Advertisement

European Food Research and Technology

, Volume 234, Issue 4, pp 655–662 | Cite as

Preparation of immobilized Trametes pubescens laccase on a cryogel-type polymeric carrier and application of the biocatalyst to apple juice phenolic compounds oxidation

  • Michaela Dina Stanescu
  • Simona Gavrilas
  • Roland Ludwig
  • Dietmar Haltrich
  • Vladimir I. Lozinsky
Original Paper

Abstract

A new biocatalyst was prepared by the immobilization of a Trametes pubescens laccase, into a wide-pore poly(vinyl alcohol) cryogel. The known enzyme was produced and purified by the previously described procedure. The resulted laccase (yield 40%) has an activity of 46.4 U mg−1 and 12.51 mg mL−1 protein content. The enzyme was subsequently immobilized in a functionalized macroporous cryogel beads by a covalent immobilization technique. The time dependence of the immobilization process and the enzyme loading of the carrier material (5.2 mg g−1 cryogel) were determined by measuring the decrease of protein amount in the enzyme solution. In conversion experiments, a higher stability of the immobilized biocatalyst compared to the free enzyme was evidenced. Steady-state kinetic characterization of four phenols (catechol, caffeic and chlorogenic acids, and catechin) has been performed with free and immobilized laccase, the catalytic parameters being determined and compared. The effect of both laccases (free and immobilized) on the phenol content of retailed apple juice samples, having the same initial composition, was also investigated by working in batch conversion. The variation in phenolic compound content has been compared with that of an untreated apple juice sample having initially the same content of phenolic compounds. A number of advantages resulted in using the immobilized laccase for the apple juice treatment (conservation to some extent of enzyme activity, higher content of phenols preserved, easy separation of the enzyme from the apple juice, therefore avoiding the possible unhealthy effects due to the remaining protein, etc.).

Keywords

Immobilized laccase Poly(vinyl alcohol) cryogel carrier Oxidation kinetics of phenolic compounds Apple juice treatment 

Notes

Acknowledgments

The authors thank European Science Foundation for the financial support of GS for a STMS at Boku University through COST 928 Programme “Control and Exploitation of Enzymes for Added-Value Food Products” as well as to the Romanian Academy (grant 1R-2008), and the Russian Foundation for Basic Research (Project # 07-03-91682_RA_a).

References

  1. 1.
    Zika E, Papatryfon I, Wolf O, Gomez-Barbero M, Stein AJ, Bock A-K (2007) Consequence, opportunities and challenges of modern biotechnology, JRC reference report EUR 22728 EN. Office for Official Publications of the European Communities, LuxembourgGoogle Scholar
  2. 2.
    Mukherjee M, Das N (2009) Fungal laccase: a biotechnologically potential enzyme. In: Mishra CSK, Champagne P (eds) Biotechnology applications. IK International Publishing House Pvt Ltd, New Delhi, pp 70–108Google Scholar
  3. 3.
    Kunamneni A, Ballesteros A, Plou FJ, Alcalde M (2007) Fungal laccase—a versatile, enzyme for biotechnological applications. In: Mendez-Vilas A (ed) Communicating current research and educational topics and trends in applied microbiology. Formatex, Zurbaran, pp 233–245Google Scholar
  4. 4.
    Shraddaha RV, Sehgal S, Kamthania M, Kumar A (2011) Laccase: microbial sources, production, purification, and potential biotechnological applications. Enzyme Res. doi: 10.4061/2011/217861 Google Scholar
  5. 5.
    Maciel MJ, Castro e Silva A, Ribeiro HCT (2010) Industrial and biotechnological applications of ligninolytic enzymes of the basidiomycota: a review. Electron J Biotechnol 13(6) http://dx.doi.org/. doi: 10.2225/vol13-issue6-fulltext-2
  6. 6.
    Brijwani K, Rigdon A, Vadlani PV (2010) Fungal laccases: production, function, and applications in food processing. Enzyme Res. doi: 10.4061/2010/149748 Google Scholar
  7. 7.
    Fernandes P (2010) Enzymes in food processing: a condensed overview on strategies for better biocatalysts. Enzyme Res. doi: 10.4061/2010/862537 Google Scholar
  8. 8.
    Couto SR, Toca Herrera JL (2006) Industrial and biotechnological applications of laccases: a review. Biotechnol Adv 24:500–513CrossRefGoogle Scholar
  9. 9.
    Minussi RC, Pastore GM, Duran N (2002) Potential applications of laccase in the food industry. Trends Food Sci Technol 13:205–216CrossRefGoogle Scholar
  10. 10.
    Osma JF, Toca-Herrera JL, Rodrıguez-Couto S (2010) Uses of laccases in the food industry. Enzyme Res. doi: 10.4061/2010/918761 Google Scholar
  11. 11.
    Dwivedi UN, Singh P, Pandey VP, Kumar A (2011) Structure–function relationship among bacterial, fungal and plant laccases. J Mol Catal B-Enzym 68:111–128CrossRefGoogle Scholar
  12. 12.
    Chiacchierini E, Restuccia D, Vinci G (2004) Bioremediation of food industry effluents: recent applications of free and immobilised polyphenoloxidases. Food Sci Technol Int 10:373–382CrossRefGoogle Scholar
  13. 13.
    Madhavi V, Lele SS (2009) Laccase properties, use. BioResources 4:1694–1717Google Scholar
  14. 14.
    Gil DMA, Rebelo MJF (2010) Evaluating the antioxidant capacity of wines: a laccase based biosensor approach. Eur Food Res Technol 231:303–308CrossRefGoogle Scholar
  15. 15.
    Duran N, Rosa MA, D’Annibale A, Gianfreda L (2002) Applications of laccases and tyrosinases (phenoloxidases) immobilized on different supports: a review. Enzyme Microb Technol 31:907–931CrossRefGoogle Scholar
  16. 16.
    Makas GY, Kalkan NA, Aksoy S, Altinok H, Hasirci N (2010) Immobilization of laccase in k-carrageenan based semi-interpenetrating polymer networks. J Biotechnol 148:216–220CrossRefGoogle Scholar
  17. 17.
    Piacquadio P, De Stefano G, Sammartino M, Sciancalepore V (1997) Phenols removal from apple juice by laccase immobilized on Cu2+-chelate regenerable carrier. Biotechn Techn 11:515–517CrossRefGoogle Scholar
  18. 18.
    Schroeder M, Pöllinger-Zierler B, Aichernig N, Siegmund B, Guebitz GM (2008) Enzymatic removal of off-flavors from apple juice. J Agric Food Chem 56:2485–2489CrossRefGoogle Scholar
  19. 19.
    Cliffe S, Fawer MS, Maier G, Takata K, Ritter G (2004) Enzyme assays for the phenolic content of natural juices. J Agric Food Chem 42:1824–1828CrossRefGoogle Scholar
  20. 20.
    Solomon EI, Sundaram UM, Machonkin TE (1996) Multicopper oxidases and oxygenases. Chem Rev 96:2563–2605CrossRefGoogle Scholar
  21. 21.
    Desai SS, Nityanand C (2011) Microbial laccases and their applications: a review. Asian J Biotech 3(2):98–124CrossRefGoogle Scholar
  22. 22.
    Morozova OV, Shumakovich GP, Gorbacheva MA, Shleev SV, Yaropolov AI (2007) “Blue” laccases. Biochemistry (Moscow) 72:1136–1150CrossRefGoogle Scholar
  23. 23.
    Cheynier V (2005) Polyphenols in foods are more complex than often thought. Am J Clin Nutr 81(sppl):223S–229SGoogle Scholar
  24. 24.
    Pokorny J (2003) Introduction. In: Pokorny J, Yanishlieva N, Gordon MH (eds) Antioxidants in food: practical applications. Woodhead Publishing Ltd., Boca Raton, pp 311–330Google Scholar
  25. 25.
    Alcade M (2007) Laccase: biological functions, molecular structure and industrial applications. In: Polaina J, MacCabe AP (eds) Industrial enzymes structure, function and application. Springer, Dordrecht, pp 461–477Google Scholar
  26. 26.
    Galhaup C, Wagner H, Hinterstoisser B, Haltrich D (2002) Increased production of laccase by the wood-degrading basidiomycete Trametes pubescens. Enzyme Microb Technol 30:529–536CrossRefGoogle Scholar
  27. 27.
    Lozinsky VI (2008) New generation of macroporous and supermacroporous materials of biotechnological interest—polymeric cryogels. Russ Chem Bull 57:1015–1032CrossRefGoogle Scholar
  28. 28.
    Amaki K, Saito E, Taniguchi K, Joshita K, Murata M (2007) Role of chlorogenic acid quinone and of chlorogenic acid quinine and catechins in the enzymatic browning of apple. Biosci Biotechnol Biochem 75(5):829–832CrossRefGoogle Scholar
  29. 29.
    Peng Y, Liu F, Peng Yu, Ye J (2005) Determination of polyphenols in apple juice and cider by capillary electrophoresis with electrochemical detection. Food Chem 92:169–175CrossRefGoogle Scholar
  30. 30.
    Van der Sluis AA, Dekker M, de Jager A, Jongen WMF (2001) Activity and concentration of polyphenolic antioxidants in apple; effect of cultivar; harvest year and storage conditions. J Agric Food Chem 49:3606–3613CrossRefGoogle Scholar
  31. 31.
    Bourbonnais R, Paice MG, Reid ID, Lanthier P, Yaguchi M (1995) Lignin Oxidation by Laccase Isozymes from Trametes versicolor and role of the mediator 2,2′-Azinobis(3-Ethylbenzthiazoline-6-Sulfonate) in Kraft Lignin depolymerization. Appl Environ Microbiol 61:1876–1880Google Scholar
  32. 32.
    Bradford MM (1976) A dye binding assay for protein. Anal Biochem 72:248–254CrossRefGoogle Scholar
  33. 33.
    Bacheva AV, Plieva FM, Lysogorskaya E, Filippova IYu, Lozinsky VI (2001) Peptide synthesis in organic media with subtilisin 72 immobilized on Poly(vinyl alcohol)-Cryogel carrier. Bioorg Med Chem Lett 11:1005–1008CrossRefGoogle Scholar
  34. 34.
    Stanescu MD, Fogorasi M, Shaskolskiy BL, Gavrilas S, Lozinsky VI (2010) New potential biocatalysts by laccase immobilization in PVA cryogel type carrier. Appl Biochem Biotechnol 60:1947–1954CrossRefGoogle Scholar
  35. 35.
    Khatiwora E, Adsul VB, Kulkarni MM, Deshpande NR, Kashalkar RV (2010) Spectroscopic determination of total phenol and flavonoid contents of Ipomoea carnea. Int J Chem Tech Res 2:1698–1701Google Scholar
  36. 36.
    Zhishen J, Mengcheng T, Jianming W (1999) The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chem 64:555–559CrossRefGoogle Scholar
  37. 37.
    Ianculov I, Modoran D (1997) Characterization of polyphenols from vegetables. Agroprint, TimişoaraGoogle Scholar
  38. 38.
    Lozinsky VI, Damshkaln LG (2001) Study of cryostructuration of polymer systems. XX. Foamed poly(vinyl alcohol) cryogels. J Appl Polym Sci 82:1609–1619CrossRefGoogle Scholar
  39. 39.
    Baur X (2005) Enzymes as occupational and environmental respiratory sensitisers. Int Arch Occup Environ Health 78(4):279–286CrossRefGoogle Scholar
  40. 40.
    Vandenplas O (2011) Occupational asthma: etiologies and risk factors. Allergy Asthma Immunol Res 3(3):157–167CrossRefGoogle Scholar
  41. 41.
    Gerhauser C (2008) Cancer chemiopreventive potential of apples, apple juice and apple components. Planta Med 74:1608–1624CrossRefGoogle Scholar
  42. 42.
    Akiyama H, Sakushima J-I, Taniuchi S, Kanda T, Yanagida A, Kojima T, Teshima R, Kobayashi Y, Goda Y, Toyoda M (2000) Antiallergic effect of apple polyphenols on the allergic model mouse. Biol Pharm Bull 23:1370–1373CrossRefGoogle Scholar
  43. 43.
    Miura D, Miura Y, Yagasaki K (2007) Effect of apple polyphenol extract on hepatoma proliferation and invasion in culture and on tumor growth, metastasis, and abnormal lipoprotein profiles in hepatoma-bearing rats. Biosci Biotechnol Biochem 71(11):2743–2750CrossRefGoogle Scholar
  44. 44.
    Giovanelli G, Ravasini G (1993) Apple juice stabilization by combined enzyme-membrane filtration process. LWT Food Sci Technol 26:1–7CrossRefGoogle Scholar
  45. 45.
    Will F, Zessner H, Becker H, Dietrich H (2007) Semi-preparative isolation and physico-chemical characterization of 4-coumaroylquinic acid and phloretin-20-xyloglucoside from laccase-oxidized apple juice. LWT Food Sci Technol 40:1344–1351CrossRefGoogle Scholar
  46. 46.
    Nicotra S, Cramarossa MR, Mucci A, Pagnoni UM, Riva S, Forti L (2004) Biotransformation of resveratrol: synthesis of trans-dehydrodimers catalyzed by laccasse from Mycelyopthora thermophyla and from Trametes pubescens. Tetrahedron 60:595–600CrossRefGoogle Scholar
  47. 47.
    Aktas N, Sahiner N, Kantoglu O, Salih B, Tanyolac A (2003) Biosynthesis and characterization of laccase catalyzed Poly(Catechol). J Polym Environ 11(3):123–128CrossRefGoogle Scholar
  48. 48.
    De Stefano G, Piacquadio P, Sciancalepore V (1996) Metal-chelate regenerable carriers in food processing. Biotechn Techn 10:857–860CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2012

Authors and Affiliations

  • Michaela Dina Stanescu
    • 1
  • Simona Gavrilas
    • 1
  • Roland Ludwig
    • 2
  • Dietmar Haltrich
    • 2
  • Vladimir I. Lozinsky
    • 3
  1. 1.Department of Chemical and Biological SciencesAurel Vlaicu UniversityAradRomania
  2. 2.Food Biotechnology Laboratory, Department of Food Sciences and TechnologyBOKU-University of Natural Resources and Applied Life SciencesWienAustria
  3. 3.A.N. Nesmeyanov Institute of Organoelement CompoundsRussian Academy of SciencesMoscowRussian Federation

Personalised recommendations