Purification and Characterization of the Laccase Enzyme from the Basidiomycete Agaricus nevoi in Al-Alam in Salahaddin/ Iraq
DOI:
https://doi.org/10.32792/utq/utjsci/v13i1.1516Keywords:
laccase enzyme, Purification, fungal isolates, ammonium sulfateAbstract
Laccase (EC 1.10.3.2) is a type of enzyme known as a multicopper oxidase, which can oxidize a wide array of phenolic and non-phenolic substances, utilizing molecular oxygen to accept electrons. In our research, we assessed the capability of three macrofungal isolates—HM001, IN002, and SH003—to produce laccase. Initial tests showed that isolates HM001 and SH003 demonstrated the most significant activity, producing decolorization zones measuring 45 mm and 16 mm in diameter, respectively. Further quantitative tests revealed that isolate HM001 had the highest enzyme activity, reaching 50 U/mL by the twentieth day of incubation, making it the focal point for more detailed studies. Molecular analysis confirmed that this isolate was Agaricus nevoi, marking a novel discovery for Salahaddin Province, Iraq. We purified the laccase enzyme using a process that included ammonium sulfate precipitation at 70% saturation, followed by ion-exchange chromatography on DEAE-cellulose and size-exclusion chromatography using Sephadex G-150, enhancing the specific activity of the enzyme from 100 to 1000 U/mg. The purified laccase worked best at 37°C and a pH of 7–8, maintaining its stability within a temperature range of 32–37°C. We estimated the molecular weight of the enzyme to be 50 kDa. In conclusion, our findings indicate that Agaricus nevoi is a valuable new source of laccase, exhibiting robust enzymatic performance and stability under moderately warm and neutral pH conditions. This successful purification and characterization of the laccase underscore its potential for future use in biotechnology and industry, especially for the efficient oxidation of various phenolic substances
References
Y. Yang, H. R. Hebron, and J. Hang, “High performance DNA purification using a novel ion-exchange matrix”. Journal of Biomolecular Techniques, 19(4), 205–212. 2008.
S. Sondhi and S. Saini, “Response surface based optimization of laccase production from Bacillus sp. MSK01 using fruit juice waste as an effective substrate,” PLOS ONE, pp. 1–17, 2019, doi:10.1371/journal.pone.0229968.
G. Janusz et al., “Laccase properties, physiological functions, and evolution,” International Journal of Molecular Sciences, vol. 21, no. 3, p. 966, 2020, doi:10.3390/ijms21030966.
P. Aza and S. Camarero, “Fungal laccases: Fundamentals, engineering and classification update,” Biomolecules, vol. 13, no. 12, p. 1716, 2023, doi:10.3390/biom13121716.
J. R. Whitaker, A. G. J. Voragen, and D. W. S. Wong, “Handbook of Food Enzymology,”. New York, USA: Marcel Dekker, 2003.
D. L. Nelson and M. M. Cox, “Lehninger Principles of Biochemistry,” Recording for the Blind & Dyslexic New York, 7th ed. New York, USA: W. H. Freeman, 2017.
S. P. Wasser, “Species diversity in the tribe Agariceae Pat. (Higher Basidiomycetes) of Israel mycobiota,” Israel Journal of Plant Sciences, vol. 46, no. 4, pp. 315–330, 1998.
R. A. Chechan, E. M. Farhan, M. M. Muslat, and Z. M. Abdul-Qader, “Morphological characterization, molecular diagnosis, and enzymatic activity of some wild mushrooms in Baghdad Province, Iraq,” Plant Archives , vol. 20, no 2, pp. 7437-7445, 2020.
Cromushrooms, “Species profile: Agaricus nevoi Wasser,” European Mycological Database, 2025.
R. K. Pundir, V. K. Mishra, S. Rana, and M. Lakhani, “Screening of laccase producing fungi from soil samples—An in vitro study,” Electronic Journal of Biology, vol. 12, no. 3, pp. 254–257, 2016.
M. M. Bradford, “A rapid and sensitive method for the quantitation of microgram quantities of protein,” Analytical Biochemistry, vol. 72, pp. 248–254, 1976.
R. Chaudhary, S. Hooda, S. Beniwal, and A. Sindhu, “Partial purification and immobilization of laccase isolated from medicinal mushroom (Ganoderma lucidum),” Indian Journal of Chemical Technology, vol. 23, no. 4, pp. 313–317, 2016.
T. Saito et al., “Purification and characterization of an extracellular laccase from a soil fungus,” Enzyme and Microbial Technology, vol. 33, pp. 520–526, 2003.
L.-L. Kiiskinen, M. Rättö, and K. Kruus, “Screening for novel laccase-producing microbes,” Journal of Applied Microbiology, vol. 97, no. 3, pp. 640–646, 2004.
A. B. Vantamuri and B. B. Kaliwal, “Isolation, screening and identification of laccase producing fungi,” International Journal of Current Microbiology and Applied Sciences, vol. 4, no. 4, pp. 1014–1021, 2015.
K. Amit et al., “Gel-based purification and biochemical study of laccase isozymes from Ganoderma sp.,” Frontiers in Microbiology, vol. 8, p. 674, 2017, doi:10.3389/fmicb.2017.00674.
Q. Wang, Y. Qian, Y. Ma, and C. Zhu, “A preliminary study on newly isolated high laccase-producing fungi,” Open Life Sciences, vol. 13, pp. 463–469, 2018.
S. M. Mustafa, “Production of pure isolates for cultivated mushroom (Agaricus bisporus) using tissue culture technology,” 2018.
H. Moallaei et al., “Enzymatic activity and molecular characterization of a secreted protease,” Iranian Journal of Public Health, vol. 38, no. 1, pp. 25–33, 2009.
C. U. Uma, “Production, purification and characterization of protease by Aspergillus flavus,” 2011..
P. Baldrian, “Increase of laccase activity during interspecific interactions of white-rot fungi,” FEMS Microbiology Ecology, vol. 50, no. 3, pp. 245–253, 2004.
A. A. Green and W. L. Hughes, “Protein fractionation on the basis of solubility,” Methods in Enzymology, vol 1, pp. 67-90, 1955.
P. T. Wingfield, “Protein precipitation using ammonium sulfate,” Current Protocols in Protein Science, vol. 84, no. 1, 2016.
F. B. Salisbury and C. W. Ross, “Plant Physiology, ” Annals of Botany, vol. 71, pp. 91-94, 1993, USA: Ancestry Publishing.
R. K. Scopes, “Protein Purification: Principles and Practice, 3rd ed,” Springer, New York, USA, 1992.
S. A.Hanan, “Al-Muthaffar,Biochemistry. Part One, Second Edition, ” University of Baghdad, Ministry of Higher Education and Scientific Research. Page 492. 1990.
M. Asgher, M. Irshad, and H. M. N. Iqbal, “Purification and characterization of lignin peroxidase,” BioResources, vol. 7, no. 3, pp. 4012–4021, 2012.
K. Miyanaga and H. Unno, “Reaction kinetics and stoichiometry,” in Comprehensive Biotechnology, 2nd ed., vol. 2. Elsevier, 2011, pp. 33–46.
V. W. Rodwell, “Structure and function of proteins and enzymes,” in Biochemistry, 24th ed. USA: Prentice-Hall, 1996.
K. M. Park and S. S. Park, “Purification and characterization of laccase from Fomitella fraxinea,” Journal of Microbiology and Biotechnology, vol. 18, no. 4, pp. 670–675, 2008.
F. Xu et al., “Differences in redox potential and substrate specificity of fungal laccases,” Biochimica et Biophysica Acta, vol. 1292, pp. 303–311, 1996.
A. G. Contato et al., “Solid-state fermentation with orange waste for laccase production,” Acta Scientiarum Biological Sciences, vol. 42, pp. 1–9, 2020.
A. Lehmacher and H. Bisswanger, “Isolation of thermostable enzymes,” Microbiology, vol. 136, no. 4, pp. 679–686, 1990.
A. G. Moat, J. W. Foster, and M. P. Spector, “Microbial Physiology,” John Wiley & Sons, New York, USA: Wiley, 2002.
M. P. Deutscher, “Guide to Protein Purification,”Gulf Professional Publishing, vol. 182, 1990.
P. Sharma, R. Goel, and N. Capalash, “Bacterial laccases,” World Journal of Microbiology and Biotechnology, vol. 23, pp. 823–832, 2007.
K. Brijwani, A. Rigdon, and P. V. Vadlani, “Fungal laccase: Production and applications,” Enzyme Research, p. 149748, 2010.
A. Góralczyk-Bińkowska et al., “Laccase activity of Nectriella pironii,” PLOS ONE, vol. 15, no. 4, e0231453, 2020
A. K. Sitarz et al., “Identification of a laccase from Ganoderma lucidum,” Enzyme and Microbial Technology, vol. 53, no. 6–7, pp. 378–385, 2013.
J. M. Chesworth, T. Stuchbury, and J. R. Scaife, “Amino acids and proteins,” in An Introduction to Agricultural Biochemistry. Springer, 1998.
A. M. Abdel-Azeem and F. M. Salem, “Biodiversity of laccase producing fungi in Egypt,” Mycosphere, vol. 3, no. 5, pp. 900–920, 2012.
M. M. Gyosheva et al., “Red list of fungi in Bulgaria,” Mycologia Balcanica, vol. 3, no. 1, pp. 81–87, 2006
H. Patel, A. Gupte, and S. Gupte, “Effect of culture conditions on laccase production,” BioResources, vol. 4, no. 1, pp. 268–284, 2009.
R. K. Sharma and D. S. Arora, “Production of lingocellulolytic enzymes,” Bioresource Technology, vol. 101, no. 23, pp. 9248–9253, 2010.
J. R. Whitaker, “Principles of Enzymology for the Food Sciences,” FOOD SCIENCE AND TECHNOLOGY, vol. 61, New York, USA: Marcel Dekker, 1972
Downloads
Published
License
Copyright (c) 2026 University of Thi-Qar Journal of Science
This work is licensed under a Creative Commons Attribution 4.0 International License.
