Journal of Biosciences

, Volume 31, Issue 3, pp 355–362 | Cite as

The effect of some osmolytes on the activity and stability of mushroom tyrosinase

  • N. Gheibi
  • A. A. Saboury
  • K. Haghbeen
  • A. A. Moosavi-Movahedi


The thermodynamical stability and remained activity of mushroom tyrosinase (MT) fromAgaricus bisporus in 10 mM phosphate buffer, pH 6.8, stored at two temperatures of 4 and 40°C were investigated in the presence of three different amino acids (His, Phe and Asp) and also trehalose as osmolytes, for comparing with the results obtained in the absence of any additive. Kinetics of inactivation obeye the first order law. Inactivation rate constant (kinact) value is the best parameter describing effect of osmolytes on kinetic stability of the enzyme. Trehalose and His have the smallest value of kinact(0.7×10−4s−1) in comparison with their absence (2.5×10−4s−1). Moreover, to obtain effect of these four osmolytes on thermodynamical stability of the enzyme, protein denaturation by dodecyl trimethylammonium bromide (DTAB) and thermal scanning was investigated. Sigmoidal denaturation curves were analysed according to the two states model of Pace theory to find the Gibbs free energy change of denaturation process in aqueous solution at room temperature, as a very good thermodynamic criterion indicating stability of the protein. Although His, Phe and Asp induced constriction of MT tertiary structure, its secondary structure had not any change and the result was a chemical and thermal stabilization of MT. The enzyme shows a proper coincidence of thermodyanamic and structural changes with the presence of trehalose. Thus, among the four osmolytes, trehalose is an exceptional protein stabilizer.


Amino acids denaturation mushroom tyrosinase osmolyte stability, trehalose 

Abbreviations used


Aspartic acid


caffeic acid


dodecyl trimethylammonium bromide His, histidine


meanamino acid residue weight


mushroom tyrosinase


phosphate buffer solution; Phe, phenylalanine


protein melting point




Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.


  1. Ajlouni S O, Beelman R B and Thompson D B 1993 Influence of gamma radiation on quality characteristics, sugar content, and respiration rate of mushrooms during postharvest storage; inFood flavors, ingredients and composition (ed.) G Charalambous (Elsevier Science Publishers:Dev. Food Sci. 32 103–121)Google Scholar
  2. Almagor A, Yedgar S and Gavish B 1992 Viscous cosolvent effect on the ultrasonic absorption of bovine serum albumin;Biophys. J. 61 480–486PubMedGoogle Scholar
  3. Arakawa T and Timasheff S N 1983 Preferential interactions of proteins with solvent components in aqueous amino acid solutions;Arch. Biochem. Biophys. 224 169–177PubMedCrossRefGoogle Scholar
  4. Back J F, Oakenfull D and Smith M B 1978 Increased thermal stability of proteins in presence of sugar;Biochemistry 18 5191–5196CrossRefGoogle Scholar
  5. Bolen D W and Baskakvo I V 2001 The Osmophobic effect: Natural Selection of a Thermodynamic Force in Protein Folding;J. Mol. Biol. 310 955–963PubMedCrossRefGoogle Scholar
  6. Brown A D and Simpson J R 1972 Water relations of sugar-tolerant yeasts: the role of intracellular polyols;J. Gen. Microbiol. 72 589–591PubMedGoogle Scholar
  7. Carnici P, Nishiyama Y, Westover A, Itoh M, Nagaoka S, Sasaki N, Okazaki Y, Muramatsu M and Hayashizaki Y 1998 Thermo-stabilization and thermoactivation of thermolabile enzymes by trehalose and its application for the synthesis of full length cDNA;Proc. Natl. Acad. Sci. USA 95 520–524CrossRefGoogle Scholar
  8. Colaco C, Sen S, Tangavelu M, Pinder S and Roser B 1992 Extraordinary stability of enzymes dried in trehalose: simplified molecular biology;Biotechnology 10 1007–1011.PubMedCrossRefGoogle Scholar
  9. Crowe J, Crowe L and Chapman D 1984 Preservation of membranes in anhydrobiotic organisms. The role of trehalose;Science 223 209–217CrossRefGoogle Scholar
  10. Elbein A D, Pan Y T, Pastuszak I and Carroll D 2003 New insights on trehalose: a multifunctional molecule;Glycobiology 13 17R-27RPubMedCrossRefGoogle Scholar
  11. Evansand D R and Dethier V G 1957 The regulation of taste thresholds for sugars in the blowfly;J. Insect Physiol 1 3–17CrossRefGoogle Scholar
  12. Fan-Guo M, Yong-Doo P and Hai-Meng Z 2001 Role of proline, glycerol, and heparin as protein folding aids during refolding of rabbit muscle creatine kinase;Int. J. Biochem. Cell Biol. 33 701–709CrossRefGoogle Scholar
  13. Gheibi N, Saboury A A, Mansuri-Torshizi H, Haghbeen K and Moosavi-Movahedi A A 2005 The inhibition effect of some n-alkyl dithiocarbamates on mushroom tyrosinase;J. Enz. Inh. Med. Chem. 20 393–399CrossRefGoogle Scholar
  14. Gheibi N, Saboury A A, Haghbeen K and Moosavi-Movahedi A A 2005 Activity and structural changes of mushroom tyrosinase induced by n-alkyl sulfates;Colloids Surf. B: Biointerfaces 45 104–107CrossRefGoogle Scholar
  15. Gonnelli M and Strambini G B 1993 Glycerol effects on protein flexibility: a tryptophan phosphorescence study;Biophys. J. 65 131–137PubMedGoogle Scholar
  16. Janovitz-Klapp A, Richard F and Nicolas J 1989 Polyphenol oxidase from apple. Partial purification and some properties;Phytochemistry 28 2903–2907CrossRefGoogle Scholar
  17. Janovitz-Klapp A, Richard F, Goupy P and Nicolas J 1990 Inhibition studies on apple polyphenol oxidase;J. Agric. Food Chem. 38 926–931CrossRefGoogle Scholar
  18. Karbassi F, Haghbeen K, Saboury A A, Ranjbar B and Moosavi-Movahedi A A 2003 Activity, structural and stability changes of mushroom tyrosinase by sodium dodecyl sulfate;Colloids Surf. B: Biointerfaces 32 137–143CrossRefGoogle Scholar
  19. Karbassi F, Saboury A A, Hassan-Khan M T, Iqbal-Choudhary M and Saifi Z S 2004 Mushroom tyrosinase inhibition by two potent uncompetitive inhibitors;J. Enz. Inhib. Med. Chem. 19 349–353Google Scholar
  20. Lee J C and Timasheff S N 1981 The stabilization of proteins by sucrose.J. Biol. Chem. 256 7193–7201PubMedGoogle Scholar
  21. Lerch K 1981 Copper monoxygenases: tyrosinase and dopamine beta-monoxygenase; inMetal ions in biological systems (ed.) H Sigel (New York: Marcel Dekker) vol.13, pp 143–186Google Scholar
  22. Liu Y and Bolen D W 1995 The peptide backbone plays a dominant role in protein stabilization by naturally occurring osmolytes;Biochemistry 34 12884–12891PubMedCrossRefGoogle Scholar
  23. Mattila P, Salo-Vaananen P, Konko K and Aro H 2002 Basic composition and amino acid contents of mushrooms cultivated in Finland;J. Agric. Food Chem. 50 6419–6422PubMedCrossRefGoogle Scholar
  24. Moosavi-Movahedi A A, Nazari K and Saboury A A 1997 Thermodynamic denaturation of horseradish peroxidase with sodium n-dodecyl sulphate and n-dodecyl trimethylammonium bromide;Colloids Surf. B: Biointerfaces 9 123–130CrossRefGoogle Scholar
  25. Murphy P K 2001Methods in molecular biology: protein structure, stability and folding (New Jersey: Humana Press) vol. 168, pp 17–36Google Scholar
  26. Nwaka S and Holzer H 1998 Molecular biology of trehalose and trehalases in the yeast,Saccharomyces cerevcesiae;Prog. Nucleic Acid. Res. Mol. Biol. 58 197–237Google Scholar
  27. Ozaki S I, Inda Y and Watanabe Y 1998 Characterization of polyethylene glycolated horseradish peroxidase in organic solvents: generation and stabilization of transient catalytic intermediates at low temperature;J. Am. Chem. Soc. 120 8020–8025CrossRefGoogle Scholar
  28. Pace C N, Shiley B A and Thomson J A 1990 inProtein structure: a practical approach (ed.) T E Creighton (Oxford: IRL Press) pp 311–330Google Scholar
  29. Pifferi P G, Baldassari L and Cultera R 1974 Inhibition by carboxylic acids of an o-diphenol oxidase fromPrunus avium fruits;J. Sci. FoodAgric. 25 263–270CrossRefGoogle Scholar
  30. Pollard A and Wyn Jones R G 1979 Metabolic engineering of glycine betaine synthesis;Planta 144 291–298CrossRefGoogle Scholar
  31. Qu Y, Bolen C L and Bolen D W 1998 Osmolyte-driven contraction of a random coil protein;Proc. Natl. Acad. Sci. USA 95 9268–9273PubMedCrossRefGoogle Scholar
  32. Ryan O, Smyth M R and Fagain O C 1994 Thermostabilized chemical derivatives of horseradish peroxidase;Enzyme Microb. Technol. 16 501–505PubMedCrossRefGoogle Scholar
  33. Robb D A 1984 Tyrosinase; inCopper proteins and copper enzymes (ed.) R Lontie (Boca Raton: CRC Press) vol. 2, pp 207–240Google Scholar
  34. Saboury A A, Miroliaei M, Nemat-Gorgani M and Moosavi-Movahedi A A 1999 Kinetics denaturation of yeast alcohol dehydrogenase and the effect of temperature and trehalose. An isothermal microcalorimetry study;Thermochim. Acta 326 127–131CrossRefGoogle Scholar
  35. Saboury A A. and Karbassi F 2000 Thermodynamic studies on the interaction of calcium ions with alpha-amylase;Thermochim. Acta 362 121–129CrossRefGoogle Scholar
  36. Saboury A A, Karbassi F, Haghbeen K, Ranjbar B, Moosavi-Movahedi A A and Farzami B 2004 Stability, structural and suicide inactivation changes of Mushroom tyrosinase after acetylation by N-acetylimidazole;Int. J. Biol. Macromol. 34 257–262PubMedCrossRefGoogle Scholar
  37. Schein C H 1990 Solubility as a function of protein structure and solvent components;Bio/Technology 8 308–317PubMedCrossRefGoogle Scholar
  38. Shahjee HMD, Banerjee K and Ahmad F 2002 Comparative analysis of naturally occurring L-amino acid osmolytes and their D-isomers on protection ofEscherichia coli against environmental stresses;J. Biosci. 27 515–520PubMedCrossRefGoogle Scholar
  39. Singer M A and Lindquist S 1998 Thermotolerance inSaccharomyces cerevisiae: the Yin and Yang of trehalose;Trends Biotechnol. 16 460–468PubMedCrossRefGoogle Scholar
  40. Sola-Pena M, Ferreira-Pereira A, Lemos A P and Meyer-Fernandes J R 1997 Carbohydrate protection of enzyme structure and function against guanidinium chloride treatment depends on the nature of carbohydrate and enzyme;Eur. J. Biochem. 248 24–29CrossRefGoogle Scholar
  41. Somero G N 1986 Protons, osmolytes, and fitness of internal milieu for protein function;Am. J. Physiol. Regul. Integr. Comp. Physiol. 251 R197-R213Google Scholar
  42. Stewart G R. and Lee JA 1974 The role of proline accumulation in halophytes;Planta 120 279–289CrossRefGoogle Scholar
  43. Strothkemp K J, Jolley R L and Mason H S 1976 Quaternary structure of mushroom tyrosinase;Biochem. Biophys. Res. Commun. 70 519–524CrossRefGoogle Scholar
  44. Taneja S and Ahmad F 1994 Increased thermal stability of proteins in the presence of amino acids;Biochem. J. 303 147–153PubMedGoogle Scholar
  45. Taylor L S, York P, Williams A C, Edwards H G M, Mehta V, Jackson G S, Badcoe I G and Clarke A R 1995 Sucrose reduces the efficiency of protein denaturation by a chaotropic agent;Biochim. Biophys. Acta 1253 39–46PubMedGoogle Scholar
  46. Timashef S N 1998 Control of protein stability and reactions by weakly interacting cosolvents: the simplicity of the complicated;Adv. Protein Chem. 51 355–432CrossRefGoogle Scholar
  47. Wyatt G R and Kalf G F 1957 The chemistry of insect hemolymph: Trehalose and other carbohydrates;J. Gen. Physiol. 40 833–846PubMedCrossRefGoogle Scholar
  48. Xie G and Timasheff S N 1997 The thermodynamic mechanism of protein stabilization by trehalose;Biphys. Chem. 64 25–43CrossRefGoogle Scholar
  49. Yancey P H, Clark M E, Hand S C, Bowlus R D and Somero G N 1982 Living with water stress: evolution of osmolyte systems;Science 217 1214–1222PubMedCrossRefGoogle Scholar

Copyright information

© Indian Academy of Sciences 2006

Authors and Affiliations

  • N. Gheibi
    • 1
  • A. A. Saboury
    • 1
  • K. Haghbeen
    • 2
  • A. A. Moosavi-Movahedi
    • 1
  1. 1.Institute of Biochemistry and BiophysicsUniversity of TehranTehranIran
  2. 2.The National Research Center for Genetic Engineering and BiotechnologyTehranIran

Personalised recommendations