Cloning, Experession, and Characterization of a Laccase from the White Rot Fungi Pleurotus pulmonarius MPN18
Main Article Content
Abstract
Laccase (EC 1.10.3.2) is an enzyme belonging to the polyphenol oxidase groups, which plays an important role in the oxidation of a wide variety of aromatic substrates, such as lignin, phenol, polyamine, and aryl diamines, as well as a number of other phenolic compounds or inorganic ions in the presence of oxygen. Laccase is widely applied in many different fields, especially in the textile industry, dyeing, and environmental pollution treatment. In this study, we have successfully cloned and expressed cDNA coding for laccase from Pleurotus pulmonarius MPN18 (PpLac). cDNA corresponds to the gene laccase (size 1566 bp) was attached to
pET 21a(+) vector and expressed in E. coli BL21, after that the enzyme was purified through HisTrapTM sp 5mL column. The purified PpLac had an activity of 899.8 U, a 74% yield with a purity of 15.2 -fold, and was tested by SDS-PAGE electrophoresis with a molecular weight of
Mw = 55 kDa. Enzyme displayed optimal activity at 50 ºC and pH 4.0. Enzyme had optimal activity of 20-40 ºC after 120 min incubation and pH 4 after 6 h incubation. In future, the recombinant enzyme will be characterized for supplementation into enzyme cocktail in the treatment of lignocellulosic material.
Keywords:
Experession, laccase, Pleurotus pulmonarius MPN18.
References
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https://doi.org/10.1515/boku-2016-0013.
[4] S. Souza, S. Melo, R. Oliveira, Ligninolytic Enzyme Production by Ganoderma spp, Enzyme. Microb, Technol, Vol. 37, No. 3, 2005, pp. 324-329, https://doi.org/10.1016/j.enzmictec.2004.12.007.
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[8] D. Yanto, N. Auliana, S. Anita, T. Watanabe, Decolorization of Synthetic Textile Dyes by Laccase from Newly Isolated Trametes hirsuta EDN084 Mediated by Violuric Acid, Earth, Environ, Sci, Vol. 374, 2019,
https://doi.org/10.1088/1755-1315/374/1/012005.
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https://doi.org/10.3923/jest.2018.16.22.
[10] S. Kawai, T. Umezawa, T. Higuchi, Degradation Mechanisms of Phenolic β-1 Lignin Substructure Model Compounds by Laccase of Coriolus versicolor, Arch, Biochem, Biophys, Vol. 262, No. 1, 1988, pp. 99-110,
https://doi.org/10.1016/0003-9861(88)90172-5.
[11] A. Leonowicz, N. Cho, J. Luterek, A. Wilkolazka, M. W. Wasilewska, A. Matuszewska, M. Hofrichter, D. Wesenberg, J. Rogalski, Fungal Laccase: Properties and Activity on Lignin, J. Basic Microbiol, Vol. 41, No. 3, 2001, pp. 185-227, https://doi.org/10.1002/15214028(200107)41:3/4<185: aid-jobm185>3.0.co;2-t.
[12] V. Leo, A. Passari, I. K. Muniraj, S. Uthandi, A. Hashem, E. F. Abd_Allah, A. A. Alqarawi, P. Singh, Elevatedlevels of Laccase Synthesis by Pleurotus pulmonarius BPSM10 and its Potential as a Dye Decolorizing Agent, Saudi J. Biol. Sci, Vol. 26, No. 3, 2019, pp. 464-468, https://doi.org/10.1016/j.sjbs.2018.10.006.
[13] G.V. Dinh, D. T. Quynh, D. H. Nghi, Screening Carbohydrate Esterase and Oxidase Enzyme from Fungi Isolated in Cuc Phuong (Ninh Binh) and Muong Phang (Dien Bien), Proceedings of the 3rd National Scientic Conference of Vietnam Museum System, No. 57, 2021, pp. 470-479.
[14] S. Sambrook, R. D. W, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, 2003.
[15] D. Linke, H. Bouws, T. Peters, M. Nimtz, R. G. Berger, H. Zorn, Laccases of Pleurotus sapidus: Characterization and cloning, J. Agric, Food, Chem, Vol. 53, No. 24, 2005, pp. 9498-9505, https://doi.org/10.1021/jf052012f.
[16] M. Q. Ai, F. F. Wang, F. Huang, Purification and Characterization of a Thermostable Laccase from Trametes trogii and its Ability in Modification of Kraft Lignin, J. Microbiol, Biotechnol, Vol. 25, No. 8, 2015, pp. 1361-1370,
https://doi.org/10.4014/jmb.1502.02022.
[17] N. Garg, N. Bieler, T. Kenzom, M. Chhabra, M. A. Schumacher, S. Mishra, Cloning, Sequence Analysis, Expression of Cyathus bulleri Laccase in Pichia pastoris and Characterization of Recombinant Laccase, BMC, Biotech, Vol. 12, 2012, pp. 1-12, https://doi.org/10.1186/1472-6750-12-75.
[18] Q. Song, X. Deng, R. Q. Song, Expression of Pleurotus ostreatus Laccase Gene in Pichia pastoris and its Degradation of Corn Stover Lignin, Microorganisms, Vol. 8, No. 4, 2020, pp. 601, https://doi.org/10.3390/microorganisms8040601.
[19] L. Lu, T. N. Wang, T. F. Xu, J. Y. Wang, C. L. Wang, M. Zhao, Cloning and Expression of Thermo-alkali-stable Laccase of Bacillus licheniformis in Pichia pastoris and its Characterization, Bioresour, Technol, Vol. 134, 2013, pp. 81-86, https://doi.org/10.1016/j.biortech.2013.02.015.
[20] S. Couto, A. Rodríguez, R. Paterson, R. Lima, J. Teixeira, Laccase Activity from the Fungus Trametes hirsuta using an Air‐lift Bioreactor, Lett, Appl, Microbiol, Vol. 42, 2006, pp. 612-616, https://doi.org/10.1111/j.1472-765X.2006.01879.x.