Abstract
The addition of exogenous endocrine disrupting compounds (EDCs) like estrone, in the food chain through the aquatic system, disrupts steroid biosynthesis and metabolism by altering either the genomic or non-genomic pathway that eventually results in various diseases. Thus, bioremediation of these compounds is urgently required to prevent their addition and persistence in the environment. Enzymatic degradation has proven to be a knight in shining armour as it is safe and generates no toxic products. The multicopper oxidases (E.C. 1.10.3.2 benzenediol: oxygen oxidoreductase), laccase with the potential to degrade both phenolic and non-phenolic substrates has recently gained attention. In this study, the laccase was purified, characterized, and used to study estrone degradation. The culture filtrate (crude laccase) was concentrated and precipitated using cold-acetone and dialyzed against tris buffer (50 mM) giving a four-fold partially purified form, with 45.56% yield and 204.14 U/mg as specific activity and a single peak at 250–300 nm. The partially purified laccase was approximately 80 kDa as estimated by SDS-PAGE preferred ABTS as substrate. Both crude and partially purified laccase showed maximum activity at pH 3.0, 40 °C, and 4 mM ABTS. Kinetic constants (Km, Vmax) of crude and partially purified laccase were found to be 0.83 mM; 494.31 mM/min, and 0.58 mM; 480.54 mM/min respectively. Iron sulphate and sodium azide inhibited laccase maximally. Crude and partially purified laccase degradation efficiency was 87.55 and 91.35% respectively. Spirulina CPCC-695 laccase with efficient estrone degradation ability renders them promising candidates for EDCs bioremediation.
Similar content being viewed by others
References
Ting YF, Praveena SM (2017) Sources, mechanisms, and fate of steroid estrogens in wastewater treatment plants: a mini review. Environ Monit Assess 189(4):178
Reichert G, Hilgert S, Fuchs S, Azevedo JCR (2019) Emerging contaminants and antibiotic resistance in the different environmental matrices of Latin America. Environ Pollut 255:113140
Liu ZH, Kanjo Y, Mizutani S (2009) Removal mechanisms for endocrine disrupting compounds (EDCs) in wastewater treatment—physical means, biodegradation, and chemical advanced oxidation: a review. Sci Total Environ 407(2):731–748
Baldrian P (2006) Fungal laccases–occurrence and properties. FEMS Microbiol Rev 30(2):215–242
Morozova OV, Shumakovich GP, Gorbacheva MA, Shleev SV, Yaropolov AI (2007) “Blue” laccases. Biochem Mosc 72(10):1136–1150. https://doi.org/10.1134/s0006297907100112
Kudanga T, Nemadziva B, Le Roes-Hill M (2017) Laccase catalysis for the synthesis of bioactive compounds. Appl Microbiol Biotechnol 101(1):13–33
Hakulinen N, Rouvinen J (2015) Three-dimensional structures of laccases. Cell Mol Life Sci 72(5):857–868
Jones SM, Solomon EI (2015) Electron transfer and reaction mechanism of laccases. Cell Mol Life Sci 72(5):869–883
Scheiblbrandner S, Breslmayr E, Csarman F, Paukner R, Führer J, Herzog PL, Shleev SV, Osipov EM, Tikhonova TV, Popov VO, Haltrich D (2017) Evolving stability and pH-dependent activity of the high redox potential Botrytis aclada laccase for enzymatic fuel cells. Sci Rep 7(1):1–13
Claus H (2003) Laccases and their occurrence in prokaryotes. Arch Microbiol 179(3):145–150
Lu L, Zeng G, Fan C, Ren X, Wang C, Zhao Q, Zhang J, Chen M, Chen A, Jiang M (2013) Characterization of a laccase-like multicopper oxidase from newly isolated Streptomyces sp. C1 in agricultural waste compost and enzymatic decolorization of azo dyes. Biochem Eng J 72:70–76
Mayer AM, Staples RC (2002) Laccase: new functions for an old enzyme. Phytochemistry 60(6):551–565
Palanisami S, Saha SK, Lakshmanan U (2010) Laccase and polyphenol oxidase activities of marine cyanobacteria: a study with poly R-478 decolourization. World J Microbiol Biotechnol 26(1):63–69
Sami N, Fatma T (2019) Studies on estrone biodegradation potential of cyanobacterial species. Biocatal Agric Biotechnol 17:576–582
Chen H, Pan SS (2005) Bioremediation potential of spirulina: toxicity and biosorption studies of lead. J Zhejiang Univ Sci B 6(3):171–174
Afreen S, Anwer R, Singh RK, Fatma T (2018) Extracellular laccase production and its optimization from Arthrospira maxima catalyzed decolorization of synthetic dyes. Saudi J Biol Sci 25(7):1446–1453
Afreen S, Bano F, Ahmad N, Fatma T (2017) Screening and optimization of laccase from cyanobacteria with its potential in decolorization of anthraquinonic dye Remazol Brilliant Blue R. Biocatal Agric Biotechnol 10:403–410
Afreen S, Shamsi TN, Baig MA, Ahmad N, Fatima S, Qureshi MI, Hassan MI, Fatma T (2017) A novel multicopper oxidase (laccase) from cyanobacteria: purification, characterization with potential in the decolorization of anthraquinonic dye. PLoS ONE 12(4):e0175144
Sami N, Ansari S, Yasin D, Fatma T (2020) Estrone degrading enzymes of Spirulina CPCC-695 and synthesis of bioplastic precursor as a by-product. Biotechnol Rep 26:e00464
Madkour FF, Kamil AE-W, Nasr HS (2012) Production and nutritive value of Spirulina platensis in reduced cost media. Egypt J Aquat Res 38:51–57
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with the Folin phenol reagent. J Biol Chem 193:265–275
Bourbonnais R, Leech D, Paice MG (1998) Electrochemical analysis of the interactions of laccase mediators with lignin model compounds. Biochim Biophys Acta (BBA) 1379(3):381–390
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685
Zhao D, Zhang X, Cui D, Zhao M (2012) Characterisation of a novel white laccase from the deuteromycete fungus Myrothecium verrucaria NF-05 and its decolourisation of dyes. PLoS ONE 7(6):e38817
Lineweaver H, Burk D (1934) The determination of enzyme dissociation constants. J Am Chem Soc 56(3):658–666
Sei K, Takeda T, Soda SO, Fujita M, Ike M (2007) Removal characteristics of endocrine-disrupting chemicals by laccase from white-rot fungi. J Environ Sci Health Part A 43(1):53–60
Yoshida H (1883) LXIII.—chemistry of lacquer (Urushi): part I— communication from the chemical society of Tokio. J Chem Soc Trans 43:472–486
Chandra R, Chowdhary P (2015) Properties of bacterial laccases and their application in bioremediation of industrial wastes. Environ Sci Process Impacts 17(2):326–342
Otto B, Schlosser D (2014) First laccase in green algae: purification and characterization of an extracellular phenol oxidase from Tetracystis aeria. Planta 240(6):1225–1236
Otto B, Beuchel C, Liers C, Reisser W, Harms H, Schlosser D (2015) Laccase-like enzyme activities from chlorophycean green algae with potential for bioconversion of phenolic pollutants. FEMS Microbiol Lett. https://doi.org/10.1093/femsle/fnv072
Xu F, Kulys JJ, Duke K, Li K, Krikstopaitis K, Deussen HJW, Abbate E, Galinyte V, Schneider P (2000) Redox chemistry in laccase-catalyzed oxidation of N-hydroxy compounds. Appl Environ Microbiol 66(5):2052–2056
Mukhopadhyay M, Banerjee R (2015) Purification and biochemical characterization of a newly produced yellow laccase from Lentinus squarrosulus MR13. 3 Biotech 5(3):227–236
Aslam MS, Aishy A, Samra ZQ, Gull I, Athar MA (2012) Identification, purification and characterization of a novel extracellular laccase from Cladosporium cladosporioides. Biotechnol Biotechnol Equip 26(6):3345–3350
More SS, Renuka PS, Pruthvi K, Swetha M, Malini S, Veena SM (2011) Isolation, purification, and characterization of fungal laccase from Pleurotus sp. Enzyme Res 2011:1–7
Reinhammar B (1984) Copper proteins and copper enzymes, vol 3. CRC Press, Boca Raton
Atalla MM, Zeinab HK, Eman RH, Amani AY, Abeer AAEA (2013) Characterization and kinetic properties of the purified Trematosphaeria mangrovei laccase enzyme. Saudi J Biol Sci 20(4):373–381
Agrawal K, Verma P (2020) Multicopper oxidase laccases with distinguished spectral properties: a new outlook. Heliyon 6(5):e03972
Palmieri G, Giardina P, Bianco C, Scaloni A, Capasso A, Sannia G (1997) A novel white laccase from Pleurotus ostreatus. J Biol Chem 272(50):31301–31307
Zhou P, Fu C, Fu S, Zhan H (2014) Purification and characterization of white laccase from the white-rot fungus Panus conchatus. BioResources 9(2):1964–1976
Xu F (1996) Oxidation of phenols, anilines, and benzenethiols by fungal laccases: correlation between activity and redox potentials as well as halide inhibition. Biochemistry 35(23):7608–7614
Chefetz B, Chen Y, Hadar Y (1998) Purification and characterization of laccase from Chaetomium thermophilium and its role in humification. Appl Environ Microbiol 64(9):3175–3179
Kim Y, Yeo S, Kim MK, Choi HT (2008) Removal of estrogenic activity from endocrine-disrupting chemicals by purified laccase of Phlebia tremellosa. FEMS Microbiol Lett 284(2):172–175
Shin EH, Choi HT, Song HG (2007) Biodegradation of endocrine-disrupting bisphenol A by white rot fungus Irpex lacteus. J Microbiol Biotechnol 17(7):1147–1151
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(6):700–707
Prasad KK, Mohan SV, Bhaskar YV, Ramanaiah SV, Babu VL, Pati BR, Sarma PN (2005) Laccase production using Pleurotus ostreatus 1804 immobilized on PUF cubes in batch and packed bed reactors: influence of culture conditions. J Microbiol 43(3):301–307
Ferraroni M, Westphal AH, Borsari M, Tamayo-Ramos JA, Briganti F, de Graaff LH, van Berkel WJ (2017) Structure and function of Aspergillus niger laccase McoG. Biocatalysis 3(1):1–21
Gaur N, Narasimhulu K, Setty YP (2018) Extraction of ligninolytic enzymes from novel Klebsiella pneumoniae strains and its application in wastewater treatment. Appl Water Sci 8(4):1–17
Garg N, Bieler N, Kenzom T, Chhabra M, Ansorge-Schumacher M, Mishra S (2012) Cloning, sequence analysis, expression of Cyathus bulleri laccase in Pichia pastoris and characterization of recombinant laccase. BMC Biotechnol 12(1):1–12
Palanisami S, Lakshmanan U (2011) Role of copper in poly R-478 decolorization by the marine cyanobacterium Phormidium valderianum BDU140441. World J Microbiol Biotechnol 27(3):669–677
Munoz C, Guillen F, Martinez AT, Martinez MJ (1997) Induction and characterization of laccase in the ligninolytic fungus Pleurotus eryngii. Curr Microbiol 34(1):1–5
Xu F (1997) Effects of redox potential and hydroxide inhibition on the pH activity profile of fungal laccases. J Biol Chem 272(2):924–928
Karamyshev AV, Shleev SV, Koroleva OV, Yaropolov AI, Sakharov IY (2003) Laccase-catalyzed synthesis of conducting polyaniline. Enzyme Microb Technol 33(5):556–564
Park KM, Park SS (2008) Purification and characterization of laccase from basidiomycete Fomitella fraxinea. J Microbiol Biotechnol 18(4):670–675
Shin KS, Lee YJ (2000) Purification and characterization of a new member of the laccase family from the white-rot basidiomycete Coriolus hirsutus. Arch Biochem Biophys 384(1):109–115
Yuan X, Tian G, Zhao Y, Zhao L, Wang H, Ng TB (2016) Biochemical characteristics of three laccase isoforms from the basidiomycete Pleurotus nebrodensis. Molecules 21(2):203
Moreira-Neto SL, Matheus DR, Machado KMG (2009) Influence of pH on the growth, laccase activity and RBBR decolorization by tropical basidiomycetes. Braz Arch Biol Technol 52:1075–1082
Palonen H, Saloheimo M, Viikari L, Kruus K (2003) Purification, characterization and sequence analysis of a laccase from the ascomycete Mauginiella sp. Enzyme Microb Technol 33(6):854–862
Irshad M, Asgher M, Sheikh MA, Nawaz H (2011) Purification and characterization of laccase produced by Schyzophylum commune IBL-06 in solid state culture of banana stalks. BioResources 6(3):2861–2873
Paranjpe PS, Karve MS, Padhye SB (2003) Characterization of tyrosinase and accompanying laccase from Amorphophallus campanulatus. Indian J Biochem Biophys 40(1):40–45
Abou-Mansour E, Polier J, Pezet R, Tabacchi R (2009) Purification and partial characterisation of a 60 KDa laccase from Fomitiporia mediterranea. Phytopathol Mediterr 48(3):447–453
Rezaei S, Shahverdi AR, Faramarzi MA (2017) Isolation, one-step affinity purification, and characterization of a polyextremotolerant laccase from the halophilic bacterium Aquisalibacillus elongatus and its application in the delignification of sugar beet pulp. Biores Technol 230:67–75
Muthukumarasamy NP, Jackson B, Joseph Raj A, Sevanan M (2015) Production of extracellular laccase from Bacillus subtilis MTCC 2414 using agroresidues as a potential substrate. Biochem Res Int 2015:1–9
Yang X, Wu Y, Zhang Y, Yang E, Qu Y, Xu H, Chen Y, Irbis C, Yan J (2020) A thermo-active laccase isoenzyme from Trametes trogii and its potential for dye decolorization at high temperature. Front Microbiol 11:241
Zhang Y, Rochefort D (2011) Activity, conformation and thermal stability of laccase and glucose oxidase in poly (ethyleneimine) microcapsules for immobilization in paper. Process Biochem 46(4):993–1000
Wang Q, Ding L, Zhu C (2018) Characterization of laccase from a novel isolated white-rot fungi Trametes sp. MA-X01 and its potential application in dye decolorization. Biotechnol Biotechnol Equip 32(6):1477–1485
Karshikoff A, Nilsson L, Ladenstein R (2015) Rigidity versus flexibility: the dilemma of understanding protein thermal stability. FEBS J 282(20):3899–3917
Chakroun H, Mechichi T, Martinez MJ, 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(4):507–513
Robles A, Lucas R, Martı́nez-Cañamero M, Omar NB, Pérez R, Gálvez A (2002) Characterisation of laccase activity produced by the hyphomycete Chalara (syn Thielaviopsis) paradoxa CH32. Enzyme Microb Technol 31(4):516–522
Martins LO, Soares CM, Pereira MM, Teixeira M, Costa T, Jones GH, Henriques AO (2002) Molecular and biochemical characterization of a highly stable bacterial laccase that occurs as a structural component of the Bacillus subtilis endospore coat. J Biol Chem 277(21):18849–18859
Koroleva OV, Gavrilova VP, Stepanova EV, Lebedeva VI, Sverdlova NI, Landesman EO, Yavmetdinov IS, Yaropolov AI (2002) Production of lignin modifying enzymes by co-cultivated white-rot fungi Cerrena maxima and Coriolus hirsutus and characterization of laccase from Cerrena maxima. Enzyme Microb Technol 30(4):573–580
Zouari-Mechichi H, Mechichi T, Dhouib A, Sayadi S, Martinez AT, Martinez MJ (2006) Laccase purification and characterization from Trametes trogii isolated in Tunisia: decolorization of textile dyes by the purified enzyme. Enzyme Microb Technol 39(1):141–148
Dantán-González E, Vite-Vallejo O, Martínez-Anaya C, Méndez-Sánchez M, González MC, Palomares LA, Folch-Mallol J (2008) Production of two novel laccase isoforms by a thermotolerant strain of Pycnoporus sanguineus isolated from an oil-polluted tropical habitat. Int Microbiol 11(3):163–169
Ibrahim V, Mendoza L, Mamo G, Hatti-Kaul R (2011) Blue laccase from Galerina sp: properties and potential for Kraft lignin demethylation. Process Biochem 46(1):379–384
Park N, Park SS (2014) Purification and characterization of a novel laccase from Fomitopsis pinicola mycelia. Int J Biol Macromol 70:583–589
Olajuyigbe FM, Fatokun CO (2017) Biochemical characterization of an extremely stable pH-versatile laccase from Sporothrix carnis CPF-05. Int J Biol Macromol 94:535–543
Ezike TC, Udeh JO, Joshua PE, Ezugwu AL, Isiwu CV, Eze SO, Chilaka FC (2021) Substrate specificity of a new laccase from Trametes polyzona WRF03. Heliyon 7(1):e06080
Johannes C, Majcherczyk A (2000) Natural mediators in the oxidation of polycyclic aromatic hydrocarbons by laccase mediator systems. Appl Environ Microbiol 66(2):524–528
D’annibale A, Celletti D, Felici M, Di Mattia E, Giovannozzi-Sermanni G (1996) Substrate specificity of laccase from Lentinus edodes. Acta Biotechnol 16(4):257–270
Kersten PJ, Kalyanaraman B, Hammel KE, Reinhammar B, Kirk TK (1990) Comparison of lignin peroxidase, horseradish peroxidase and laccase in the oxidation of methoxybenzenes. Biochem J 268(2):475–480
Niladevi KN, Jacob N, Prema P (2008) Evidence for a halotolerant-alkaline laccase in Streptomyces psammoticus: purification and characterization. Process Biochem 43(6):654–660
Abdelgalil SA, Attia AR, Reyed RM, Soliman NA (2020) Partial purification and biochemical characterization of a new highly acidic NYSO laccase from Alcaligenes faecalis. J Genet Eng Biotechnol 18(1):1–11
Sadhasivam S, Savitha S, Swaminathan K, Lin FH (2008) Production, purification and characterization of mid-redox potential laccase from a newly isolated Trichoderma harzianum WL1. Process Biochem 43(7):736–742
Zhou C, Dong A, Wang Q, Yu Y, Fan X, Cao Y, Li T (2017) Effect of common metal ions and anions on laccase catalysis of guaiacol and lignocellulosic fiber. BioResources 12(3):5102–5117
Wang X, Hu J, Liang Y, Zhan H (2011) Effects of metal ions on laccase activity. Asian J Chem 23(12):5422
Ryan S, Schnitzhofer W, Tzanov T, Cavaco-Paulo A, Gübitz GM (2003) An acid-stable laccase from Sclerotium rolfsii with potential for wool dye decolourization. Enzyme Microb Technol 33(6):766–774
Tadkaew N, Hai FI, McDonald JA et al (2011) Removal of trace organics by MBR treatment: the role of molecular properties. Water Res 45:2439–2451
Yang S, Hai FI, Nghiem LD et al (2013) Understanding the factors controlling the removal of trace organic contaminants by white-rot fungi and their lignin modifying enzymes: a critical review. Biores Technol 141:97–108
Acknowledgements
Authors are thankful to Culture Collection Centres of India, University of Madras, for providing the Spirulina CPCC-695 species.
Author information
Authors and Affiliations
Contributions
NS and TF designed the experiment, BA helped in the protein purification, DY helped in data curation. NS carried out the lab work and wrote the initial manuscript that was corrected and finalised by TF.
Corresponding author
Ethics declarations
Competing interest
The authors declare that they have no known competing interests.
Additional information
Publisher's Note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Rights and permissions
Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.
About this article
Cite this article
Sami, N., Afzal, B., Yasin, D. et al. Biochemical Characterization of Laccase from Spirulina CPCC-695 and Their Role in Estrone Degradation. Protein J 43, 115–128 (2024). https://doi.org/10.1007/s10930-023-10169-7
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s10930-023-10169-7