Abstract
Laccase, an oxidoreductive enzyme, is important in bioremediation. Although marine fungi are potential sources of enzymes for industrial applications, they have been inadequately explored. The fungus MTCC 5159, isolated from decaying mangrove wood and identified as Cerrena unicolor based on the D1/D2 region of 28S and the 18S ribosomal DNA sequence, decolorized several synthetic dyes. Partially purified laccase reduced lignin content from sugarcane bagasse pulp by 36% within 24 h at 30°C. Laccase was the major lignin-degrading enzyme (~24,000 U L−1) produced when grown in low-nitrogen medium with half-strength seawater. Three laccases, Lac I, Lac II, and Lac III, of differing molecular masses were produced. Each of these, further resolved into four isozymes by anion exchange chromatography. The N-terminal amino acid sequence of the major isozyme, Lac IId showed 70–85% homology to laccases from basidiomycetes. It contained an N-linked glycan content of 17%. The optimum pH and temperature for Lac IId were 3 and 70°C, respectively, the half-life at 70°C being 90 min. The enzyme was most stable at pH 9 and retained >60% of its activity up to 180 min at 50°C and 60°C. The enzyme was not inhibited by Pb, Fe, Ni, Li, Co, and Cd at 1 mmol. This is the first report on the characterization of thermostable metal-tolerant laccase from a marine-derived fungus with a potential for industrial application.
Similar content being viewed by others
References
Annonymous (1988) Kappa number of pulp-T 236-cm-85. In: TAPPI test methods, vol 1. TAPPI, Atlanta, pp 1–3
Asgher M, Bhatti HN, Ashraf M, Legge RL (2008) Recent developments in biodegradation of industrial pollutants by white-rot fungi and their enzyme system. Biodegradation 19:771–783
Baldrian P (2003) Interactions of heavy metals with white-rot fungi. Enzyme Microb Technol 32:78–91
Baldrian P (2006) Fungal laccases: occurrence and properties. FEMS Microbial Rev 30:215–242
Baldrian P, Gabriel J (1997) Effect of heavy metals on the growth of selected wood-rotting basidiomycetes. Folia Microbiol 42:521–523
Baldrian P, Gabriel L, Nerud F (1996) Effect of cadmium on the ligninolytic activity of Stereum hirsutum and Phanerochaete chrysosporium. Folia Microbiol 41:363–367
Bucher VVC, Hyde KD, Pointing SB, Reddy CA (2004) Production of wood decay enzymes, mass loss and lignin solubilization in wood by marine ascomycetes and their anamorphs. Fungal Divers 15:1–14
Coll PM, Fernández-Abalos JM, Villanueva JR, Santamaria R, Pérez P (1993) Purification and characterization of a phenoloxidase (laccase) from the lignin-degrading basidiomycete PM1 (CECT 2971). Appl Environ Microbiol 59:2607–2613
Das N, Chakraborty TK, Mukherjee M (2001) Purification and characterization of a growth-regulating laccase from Pleurotus florida. J Basic Microbiol 41:261–267
Dawley RM, Flurkey WH (1993) Differentiation of tyrosinase and laccase using 4-hexyl-resorcinol, a tyrosinase inhibitor. Phytochemistry 33:281–284
D’Souza DT, Tiwari R, Sah AK, Raghukumar C (2006) Enhanced production of laccase by a marine fungus during treatment of colored effluents and synthetic dyes. Enzyme Microb Technol 38:504–511
D’Souza-Ticlo D, Verma AK, Mathew M, Raghukumar C (2006) Effect of nutrient nitrogen on laccase production, its isozyme pattern and effluent decolorization by the fungus NIOCC #2a isolated from mangrove wood. Indian J Mar Sci 35:364–372
Eggert C, Temp U, Eriksson K-EL (1996) The ligninolytic systems of the white-rot fungus Pycnoporus cinnabarinus: purification and characterization of the laccase. Appl Environ Microbiol 62:1151–1158
Gianfreda L, Sannino F, Filazzola MT, Leonowicz A (1998) Catalytic behavior and detoxifying ability of a laccase from the fungal strain Cerrena unicolor. J Mol Catal B: Enzymatic 4:13–23
Heinzkill M, Bech L, Halkie T, Schneider P, Anke T (1998) Characterization of laccases and peroxidases from wood-rotting fungi (family Coprinaceae). Appl Environ Microbiol 64:601–606
Heukeshoven J, Dernick R (1985) Simplified method for silver staining of proteins in polyacrylamide gels and the mechanism of silver staining. Electrophoresis 6:103–112
Howard RL, Abotsi E, Jansen van Rensburg EL, Howard S (2003) Lignocellulose biotechnology: issues of bioconversion and enzyme production. African J Biotechnol 2:602–619
Jensen PR, Fenical W (2000) Marine microorganisms and drug discovery: current status and future potential. In: Fusetani N (ed) Drugs from the sea. Karger, Basel, pp 6–29
Jordaan J, Pletschke BI, Leukes WD (2004) Purification and partial characterization of a thermostable laccase from an unidentified basidiomycete. Enzyme Microb Technol 34:635–641
Johannes C, Majcherczyk A (2000) Natural mediators in the oxidation of polycyclic aromatic hydrocarbons by laccase mediator systems. Appl Environ Microbiol 66:524–528
Kim Y, Cho N-S Eom T-J, Shin W (2002) Purification and characterization of a laccase from Cerrena unicolor and its reactivity in lignin degradation. Bull Korean Chem Soc 23:985–989
Kohlmeyer J, Kohlmeyer E (1979) Marine mycology. The higher fungi, 1st edn. Academic, New York
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head bacteriophage T4. Nature 227:680–685
Michniewiez A, Ulrich R, Ledakowiez S, Hofrichter M (2006) The white-rot fungus Cerrena unicolor strain 137 produces two laccase isoforms with different physico-chemical and catalytic properties. Appl Microbiol Biotechnol 69:682–688
Nelson DL, Cox MM (2004) Lehninger principles of biochemistry, 4th edn. Freeman, New York
Niku-Paavola ML, Karhuner E, Salola P, Raunio V (1988) Lignolytic enzymes of the white-rot fungus Phlebia radiata. Biochem J 254:877–884
Pointing SB, Hyde KD (2000) Lignocellulose-degrading marine fungi. Biofouling 15:221–229
Pointing SB, Vrijmoed LLP, Jones EBG (1998) A qualitative assessment of lignocellulose degrading enzyme activity in marine fungi. Bot Mar 41:293–298
Pointing SB, Buswell JA, Jones EBG, Vrijmoed LLP (1999) Extracellular cellulolytic enzyme profiles of five lignicolous mangrove fungi. Mycol Res 103:696–700
Raghukumar C (2002) Bioremediation of colored pollutants by terrestrial versus facultative marine fungi. In: Hyde KD (ed) Fungi in marine environment. Fungal biodiversity research series 7. Fungal Diversity Press, Hong Kong, pp 317–344
Raghukumar C (2008) Marine fungal biotechnology: an ecological perspective. Fungal Divers 31:19–35
Raghukumar C, Raghukumar S, Chinnaraj A, Chandramohan D, D’Souza TM, Reddy CA (1994) Laccase and other lignocellulose modifying enzymes of marine fungi isolated from the coast of India. Bot Mar 37:515–523
Raghukumar C, D’Souza TM, Thorn RG, Reddy CA (1999) Lignin modifying enzymes of Flavodon flavus, a basidiomycete isolated from a coastal marine environment. Appl Environ Microbiol 65:2103–2111
Raghukumar C, D’Souza-Ticlo D, Verma AK (2008) Treatment of colored effluents with lignin-degrading enzymes: an emerging role of marine-derived fungi. Crit Rev Microbiol 34:189–206
Stepanova EV, Pegasova TV, Gavrilova VP, Landesman EO, Koroleva OV (2003) Extracellular laccases from Cerrena unicolor 059, C. unicolor 0784 and Pleurotus ostreatus 0432: a comparative assay. Appl Biochem Microbiol 39:375–381
Tien M, Kirk TK (1988) Lignin peroxidase of Phanerochaete chrysosporium. Methods Enzymol 161:238–249
White TJ, Bruns TD, Lee SB, Taylor JW (1990) Amplification and direct sequencing of fungal ribosomal RNA genes for phylogenetics. In: Innis N, Gelfand D, Sninsky J, White TJ (eds) PCR-protocols and applications—a laboratory manual. Academic, New York, pp 315–322
Wong KKY, Richardson JD, Mansfield SD (2000) Enzymatic treatment of mechanical pulp fibres for improving papermaking properties. Biotechnol Prog 16:1025–1029
Xiao YZ, Tu XM, Wang J, Zhang M, Cheng Q, Zeng WY, Shi YY (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
Xu F, Shin W, Brown SH, Wahlleithner JA, Sundaram UM, Solomon EI (1996) A study of recombinant fungal laccases and bilirubin oxidase that exhibit significant differences in redox potential, substrate specificity and stability. Biochim Biophys Acta 1292:303–311
Acknowledgments
We are grateful to Director N.I.O. for valuable support and encouragement. Donna D’Souza-Ticlo and Deepak Sharma are thankful to CSIR for a Senior Research Fellowship and DBT for a Post-doctoral Fellowship, respectively. Raghukumar wishes to thank the Department of Biotechnology, New Delhi for the research grant no. BT/PR 3380/PID/06/166/2002 under which a part of this work was carried out. We are grateful to Dr. D. M Salunke, National Institute of Immunology, New Delhi for N-terminal AA sequencing. We acknowledge the technical advice and help of Dr Somdutta Sen and Ms Reena Arora, Proteomics Facility, TCGA, New Delhi. NIO’s contribution No.4502
Author information
Authors and Affiliations
Corresponding author
Electronic supplementary material
Below is the link to the electronic supplementary material.
ESM 1
Supplementary Table comparing marine-derived C. unicolor MTCC 5159 with terrestrial isolates of C. unicolor (DOC 35.0 kb)
Rights and permissions
About this article
Cite this article
D’Souza-Ticlo, D., Sharma, D. & Raghukumar, C. A Thermostable Metal-Tolerant Laccase with Bioremediation Potential from a Marine-Derived Fungus. Mar Biotechnol 11, 725–737 (2009). https://doi.org/10.1007/s10126-009-9187-0
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10126-009-9187-0