. http://dx.doi.org/10.1016/S0065-3233(08)60553-7
[25] Gohel, V. and Naseby, D.C. (2007) Thermalstabilization of Chitinolytic Enzymes of Pantoea dispersa. Biochemical Engineering Journal, 35, 150-157. http://dx.doi.org/10.1016/j.bej.2007.01.009
[26] D’Amico, S., Marx, J.C., Gerday, C. and Feller, G. (2003) Activity-Stability Relationships in Extremophilic Enzymes. Journal of Molecular Biology, 278, 7891-7896.
[27] Ternstrom, T., Svendsen, A., Akke, M. and Adlercreutz, P. (2005) Unfolding and Inactivation of Cutinases by AOT and Guanidine Hydrochloride. Biochimica et Biophysica Acta (BBA), Proteins and Proteomics, 1748, 74-83.
[28] Loladze, V.V., Ibarra-Molero, B., Sanchez-Ruiz, J.M. and Makhatadze, G.I. (1999) Engineering a Thermostable Protein via Optimization of Charge-Charge Interactions on the Protein Surface. Biochemistry, 38, 16419-16423.
[29] Declerck, N., Machius, M., Joyet, P., Wiegand, G., Huber, R. and Gaillardin, C. (2002) Hyperthermostabilization of Bacillus licheniformis α-Amylase and Modulation of Its Stability over a 50?C Temperature Range. Protein Engineering, Design and Selection, 16, 287-293.
[30] Gummadi, S.N. (2003) What Is the Role of Thermodynamics on Protein Stability? Biotechnology and Bioprocess Engineering, 8, 9-18. http://dx.doi.org/10.1007/BF02932892
[31] Foster, R.L. (1980) Modification of Enzyme Activity. Croom Helm, London.
[32] Voordouw, G., Milo, C. and Roche, R.S. (1976) Role of Bound Calcium Ions in Thermostable, Proteolytic Enzymes. Separation of Intrinsic and Calcium Ion Contributions to the Kinetic Thermal Stability. Biochemistry, 15, 3716-3724.

Copyright © 2021 by authors and Scientific Research Publishing Inc.

Creative Commons License

This work and the related PDF file are licensed under a Creative Commons Attribution 4.0 International License.