Springer Nature is making Coronavirus research free. View research | View latest news | Sign up for updates

Carbon-oxygen bond formation by fungal laccases: cross-coupling of 2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide with the solvents water, methanol, and other alcohols

  • 248 Accesses

  • 19 Citations


Laccase-catalyzed reactions lead to oxidation of the substrate via a cation radical, which has been described to undergo proton addition to form a quinonoid derivative or nucleophilic attack by itself producing homomolecular dimers. In this study, for the substrate 2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide, we show that, besides the quinonoid form of substrate, all products formed are nonhomomolecular ones. Indeed, without addition of a reaction partner, heteromolecular products are formed from the quinonoid form of the laccase-substrate and the solvents water or methanol present in the incubation assay. Consequently, in laccase catalyzed syntheses performed in aqueous solutions or in the presence of methanol or other alcohols, undesirable heteromolecular coupling reactions between the laccase substrate and solvents must be taken into account. Additionally, it could be shown at the example of methanol and other alcohols that C-O-bound cross-coupling of dihydroxylated aromatic substances with the hydroxyl group of aliphatic alcohols can be catalyzed by fungal laccases.

This is a preview of subscription content, log in to check access.

Fig. 1
Fig. 2
Fig. 3
Fig. 4


  1. Aktas N, Tanyolac A (2003) Kinetics of Laccase-catalyzed oxidative polymerization of catechol. J Mol Catal B-Enzym 22:61–69

  2. Amster IJ (1996) Fourier-transform mass spectrometry. J Mass Spectrom 31:1325–1337

  3. Balakshin MY, Chen CL, Gratzl JS, Kirkman AG, Jakob H (2001) Biobleaching of pulp with dioxygen in the laccase-mediator system. Holzforschung 54:390–396

  4. Bialk HM, Simpson AJ, Pedersen JA (2005) Cross-coupling of sulfonamide antimicrobial agents with model humic constituents. Environ Sci Technol 39:4463–4473

  5. Bollag JM (1992a) Decontaminating soil with enzymes. Environ Sci Technol 26:1876–1881

  6. Bollag JM (1992b) Enzymes catalyzing oxidative coupling reactions of pollutants. In: Sigel H, Sigel A (eds) Metal ions in biological systems. Marcel Dekker, New York, pp 205–217

  7. Bollag JM, Bollag WB (1990) A model for enzymatic binding of pollutants in the soil. Int J Environ Anal Chem 39:147–157

  8. Bourbonnais R, Paice MG (1990) Oxidation of non-phenolic substrates—an expanded role for laccase in lignin biodegradation. FEBS Lett 267:99–102

  9. 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 61:1876–1880

  10. Braun-Lüllemann A, Majcherczyk A, Huttermann A (1997) Degradation of styrene by white-rot fungi. Appl Microbiol Biotechnol 47:150–155

  11. Brown BR (1967) Biochemical aspects of oxidative coupling of phenols. In: Tylor WJ, Battersby AR (eds) Oxidative coupling of phenols. Marcel Dekker, New York, pp 167–201

  12. Burton SG (2003) Laccases and phenol oxidases in organic synthesis—a review. Curr Org Chem 7:1317–1331

  13. Chakar FS, Ragauskas AJ (2004) Biobleaching chemistry of laccase-mediator systems on high-lignin-content kraft pulps. Can J Chem-Rev Can Chim 82:344–352

  14. Chung JE, Kurisawa M, Uyama H, Kobayashi S (2003) Enzymatic synthesis and antioxidant property of gelatin-catechin conjugates. Biotechnol Lett 25:1993–1997

  15. Ciecholewski S, Hammer E, Manda K, Bose G, Nguyen VTH, Langer P, Schauer F (2005) Laccase-catalyzed carbon–carbon bond formation: oxidative dimerization of salicylic esters by air in aqueous solution. Tetrahedron 61:4615–4619

  16. Duran N, Rosa M, D’Annibale A, Gianfreda L (2002) Applications of laccases and tyrosinases (phenoloxidases) immobilized on different supports: a review. Enzyme Microb Technol 31:907–931

  17. Eggert C, LaFayette PR, Temp U, Eriksson KEL, Dean JFD (1998) Molecular analysis of a laccase gene from the white rot fungus Pycnoporus cinnabarinus. Appl Environ Microbiol 64:1766–1772

  18. Figueroa-Espinoza MC, Morel MH, Rouau X (1998) Effect of lysine, tyrosine, cysteine, and glutathione on the oxidative cross-linking of feruloylated arabinoxylans by a fungal laccase. J Agr Food Chem 46:2583–2589

  19. Gianfreda L, Xu F, Bollag JM (1999) Laccases: a useful group of oxidoreductive enzymes. Bioremediation J 3:1–25

  20. Heinzkill M, Bech L, Halkier T, Schneider P, Anke T (1998) Characterization of laccases and peroxidases from wood-rotting fungi (family Coprinaceae). Appl Environ Microbiol 64:1601–1606

  21. Ikeda R, Uyama H, Kobayashi S (1996) Novel synthetic pathway to a poly(phenylene oxide). Laccase-catalyzed oxidative polymerization of syringic acid. Macromolecules 29:3053–3054

  22. Ikeda R, Tsujimoto T, Tanaka H, Oyabu H, Uyama H, Kobayashi S (2000) Man-made urushi preparation of crosslinked polymeric films from renewable resources via air-oxidation processes. Proc Japan Acad 76(B):155–160

  23. Ikeda R, Tanaka H, Uyama H, Kobayashi S (2001) A new crosslinking method of vinyl polymers having a phenol moiety via oxidative coupling. Polym J 33:959–961

  24. Jonas U, Hammer E, Schauer F, Bollag JM (1998) Transformation of 2-hydroxy dibenzofuran by laccases of the white rot fungi Trametes versicolor and Pycnoporus cinnabarinus and characterization of oligomerisation products. Biodegradation 8:321–328

  25. Katase T, Bollag JM (1991) Transformation of trans-4-hydroxycinnamic acid by a laccase of the fungus Trametesversicolor: its significance in humification. Soil Sci 151:291–296

  26. Kobayashi S, Uyama H, Ikeda R (2001) Artificial urushi. Chemistry 7:4754–4760

  27. Kurisawa M, Chung JE, Uyama H, Kobayashi S (2003) Enzymatic synthesis and antioxidant properties of poly(rutin). Biomacromolecules 4:1394–1399

  28. Kurniawati S, Nicell JA (2005) Kinetic model of laccase-catalyzed oxidation of aqueous phenol. Biotechnol Bioeng 91:114–123

  29. Leonowicz A, Edgehill RU, Bollag JM (1984) The effect of pH on the transformation of syringic and vanillic acids by the laccases of Rhizoctonia praticola and Trametes versicolor. Arch Microbiol 137:89–96

  30. Liu SY, Minard RD, Bollag JM (1981) Coupling reactions of 2,4-dichlorophenol with various anilines. J Agr Food Chem 29:253–257

  31. Mai C, Schormann W, Hüttermann A (2001) Chemo-enzymatically induced copolymerization of phenolics with acrylate compounds. Appl Microbiol Biotechnol 55:177–186

  32. Manda K, Hammer E, Mikolasch A, Niedermeyer T, Dec J, Jones AD, Benesi AJ, Schauer F, Bollag JM (2005) Laccase-induced cross-coupling of 4-aminobenzoic acid with para-dihydroxylated compounds 2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide and 2,5-dihydroxybenzoic acid methyl ester. J Mol Catal B-Enzym 35:86–92

  33. Manda K, Hammer E, Mikolasch A, Gördes D, Thurow K, Schauer F (2006) Laccase-induced derivatization of unprotected amino acid l-tryptophan by coupling with p-hydroquinone 2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide. Amino Acids 31:409–419

  34. Marshall AG, Schweikhard L (1992) Fourier-transform ion-cyclotron Resonance mass-spectrometry—technique developments. Int J Mass Spectrom Ion Proc 118:37–70

  35. Marshall AG, Hendrickson CL, Jackson GS (1998) Fourier transform ion cyclotron resonance mass spectrometry: a primer. Mass Spectrom Rev 17:1–35

  36. Maruyama T, Komatsu C, Michizoe J, Ichinose H, Goto M (2006) Laccase-mediated oxidative degradation of the herbicide dymron. Biotechnol Prog 22:426–430

  37. Mayer AM, Staples CA (2002) Laccase: new functions for an old enzyme. Phytochem 60:551–565

  38. Mikolasch A, Hammer E, Jonas U, Popowski K, Stielow A, Schauer F (2002) Synthesis of 3-(3,4-dihydroxyphenyl)-propionic acid derivatives by N-coupling of amines using laccase. Tetrahedron 58:7589–7593

  39. Mikolasch A, Niedermeyer T, Lalk M, Witt S, Seefeldt S, Hammer E, Schauer F, Gesell M, Hessel S, Jülich WD, Lindequist U (2006) Penicillins synthesized by biotransformation using laccase from Trametes spec. Chem Pharm Bull 54:632–638

  40. Minussi RC, Pastore GM, Duran N (2002) Potential applications of laccase in the industry. Trends Food Sci Technol 13:205–216

  41. Nicotra S, Cramarossa MR, Mucci A, Pagnoni UM, Riva S, Forti L (2004) Biotransformation of resveratrol: synthesis of trans-dehydrodimers catalyzed by laccases from Myceliophtora thermophyla and from Trametes pubescens. Tetrahedron 60:595–600

  42. Niedermeyer THJ, Mikolasch A, Lalk M (2005) Nuclear amination catalyzed by fungal laccases: reaction products of p-hydroquinones and primary aromatic amines. J Org Chem 70:2002–2008

  43. Pilz R, Hammer E, Schauer F, Kragl U (2003) Laccase-catalysed synthesis of coupling products of phenolic substrates in different reactors. Appl Microbiol Biotechnol 60:708–712

  44. Potthast A, Rosenau T, Chen CL, Gratzl JS (1996) A novel method for the conversation of benzyl alcohols to benzaldehydes by laccase-catalyzed oxidation. J Mol Catal A-Chem 108:5–9

  45. Riva S (2006) Laccases: blue enzymes for green chemistry. Trends Biotechnol 24:219–226

  46. Schäfer A, Specht M, Hetzheim A, Francke W, Schauer F (2001) Synthesis of substituted imidazoles and dimerization products using cells and laccase from Trametes versicolor. Tetrahedron 57:7693–7699

  47. Schultz A, Jonas U, Hammer E, Schauer F (2001) Dehalogenation of chlorinated hydroxybiphenyls by fungal laccase. Appl Environ Microbiol 67:4377–4381

  48. Simmons KE, Minard RD, Bollag JM (1989) Oxidative co-oligomerization of guaiacol and 4-chloroaniline. Environ Sci Technol 23:115–121

  49. Tatsumi K, Wada S, Ichikawa H, Liu SY, Bollag JM (1992) Cross-coupling of a chloroaniline and phenolic-acids catalyzed by a fungal enzyme. Wat Sci Technol 26:2157–2160

  50. Tatsumi K, Freyer A, Minard RD, Bollag JM (1994) Enzymatic coupling of chloroanilines with syringic acid, vanillic acid and protocatechuic acid. Soil Biol Biochem 26:735–742

  51. Thurston CF (1994) The structure and function of fungal laccase. Microbiol 140:19–26

  52. Tominaga J, Michizoe J, Kamiya N, Ichinose H, Maruyama T, Goto M (2004) Factors affecting the oxidative activity of laccase towards biphenyl derivatives in homogeneous aqueous-organic systems. J Biosci Bioeng 98:14–19

  53. Uchida H, Fukuda T, Miyamoto H, Kawabata T, Suzuki M, Uwajima T (2001) Polymerization of bisphenol A by purified laccase from Trametes villosa. Biochem Biophys Res Commun 287:355–358

  54. Vianello F, Ragusa S, Cambria MT, Rigo AA (2006) High sensitivity amperometric biosensor using laccase as biorecognition element. Biosens Bioelectron 21:2155–2160

  55. Wang CJ, Thiele S, Bollag JM (2002) Interaction of 2,4,6-trinitrotoluene (TNT) and 4-amino-2,6-dinitrotoluene with humic monomers in the presence of oxidative enzymes. Arch Environ Contam Toxicol 42:1–8

  56. Wells A, Teria M, Eve T (2006) Green oxidations with laccase-mediator systems. Biochem Soc Trans 34:304–308

  57. Xu F (1999) Laccase. In: Fleckinger MC, Drew SW (eds) Encyclopedia of bioprocess technolog.: ferm., biocatalysis, and bioseparation. Wiley, New York, pp 1545–1554

  58. Yaropolov AI, Skorobogatko OV, Vartanov SS, Varfolomeyev SD (1994) Laccase—properties, catalytic mechanism, and applicability. Appl Biochem Biotechnol 49:257–280

Download references


Financial support from the government of Mecklenburg-Vorpommern, Germany, and from the European Union (Landesforschungsschwerpunkt “Neue Wirkstoffe und Biomaterialien—Innovative Screeningverfahren and Produktentwicklung”) is gratefully acknowledged.

Author information

Correspondence to Katrin Manda.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Manda, K., Gördes, D., Mikolasch, A. et al. Carbon-oxygen bond formation by fungal laccases: cross-coupling of 2,5-dihydroxy-N-(2-hydroxyethyl)-benzamide with the solvents water, methanol, and other alcohols. Appl Microbiol Biotechnol 76, 407–416 (2007). https://doi.org/10.1007/s00253-007-1024-7

Download citation


  • Laccase
  • Homomolecular product
  • Cross-coupling
  • Alcohols
  • C-O-bound