Preliminary Production and Partial Purification of Laccase from a White Rot Fungus and Its Application in Dye Degradation

Main Article Content

Chukwudi Innocent Nnamchi
Grace Adaeze Ezeofor
Chioma Onyetugo Amadi

Abstract

In this study, a laccase producing white rot fungus from the wood degrading family of dermatophytic Trichophyton sp. was isolated. The laccase which was produced via solid state fermentation using rice bran as the lignocellulosic support was partially purified before its effectiveness in degrading an azo dye (methyl orange) was examined. Time course study showed that day nine (day 9) gave the maximum enzyme production. The crude laccase was purified by ammonium sulfate precipitation and dialyzed against sodium phosphate buffer pH 7. Optimum pH and temperature of the were 4.5 and 55°C respectively as assayed using ABTS (2,2 azino bis-3-ethyle benzothiozoline-6-sulfonic acid) as substrate. Dye degradation assay was carried out by introducing 0.5 ml of the enzyme into three dye concentrations: 50 mg/L, 100 mg/L and 200 mg/L and monitoring them for up to 48 hours by taking their absorbances at intervals at 5mins, 5hours, 24hours and 48 hours after enzyme introduction. At the end of the experiment, good dye degradation by laccase from Trichophyton sp. was observed to be positive with 50 mg/L being the most effective concentration during the study. The result are positive indicators of the fact that locally isolated white rot fungi has the capacity to be used for many biotechnological dye degradation and removal studies.

Keywords:
White rot fungi, laccase, partial purification, dye degradation, enzymes, ABTS.

Article Details

How to Cite
Nnamchi, C. I., Ezeofor, G. A., & Amadi, C. O. (2020). Preliminary Production and Partial Purification of Laccase from a White Rot Fungus and Its Application in Dye Degradation. Biotechnology Journal International, 23(4), 1-9. https://doi.org/10.9734/bji/2019/v23i430089
Section
Original Research Article

References

Williamson PR. Biochemical and molecular characterization of the diphenol oxidase of cryptococcus neoformans: identification as a laccase. J Bacteriol. 1994;176(3):656–664.

Shraddha RS, Sehgal S, Kamthania M, Kumar A. Laccase: microbial sources, production, purification and potential biotechnological applications. Enzyme Res. 2011;2011:217861.

Couto SR, Toca Herrera JL, Industrial and biotechnological applications of laccases: A review. Biotechnol. Adv. 2006;24(5): 500–513.

Madhavi V, Lele SS. Laccase: Properties and applications. Bio Resources. 2009;4 (4):1694–1717.

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

Eggert C, Temp U, Dean JFD, Eriksson KEL. A fungal metabolite mediates degradation of non-phenolic lignin structures and synthetic lignin by laccase. FEBS Letters. 1996;391(1-2):144–148.

Yang J, Li W, Ng TB, Deng X, Lin J and Ye X. Laccases: Production, expression regulation and applications in pharmaceutical biodegradation. Front. Microbiol. 2017;8:832.

Tien M, Kirk TK. Lignin peroxidase of Phanerochaete chysosporium. Methods Enzymol. 1988;161:238-249.

Shin KS, Lee YJ. Purification and characterization of a new member of the laccase family from the white rot basidiomycete Coriolus hirutus. Arch Biochem. Biophys. 2000;384:109-115.

Lowry OH, Rosebrough NJ, Farr A, Randall RJ. Protein measurement with the folio phenol reagent. J Biol Chem. 1951; 193(1):265-275.

Yoshida, H. Chemistry of lacquer urushi immobilized Laccase for decolorization of reactive black 5, J. Chem. Soc. 1983;43 (3):472-486.

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

Xu F, Kulys JJ, Duke K, Li K, Krikstopaitis K, Deussen HW, et al. Redox chemistry in laccase-catalyzed oxidation of N-hydroxy compounds. Appl. Environ. Microbiol. 2000;66(5):2052–2056.

Cordi L, Minussi RC, Freire RS, Duran N. Fungal laccase: Copper induction, semi-purification, immobilization, phenolic effluent treatment and electrochemical measurement. Afr. J. Biotechnol. 2007;6: 1255-1259.

More SS, Renuka PS, Pruthvi K, Swetha M, Malini S, Veena SM. Isolation, purification and characterization of fungal laccase from Pleurotus sp. Enzyme Res. 2011;1–7.

Pandey A, Selvakumar P, Soccol CR, Nigam P. Solid state fermentation for the production of industrial enzymes. Current Sci. 1999;77:149–162.

Couto SR, Sanromán MAA. Application of solid-state fermentation to ligninolytic enzyme production. Biochem Eng. 2005; 22:211–219.

Brijwani K, Oberoi HS, Vadlani PV. Production of a cellulolytic enzyme system in mixed-culture solid-state fermentation of soybean hulls supplemented with wheat bran. Process Biochem. 2010;45:120–128.

Baldrian, P. Fungal laccases – occurrence and properties. FEMS Microbiol. Rev. 2006;30(2):215-242.

Ko EM, Leem YE, Choi HT. Purification and characterization of laccase isozymes from the white-rot basidiomycete Ganoderma lucidum. Appl Microbiol Biotechnol. 2001;57:98–102.

Jung H, Xu F, Li K. Purification and characterization of laccase from wood-degradingfungus Trichophyton rubrum LKY-7. Enzyme Microb. Technol. 2002;30: 161–168.

Robinson PK. Enzymes: Principles and biotechnological applications. Essays Biochem. 2015;59:1–41.

Teixeira RSS, Pereira PM, Ferreira-Leitao VS. Extraction and application of laccases from shimeji mushrooms (Pleurotus ostreatus) residues in decolourisation of reactive dyes and a comparative study using commercial laccase from Aspergillus oryzae. Enzyme Res. 2010;905896.