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Comparative Structural Changes and Inactivation Kinetics of Pectin Methylesterases from Different Orange Cultivars Processed by High Pressure

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Abstract

Pectin methylesterases (PMEs) from two different orange sources, Valencia and Navel cv., were extracted and purified using cation exchange, heparin chromatography, and finally, gel filtration chromatography, yielding a single peak corresponding to a protein of molecular weight 34 and 35 kDa for Valencia and Navel PMEs, respectively. Effects of high pressure (HP) and thermal processing for various treatment durations on the activity of PMEs in Tris–HCl buffer solution (pH 7.5) were explored. Higher levels of pressure, temperature, and treatment duration resulted in an analogous reduction of the PME residual activity. HP-induced inactivation of both purified PMEs was described by a first-order kinetic model. Kinetic parameters were estimated and a multiparameter equation was developed to predict the PME inactivation rate constant at any combination of pressure and temperature conditions for both enzymes. The PME from Valencia orange peel appeared to be more heat- and pressure-sensitive compared to Navel PME. HP-induced conformational changes of the PME molecules were also investigated using circular dichroism (CD) spectroscopy. A direct comparison of the CD results for treated and untreated proteins reveals that pressure treatment has negligible effects upon far-UV CD spectra, while significant irreversible changes are depicted in near UV for both PMEs. It is, thus, evidenced that exposure to HP may lead to a structurally molten globulelike state, where the PME maintains a secondary structure of untreated protein molecules, while a tertiary structure is substantially affected bearing subsequent impact on substrate–enzyme binding interaction, leading to reduction of enzyme activity.

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References

  • Balan, A., Santa-Cruz, C. P., Moutran, A., Ferreira, R. C. C., Medrano, F. J., Pérez, C. A., et al. (2006). The molybdate-binding protein (ModA) of the phytopathogen Xanthomonas axonopodis pv citri. Protein Expression and Purification, 50(2), 215–222.

    Article  CAS  Google Scholar 

  • Balogh, T., Smout, C., Ly Nguyen, B., Van Loey, A., & Hendrickx, M. (2004). Thermal and high pressure inactivation kinetics of carrot pectinmethylesterase (PME): From model systems to real foods. Innovative Food Science & Emerging Technologies, 5(4), 429–436.

    Article  CAS  Google Scholar 

  • Barbosa-Cánovas, G. V. (1999). Preservation of foods with pulsed electric fields. London: Academic.

    Google Scholar 

  • Böhm, G., Muhr, R., & Jaenicke, R. (1992). Quantitative analysis of protein far UV circular dichroism spectra by neural networks. Protein Engineering, 5(3), 191–195.

    Article  Google Scholar 

  • Boulekou, S., Katsaros, G., & Taoukis, P. (2010). Inactivation kinetics of peach pulp pectin methylesterase as a function of high hydrostatic pressure and temperature process conditions. Food and Bioprocess Technology, 3(5), 699–706.

    Article  CAS  Google Scholar 

  • Bradford, M. (1976). A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein dye binding. Analytical Biochemistry, 72, 248–254.

    Article  CAS  Google Scholar 

  • Cameron, R. G., Baker, R. A., & Grohmann, K. (1996). Citrus tissue extracts affect juice cloud stability. Journal of Food Science, 62, 242–245.

    Article  Google Scholar 

  • Cameron, G. R., Savary, B. J., Hotchkiss, A. T., & Fishman, M. L. (2005). Isolation, characterization and pectin-modifying properties of a thermally tolerant pectin methylesterase from Citrus sinensis var. Valencia. Journal of Agricultural and Food Chemistry, 53(6), 2255–2260.

    Article  CAS  Google Scholar 

  • Castro, S., Saraiva, J., Lopes-da-Silva, J., Delgadillo, I., Van Loey, A., Smout, C., et al. (2008). Effect of thermal blanching and of high pressure treatments on sweet green and red bell pepper fruits (Capsicum annuum L.). Food Chemistry, 107(4), 1436–1449.

    Article  CAS  Google Scholar 

  • Castro, A. J., Barbosa-Cánovas, G. V., & Swanson, B. G. (1993). Microbial inactivation of foods by pulsed electric fields. Journal of Food Processing and Preservation, 17(1), 47–73.

    Article  Google Scholar 

  • Chang, C. T., Wu, C.-S. C., & Yang, J. T. (1978). Circular dichroic analysis of protein conformation: Inclusion of the β-turns. Analytical Biochemistry, 91(1), 13–31.

    Article  CAS  Google Scholar 

  • Eagerman, B. A., & Rouse, A. H. (1976). Heat inactivation temperature–time relationships for pectin esterase inactivation in citrus juices. Journal of Food Science, 41(6), 1396–1397.

    Article  CAS  Google Scholar 

  • Foguel, D., & Silva, J. L. (1994). Cold denaturation of a repressor–operator complex: The role of entropy in protein-DNA recognition. Proceedings of the National Academy of Sciences, USA, 91(17), 8244–8247.

    Article  CAS  Google Scholar 

  • Gervilla, R., Ferragut, V., & Guamis, B. (2000). High pressure inactivation of microorganisms inoculated into ovine milk of different fat contents. Journal of Dairy Science, 83(4), 674–682.

    Article  CAS  Google Scholar 

  • Goto, Y., & Fink, A. L. (1989). Conformational states of beta-lactamase: Molten-globule states at acidic and alkaline pH with high salt. Biochemistry, 28(3), 945–952.

    Article  CAS  Google Scholar 

  • Guiavarc'h, Y., Segovia, O., Hendrickx, M., & Van Loey, A. (2005). Purification, characterization, thermal and high-pressure inactivation of a pectin methylesterase from white grapefruit (Citrus paradisi). Innovative Food Science and Emerging Technologies, 6(4), 363–371.

    Article  Google Scholar 

  • Hendrickx, M., Ludikhuyze, L., Van den Broeck, I., & Weemaes, C. (1998). Effects of high pressure on enzymes related to food quality.Trends of. Food Science and Technology, 9(5), 107–203.

    Article  Google Scholar 

  • Hendrickx M & Knorr D (2002) Ultra high pressure treatment of foods. Kluwer, New York, Aspen food engineering series.

  • Heremans, K. (1982). High pressure effects on proteins and other biomolecules. Annual Review of Biophysics and Bioengineering, 11, 1–21.

    Article  CAS  Google Scholar 

  • Hummer, G., Garde, S., Garcia, A. E., Paulaitis, M. E., & Pratt, L. R. (1998). The pressure dependence of hydrophobic interactions is consistent with the observed pressure denaturation of proteins. Proceedings of the National Academy of Sciences of the United States of America, 95(4), 1552–1555.

    Article  CAS  Google Scholar 

  • Johansson, K., El Ahmad, M., Friemann, R., Jornvall, H., Markovic, O., & Eklund, H. (2002). Crystal structure of plant pectin methylesterase. FEBS Letters, 514(2–3), 243–249.

    Article  CAS  Google Scholar 

  • Katsaros, G., Katapodis, P., & Taoukis, P. (2009a). High hydrostatic pressure inactivation kinetics of the plant proteases ficin and papain. Journal of Food Engineering, 91(1), 42–48.

    Article  CAS  Google Scholar 

  • Katsaros, G., Giannoglou, M., & Taoukis, P. (2009b). Kinetic study of the combined effect of high hydrostatic pressure and temperature on the activity of Lactobacillus delbrueckii ssp. bulgaricus aminopeptidases. Journal of Food Science, 74(5), 219–225.

    Article  Google Scholar 

  • Katsaros, G., Tsevdou, M., Panagiotou, T., & Taoukis, P. (2010a). Kinetic study of high pressure microbial and enzyme inactivation and selection of pasteurization conditions. International Journal of Food Science and Technology, 45(6), 1119–1129.

    Article  CAS  Google Scholar 

  • Katsaros, G., Tavantzis, G., & Taoukis, P. (2010b). Production of novel dairy products using actinidin and high pressure as enzyme activity regulator. Innovative Food Science and Emerging Technologies, 11(1), 47–51.

    Article  CAS  Google Scholar 

  • Laratta, B., Masi, L. D., Minasi, P., & Giovane, A. (2008). Pectin methylesterase in Citrus bergamia R. Purification, biochemical characterization and sequence of the exon related to the enzyme active site. Food Chemistry, 110(4), 829–837.

    Article  CAS  Google Scholar 

  • Ly-Nguyen, B., Van Loey, A. M., Smout, C., ErenOzcan, S., Fachin, D., Verlent, I., & Hendrickx, M. E. (2003a). Mild heat and high-pressure inactivation of carrot pectinmethylesterase: A kinetic study. Journal of Food Science, 68(4), 1377–1383.

    Google Scholar 

  • Ly-Nguyen, B., Loey, A. M. V., Smout, C., Verlent, I., Duvetter, T., & Hendrickx, M. E. (2003b). Effect of mild-heat and high-pressure processing on banana pectin methylesterase: A kinetic study. Journal of Agricultural and Food Chemistry, 51(27), 7974–7979.

    Article  CAS  Google Scholar 

  • Menéndez, O. H., Schwarzenbolz, U. R., & Henle, T. (2006). Structural changes of microbial transglutaminase during thermal and high-pressure treatment. Journal of Agricultural and Food Chemistry, 54(5), 1716–1721.

    Article  Google Scholar 

  • Messens, W., Camp, J. V., & Huyghebaert, A. (1997). The use of high pressure to modify the functionality of food proteins. Trends in Food Science and Technology, 8(4), 107–112.

    Article  CAS  Google Scholar 

  • Mozhaev, V., Heremans, K., Frank, J., Masson, P., & Balny, C. (1994). Exploiting the effects of high hydrostatic pressure in biotechnological applications. Trends in Biotechnology, 12(12), 493–501.

    Article  CAS  Google Scholar 

  • Mozhaev, V., Heremans, K., Frank, J., Masson, P., & Balny, C. (1996). High pressure effects on protein structure and function. Proteins: Structure,Function and Bionformatics, 24(1), 81–91.

    Article  CAS  Google Scholar 

  • Nienaber, U., & Shellhammer, T. H. (2001). High-pressure processing of orange juice: Kinetics of pectinmethylesterase inactivation. Journal of Food Science, 66(2), 328–331.

    Article  CAS  Google Scholar 

  • Polydera, A., Galanou, E., Stoforos, N., & Taoukis, P. (2004). Inactivation kinetics of pectin methylesterase of Greek Navel orange juice as a function of high hydrostatic pressure and temperature process conditions. Journal of Food Engineering, 62(3), 291–298.

    Article  Google Scholar 

  • Ramos, C. H. I. (2004). A spectroscopic-based laboratory course for protein conformational studies. Biochemistry and Molecular Biology Education, 32(1), 31–34.

    Article  CAS  Google Scholar 

  • Ramos, C. H. I., Kay, M. S., & Baldwin, R. L. (1999). Putative interhelix ion pairs involved in the stability of myoglobin. Biochemistry, 38(30), 9783–9790.

    Article  CAS  Google Scholar 

  • Ramos, C. H. I., Weisbuch, S., & Jamin, M. (2007). Diffusive motions control the folding and unfolding kinetics of apomyoglobin pH 4 molten globule intermediate. Biochemistry, 46(14), 4379–4389.

    Article  CAS  Google Scholar 

  • Ravindra, R., & Winter, R. (2003). On the temperature-pressure free-energy landscape of proteins. Chem PhysChem, 4(4), 359–365.

    Article  CAS  Google Scholar 

  • Regis, W. C. B., Fattori, J., Santoro, M. M., Jamin, M., & Ramos, C. H. I. (2005). On the difference in stability between horse and sperm whale myoglobins. Archives of Biochemistry and Biophysics, 436(1), 168–177.

    Article  CAS  Google Scholar 

  • Ribeiro-Jr, E. A., & Ramos, C. H. I. (2004). Origin of the anomalous circular dichroism spectra of many apomyoglobin mutants. Analytical Biochemistry, 329(2), 300–306.

    Article  Google Scholar 

  • Rombouts, F. M., Versteeg, C., Karman, A. H., & Pilnik, W. (1982). Pectinesterases in component parts of citrus fruits related to problems of cloud loss and gelation in citrus products. In P. Dupuy (Ed.), Use of enzymes in food technology (pp. 483–487). Paris, France: Technique et documentation Lavoisier.

    Google Scholar 

  • Rouse, A. H., & Atkins, C. D. (1955). Pectinesterase and pectin in commercial orange juice as determined by methods used at the Citrus Experiment Station. Bulletin of the University of Florida Agricultural Experiment Station, 570, 1–19.

    Google Scholar 

  • Sampedro, F., Rodrigo, D., & Hendrickx, M. (2008). Inactivation kinetics of pectin methyl esterase under combined thermal–high pressure treatment in an orange juice–milk beverage. Journal of Food Engineering, 86(1), 133–139.

    Article  CAS  Google Scholar 

  • Savary, B. J., Vasu, P., Nuñez, A., & Cameron, R. G. (2010). Identification of thermolabile pectin methylesterases from sweet orange fruit by peptide mass fingerprinting. Journal of Agricultural and Food Chemistry, 58, 12462–12468.

    Article  CAS  Google Scholar 

  • Sila, D., Smout, C., Satara, Y., Truong, V., Van Loey, A., & Hendrickx, M. (2007). Combined thermal and high pressure effect on carrot pectinmethylesterase stability and catalytic activity. Journal of Food Engineering, 78(3), 755–764.

    Article  CAS  Google Scholar 

  • Tedford, L. A., Smith, D., & Schaschke, C. J. (1999). High pressure processing effects on the molecular structure of ovalbumin, lysozyme and β-lactoglobulin. Food Research International, 32(2), 101–106.

    Article  CAS  Google Scholar 

  • Thanassoulas, A., Nomikos, M., Theodoridou, M., Stavros, P., Mastellos, D., & Nounesis, G. (2011). Thermal and chemical denaturation of the BRCT functional module of human 53BP1. International Journal of Biological Macromolecules, 49(3), 297–304.

    Article  CAS  Google Scholar 

  • Tiwari, B. K., Muthukumarappan, K., O'Donnell, C. P., & Cullen, P. J. (2009). Inactivation kinetics of pectin methylesterase and cloud retention in sonicated orange juice. Innovative Food Science and Emerging Technologies, 10(2), 166–171.

    Article  CAS  Google Scholar 

  • Van den Broeck, I., Ludikhuyze, L. R., Van Loey, A. M., & Hendrickx, M. (2000a). Effect of temperature and/or pressure on tomato pectinesterase activity. Journal of Agricultural and Food Chemistry, 48(2), 551–558.

    Article  Google Scholar 

  • Van den Broeck, I., Ludikhuyze, L. R., Van Loey, A. M., & Hendrickx, M. E. (2000b). Inactivation of orange pectinesterase by combined high-pressure and -temperature treatments: A kinetic study. Journal of Agricultural and Food Chemistry, 48(5), 1960–1970.

    Article  Google Scholar 

  • Versteeg, C., Rombouts, F. M., & Pilnik, W. (1978). Purification and some characteristics of two pectinesterase isoenzymes from orange. Lebensmittel-Wissenschaft und Technologie, 11, 267–274.

    CAS  Google Scholar 

  • Vidugiris, G. J. A., & Royer, C. A. (1998). Determination of the volume changes for the pressure-induced transitions of apomyoglobin between the native, molten globule and unfolded states. Biophysical Journal, 75(1), 463–470.

    Article  CAS  Google Scholar 

  • Woody, R. W. (1995). Circular dichroism. Methods in Enzymology, 246, 34–71.

    Article  CAS  Google Scholar 

  • Zhong, K., Wu, J., Wang, Z., Chen, F., Liao, X., & Hu, X. (2007). Inactivation kinetics and secondary structural change of PEF treated POD and PPO. Food Chemistry, 100(1), 115–123.

    Article  CAS  Google Scholar 

  • Zhou, L., Wu, J., Hu, X., Zhi, X., & Liao, X. (2009). Alterations in the activity and structure of pectin methylesterase treated by high pressure carbon dioxide. Journal of Agricultural and Food Chemistry, 57(5), 1890–1895.

    Article  CAS  Google Scholar 

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Acknowledgments

The authors would like to thank Professor P. Christakopoulos and Lecturer E. Topakas for offering their expert advice and experience on enzyme purification assessment. This research has been cofinanced by the European Union (European Social Fund—ESF) and Greek national funds through the Operational Program “Education and Lifelong Learning” of the National Strategic Reference Framework (NSRF)—Research Funding Program: Heracleitus II. Investing in knowledge society through the European Social Fund.

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Authors and Affiliations

  1. Laboratory of Food Chemistry and Technology, School of Chemical Engineering, National Technical University of Athens, Zografou, 15780, Greece

    Z. Alexandrakis, G. Katsaros & P. Taoukis

  2. Biomolecular Physics Laboratory, National Centre for Scientific Research “Demokritos”, 15310, Aghia Paraskevi, Greece

    P. Stavros & G. Nounesis

  3. Biotechnology Laboratory, Department of Biological Applications and Technologies, University of Ioannina, Ioannina, Greece

    P. Katapodis

  4. 5, Iroon Polytechniou Str. Zografou, 15780, Athens, Greece

    P. Taoukis

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  1. Z. Alexandrakis
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  4. P. Katapodis
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Correspondence to P. Taoukis.

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Alexandrakis, Z., Katsaros, G., Stavros, P. et al. Comparative Structural Changes and Inactivation Kinetics of Pectin Methylesterases from Different Orange Cultivars Processed by High Pressure. Food Bioprocess Technol 7, 853–867 (2014). https://doi.org/10.1007/s11947-013-1087-7

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