Screening, Production and characterization of lacasse from fusant strain of

Aswini Kamesh



The current work emphasizes on the screening, production and characterization of laccase from improved strain of Pleurotus species. Temperature stabilities of laccases were found to be 70°C. The optimum pH of the laccase differed with the substrate used. The optimum pH for the guaiacol was 7.0. The effect of different metal ions on the activity of laccase enzyme was investigated. Results showed that all the metal ions used exhibited inhibition effects to the enzyme activity but with different degrees. FeSO4 showed complete inhibition effect on laccase enzyme. The metal ions KCl and NaCl have less effect on the enzyme activity where the enzyme retained about 71% of its initial activity. Effect of a range of potent laccase inhibitors on the enzyme activity was tested. The oxidase inhibitors sodium azide and sodium cyanide caused about 77.77 and 51.48% inhibition of laccase activity respectively. However the enzyme retained 69.04% of its activity in the presence of 1 mM SDS. Other inhibitors, EDTA and aniline have low effect on laccase activity. The effect of enzyme concentration on enzyme activity was carried out under standard conditions with varying amounts of enzyme (mg protein) added. The results showed that, the maximum laccase activity was obtained with 0.0036 mg protein of the purified enzyme per reaction mixture. The effect of substrate (guaiacol) concentration on the activity of the purified laccase enzyme was studied. Results indicated that the activity increased linearly up on increasing the guaiacol concentration to reach maximum at 40 mM and higher substrate concentrations than 40 mM resulted in a constant enzyme activity. The kinetic parameters (Km and Vmax values) of the purified laccase enzyme was determined by using guaicol as a substrate and calculated from Lineweaver and Burk plots. The results indicated that the Km value was 71.43mM and Vmax value was 142.86U/ml protein. The laccase from Pleurotus species was purified using DEAE-Cellulose and eluted with NaCl showed the highest laccase activity. The molecular mass of purified laccases was found to be approximately 43 kDa.


Key Words:  Laccase, Purification, Pleurotus, DEAE- Cellulose, Kinetic parameters


Full Text:




Archibald, F.S, Bourbonnais, R., Jurasek, C., Paice, M.G., & Reid, I.D. Kraft pulp bleaching band delignification by Trametes versicolor (1997). Journal of Biotech. 53, 215-336.

Aswini, L., Arunagirinathan, N. and Kavitha, M. (2014). Strain Improvement of Pleurotus Species by Protoplast Fusion. International Journal of Advancements in Research & Technology, Volume 3: 2278-7763.

Das, N., & Mukheerjee, M. (1995). Conditions for isolation of regenerating protoplasts from Pleurotus sajor caju. J. Basic Microbiol. 35:157-161.

Gayazov, R., & Rodakiewicz-Nowak, J. (1996). Semi-continuous production of laccase by Phlebia radiata in different culture media. Folia Microbiologica . vol. 41, no. 6, pp. 480–484.

Gold, M.H., Cheng, T.M., & Alic, M (1983). Formation, fusion, and regeneration of protoplasts from wild-type and auxotrophic strains of the white rot basidiomycete Phanerochaete chrysosporium. Appl. Environ. Microbiol. 46: 260–263.

Heinzkill, M, L. Bech, T. Halkier, P. Schneider, & Anke.T. (1998). Characterization of laccases and peroxidases from woodrotting fungi (family Coprinaceae). Applied and Environmental Microbiology. vol. 64, no. 5, pp. 1601–1606.

Huttermann, A.C., Mai, A & Kharazipour, A.(2001). Applied Microbiology and Biotechnology. 55, 387.

Jaswal, R.K., Sodhi, H.S., Kapoor, S., & Khanna, P.K. (2013).Development of high yielding morphologically improved strains of Pleurotus through interspecific hybridization. Indian J. Agric. Sci. 83:374-379.

Larraya, L., Rez, G., Iribarren, A., Blanco, J.A., Alfonso, M., Pisabarro, A.G. & Rez, L. (2001). Relationship between monokaryotic growth rate and mating type in the edible basidiomycete Pleurotus ostreatus. Appl. Environ. Microbiol. 67:3385-3390.

Leontievsky, N. Myasoedova, N. Pozdnyakova. & Golovleva,L..(1997). FEBS Letters 413, 446.

Ng, T.B. (2004) Peptides and proteins from fungi. Peptides, 25: 1055–1073.

Thurston, C.F. (1994).The structure and function of fungal laccases. Microbiology 140, 19-26.

Yoshida, H. (1883). Chemistry of lacquer (Urushi), part I. J Chem Soc;. 43: 472-486.

Zhao, J. & Chang,S.T. (1995).Intraspecific hybridization between Coprinus cinerus and Schizophyllum commune by PEG – induced protoplast fusion and electrofusion. World J. Microbiol. Biotechnol., 9: 538 – 543

Jhadav, A., Vamsi, K.K., Khairnar. Y. (2009). Int. J. Microbiol. Res. 1:9-12.

Das, N., Naskar, S., Chowdhury, P., Pasman, B., & Adhikari, D. (2001). Experimental evidence for presence of a growth regulating extracellular laccase in some

Pleurotus species. Research Journal of Microbiology, 6, 496-502.

Lowry, H. Rosebrough, N.J. Farr, A.L. & Randall, R.L. (1951). Protein measurement with the folin phenol reagent. The Journal of Biological Chemistry, vol. 193, no. 1, pp. 265–275.

Dubois, M., Gilles, K.A., Hamilton, J.K., Rebers, P.A., & Smith, F. (1956). Colorimetric method for determination of sugars and selected substances. Analyt. Chem.28:350–358.

Fields, A. (2001) Review: Protein function at thermal extremes: balancing stability and flexibility. Comp. Biochem Physiol Part A 129, 417–431.

Somero, G.N. (2004). Adaptation of enzymes to temperatures: searching for basic ‘strategies’. Comp Biochem Physiol B 139, 312–333.

Liers, C., Ullrich. R., Pecyna. M., Schlosser, D. & Hofrichter. M. (2007).Production, purification and partial enzymatic and molecular characterization of a laccase from the wood-rotting ascomycete Xylaria polymorpha. Enzyme Microb. Technol.41:785–793.

Palmieri, G., Giardina, P., Bianco, C., Scaloni, A., Capasso, A & Sannia, G. (1997). A novel white laccase from Pleurotus ostreatus. J Biol Chem 272: 31301–31307.

Palmieri, G., Giardina, P., Marzullo, L., Desiderio, B., Nitti, B., Cannio. R. & Sania, G. (1993).Stability and activity of phenoloxidase from lignolytic fungus. Pleurotus ostreatus. Appl Microbiol Biotechnol.39:632–636.

De’Souza, C. G. M., & Peralta, R. M. (2003). Purification and characterization of the main laccase produced by the white rot fungus Pleurotus pulmonarius on wheatbran solid state medium. J. Basic Microbiol.43, 278-286.

Sadhasivam, S., Savitha, S., Swaminathan K., & Lin Feng-Huei.(2008). Production, purification and characterization of mid-redox potential laccase from a newly isolated Trichoderma harzianum WL1. Process Biochem; 43:736–742.

Nagai, M., Sato, T., Watanabe, H., Saito, K., Kawata, M., & Enei H. (2002).Purification, characterization of an extracellular laccase from edible mushroom Lentinula edodes and decolourization of chemically different dyes. Applied Microbial. Biotechnol.60 (3):327-335.

Chakroun, H., Mechich, T., Martinez, M.J., Dhouib, A., & Sayadi, S. (2010). Purification and characterization of a novel laccase from the ascomycete Trichoderma atroviride: application on bioremediation of phenolic compounds. Process Biochem. 45:507–513.

Lineweaver, H. & Burk, D. (1934). The Determination of Enzyme Dissociation Constants". Journal of the American Chemical Society 56 (3): 658–666.

Banerjee, U.C., & Vohra, R.M. (1991) Production of laccase by Curvularia sp. Folia Microbial. 36:343.

Yaropolov, A.I., Skorobogatko, O.V., Vartanov, S.S., & Varfolomeyev, S, D., (1994) .Laccase-properties, catalytic mechanism, and applicability. Appl Biochem Biotechnol 49:257-280

Xiao, Y, Tu, X.,Wang, J., Zhang, M., Cheng, Q., Zeng, W., & Shi, Y. (2003). Purification, molecular characterization and reactivity with aromatic compounds of a laccase from basidiomycete Trametes sp. strain AH28-2. Appl. Microbiol. Biotechnol. 60, 700-707.


  • There are currently no refbacks.

Creative Commons License
This work is licensed under a Creative Commons Attribution 3.0 License.