Abstract
In the present study, a sequential staining process of polyphenoloxidase and phenoloxidase enzymes was designed by the zymography technique. As a first step, electrophoresis was carried out under native conditions, and later, first staining was carried out with a revealing solution of 3-methyl-2-benzothiazoline hydrazone (MBTH)—3-dimethylamino benzoic acid (DMAB) that allowed the visualization of polyphenoloxidase enzymes, and later and using the same gel, we proceeded to the differential staining of phenoloxidase, adding a solution of H2O2. The technique was standardized using commercial enzymes of laccase (T. versicolor) and horseradish. The technique was used to identify polyphenoloxidases (laccases) and phenoloxidases (lignin peroxidase) of crude extracts obtained from the growth of the basidiomycete Lentinus strigosus on Pinus radiata. The technique showed great sensitivity to detect the different enzymatic activities (1.56 Activity Unit/mL minimum) in the same gel without interference between the enzymes and the solutions used. On the other hand, the efficiency of the technique was compared with the substrates that are commonly used for the detection of this type of activities such as 2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) and guaiacol, observing greater sensitivity and minimal interference, so that the present method will allow in the same gel, and visualize polyphenoloxidase and phenoloxidase activities simultaneously facilitating expression studies.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
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. https://doi.org/10.1371/journal.pone.0175144
Berthet S, Thevenin J, Baratiny D, Demont-Caulet N, Debeaujon I, Bidzinski P, Lapierre C (2012) Role of plant laccases in lignin polymerization. Adv Bot Res Academic Press 61:145–172. https://doi.org/10.1016/B978-0-12-416023-1.00005-7
Bertrand B, Martínez-Morales F, Tinoco-Valencia R, Rojas S, Acosta-Urdapilleta L, Trejo-Hernández MR (2015) Biochemical and molecular characterization of laccase isoforms produced by the white-rot fungus Trametes versicolor under submerged culture conditions. J Mol Catal B Enzym 122:339–347. https://doi.org/10.1016/j.molcatb.2015.10.009
Bouacem K, Rekik H, Jaouadi NZ, Zenati B, Kourdali S, El Hattab M, Badis A, Annane R, Beja S, Hacene H, Bouanane-Darenfed A, Jauaadi B (2018) Purification and characterization of two novel peroxidases from the dye-decolorizing fungus Bjerkandera adusta strain CX-9. Int J Biol Macromol 106:636–646. https://doi.org/10.1016/j.ijbiomac.2017.08.061
Castillo MD, Stenstrom J, Ander P (1994) Determination of manganese peroxidase activity with 3-methyl-2-benzothiazolinone hydrazone and 3-(dimethylamino) benzoic acid. Anal Biochem 218(2):399–404. https://doi.org/10.1006/abio.1994.1198
Chen YR, Sarkanen S, Wang YY (2012) Lignin-degrading enzyme activities. In: Biomass conversion. Humana Press, Totowa, NJ. pp 251–268. Doi: https://doi.org/10.1007/978-1-61779-956-3_21
Cilerdzic J, Galic M, Vukojevic J, Brceski I, Stajic M (2017) Potential of selected fungal species to degrade wheat straw, the most abundant plant raw material in Europe. BMC Plant Biol 17(2):75–81. https://doi.org/10.1186/s12870-017-1196-y
Francoz E, Ranocha P, Nguyen-Kim H, Jamet E, Burlat V, Dunand C (2015) Roles of cell wall peroxidases in plant development. Phytochemistry 112:15–21. https://doi.org/10.1016/j.phytochem.2014.07.020
Haemmerli SD, Schoemaker HE, Schmidt HW, Leisola MS (1987) Oxidation of veratryl alcohol by the lignin peroxidase of Phanerochaete chrysosporium Involvement of activated oxygen. FEBS Lett 220(1):149–154. https://doi.org/10.1016/0014-5793(87)80893-1
Halpin B, Pressey R, Jen J, Mondy N (1989) Purification and characterization of peroxidase isoenzymes from green peas (Pisum sativum). J Food Sci 54(3):644–649. https://doi.org/10.1111/j.1365-2621.1989.tb04672.x
Hemeda HM, Klein BP (1991) Inactivation and regeneration of peroxidase activity in vegetable extracts treated with antioxidants. J Food Sci 56(1):68–71. https://doi.org/10.1111/j.1365-2621.1991.tb07977.x
Higuera BL, Martínez S, Caicedo O (2009) Inducción de actividad peroxidasa y de fenoles totales como respuesta del fruto de lulo (Solanum quitoense l.) al patógeno causal de la antracnosis. Rev Col Quim 38(1):25–42
Iracheta-Cárdenas MM, Rocha-Peña MA, Galán-Wong LJ, Arévalo-Niño K, Tovar-Herrera OE (2016) A Pycnoporus sanguineus laccase for denim bleaching and its comparison with an enzymatic commercial formulation. J Environ Manage 177:93–100. https://doi.org/10.1016/j.jenvman.2016.04.008
Jain KK, Kumar A, Shankar A, Pandey D, Chaudhary B, Sharma KK (2020) De novo transcriptome assembly and protein profiling of copper-induced lignocellulolytic fungus Ganoderma lucidum MDU-7 reveals genes involved in lignocellulose degradation and terpenoid biosynthetic pathways. Genomics 112(1):84–198. https://doi.org/10.1016/j.ygeno.2019.01.012
Janusz G, Kucharzyk KH, Pawlik A, Staszczak M, Paszczynski AJ (2013) Fungal laccase, manganese peroxidase and lignin peroxidase: gene expression and regulation. Enzyme Microb Technol 52(1):1–12. https://doi.org/10.1016/j.enzmictec.2012.10.003
Karp SG, Faraco V, Amore A, Birolo L, Giangrande C, Soccol VT, Pandey A, Soccol CR (2012) Characterization of laccase isoforms produced by Pleurotus ostreatus in solid state fermentation of sugarcane bagasse. Bioresour Technol 114:735–739. https://doi.org/10.1016/j.biortech.2012.03.058
Khozani MA, Emtiazi G, Aghaei SS, Ghasemi SM, Zolfaghari MR (2020) Application of fungal laccase for heavy metals precipitation using tannin as a natural mediator. Int J Environ Sci Technol. https://doi.org/10.1007/s13762-020-02992-7
Kumar A, Singh D, Sharma KK, Arora S, Singh AK, Gill SS, Singhal B (2017) Gel-based purification and biochemical study of laccase isozymes from Ganoderma sp. and its role in enhanced cotton callogenesis. Front Microbiol 8:674. https://doi.org/10.3389/fmicb.2017.00674
Kuwahara M, Glenn JK, Morgan MA, Gold MH (1984) Separation and characterization of two extracelluar H2O2-dependent oxidases from ligninolytic cultures of Phanerochaete chrysosporium. FEBS Lett 169(2):247–250. https://doi.org/10.1016/j.heliyon.2020.e03170
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227(5259):680–685. https://doi.org/10.1038/227680a0
Li K, Helm RF, Eriksson KEL (1998) Mechanistic studies of the oxidation of a non‐phenolic lignin model compound by the laccase/1‐hydroxybenzotriazole redox system. Biotechnol Appl Bioc 27(3):239–243. https://iubmb.onlinelibrary.wiley.com/doi/abs/. Doi: https://doi.org/10.1111/j.1470-8744.1998.tb00500.x
Liu EH, Gibson DM (1997) Visualization of peroxidase isozymes with eugenol, a noncarcinogenic substrate. Anal Biochem 79(1–2):597–601. https://doi.org/10.1016/0003-2697(77)90439-0
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. https://doi.org/10.4061/2011/248735
Navarro D, Rosso MN, Haon M, Olivé C, Bonnin E, Lesage-Meessen L, Chevret L, Coutinho PM, Henrissat B, Berrin JG (2014) Fast solubilization of recalcitrant cellulosic biomass by the basidiomycete fungus Laetisaria arvalis involves successive secretion of oxidative and hydrolytic enzymes. Biotechnol Biofuels 7(1):1–14. https://doi.org/10.1186/s13068-014-0143-5
Ngo TT, Lenhoff HM (1980) A sensitive and versatile chromogenic assay for peroxidase and peroxidase-coupled reactions. Anal Biochem 105(1):389–397. https://doi.org/10.1016/0003-2697(80)90475-3
Oliveira HP, Silva RG, Oliveira JT, Sousa DO, Pereira ML, Souza PF, Soares AA, Gomes VM, Mosteiro-Moreira ACO, Moreno FBMB, Vasconcelos IM (2017) A novel peroxidase purified from Marsdenia megalantha latex inhibits phytopathogenic fungi mediated by cell membrane permeabilization. Int J Biol Macromol 96:743–753. https://doi.org/10.1016/j.ijbiomac.2016.12.083
Oloketuyi S, Annovi G, De Marco A (2020) Peroxidase zymograms obtained by agarose native gel electrophoresis have unmet resolution and completeness. Int J Biol Macromol 156(1):869–873. https://doi.org/10.1016/j.ijbiomac.2020.04.058
Pütter J, Becker R (1983) Peroxidase. In: Methods of enzymatic analysis. Vol 3. HU Bergmeyer (Ed.). pp 286–293.
Quintanar-Gómez S, Arana-Cuenca A, Mercado-Flores Y, Gracida-Rodríguez, JN, y Téllez-Jurado A (2012) Effect of particle size and aeration on the biological delignification of corn straw using Trametes sp. 44. BioResource. 7(1):327–344. https://ojs.cnr.ncsu.edu/index.php/BioRes/article/view/BioRes_07_1_0327_QuintanrG_CFRJ_Particle_Size_Aeration_Bio_Delignification
Sawicki E, Stanley TW, Hauser TR, Elbert W, Nee JL (1961) Mass spectra of aliphatic thiols and sulfides. Anal Chem 33(6):702–722. https://doi.org/10.1021/ac60174a018
Suzuki T, Honda Y, Mukasa Y, Kim SJ (2006) Characterization of peroxidase in buckwheat seed. Phytochemistry 67(3):219–224. https://doi.org/10.1016/j.phytochem.2005.11.014
Téllez-Téllez M, Sánchez C, Díaz R, Díaz-Godínez G (2012) Zymogram patterns of extracellular laccases of Pleurotus species grown on non-inducer agar medium. Rev Mex Ing Quim 11(3):383–388. http://www.scielo.org.mx/scielo.php?script=sci_arttext&pid=S1665-27382012000300003&lng=es&tlng=en
Tonami H, Uyama Nagahata R, Kobayashi S (2004) Guaiacol oxidation products in the enzyme-activity assay reaction by horseradish peroxidase catalysis. Chem Lett 33(7):796–797. https://doi.org/10.1246/cl.2004.796
Vantamuri AB, Kaliwal BB (2016) Purification and characterization of laccase from Marasmius species BBKAV79 and effective decolorization of selected textile dyes. 3 Biotech 6(2):1–10. https://doi.org/10.1007/s13205-016-0504-9
Wang KF, Hu JH, Guo C, Liu CZ (2016) Scale-up laccase production from Trametes versicolor stimulated by vanillic acid. Bioproc Biosyst Eng 39(7):1041–1049. https://doi.org/10.1007/s00449-016-1582-0
Wilkesman J, Castro D, Contreras LM, Kurz L (2014) Guaiacol peroxidase zymography for the undergraduate laboratory. Biochem Mol Biol Edu 42(5):420–426. https://doi.org/10.1002/bmb.20809
Wong DW (2009) Structure and action mechanism of ligninolytic enzymes. Appl Biochem Biotechnol 157(2):174–209. https://doi.org/10.1007/s12010-008-8279-z
Zerva A, Zervakis GI, Christakopoulos P, Topakas E (2017) Degradation of olive mill wastewater by the induced extracellular ligninolytic enzymes of two wood-rot fungi. J Environ Manage 203:791–798. https://doi.org/10.1016/j.jenvman.2016.02.042
Acknowledgements
This work was carried out thanks to the support of the National Council of Science and Technology (CONACyT) of Mexico.
Author information
Authors and Affiliations
Contributions
YG-E: fomal analysis, methodology, investigation, and writing—original draft. YM-F: validation, conceptualization, and supervisión. MAA-R: software, formal analysis, and data curation. JÁ-C: visualization and supervisión. EA-vW: validation, formal analysis, and supervisión. AIM-R: conceptualisation and supervisión. AT-J: conceptualization, writing review and editing, resources, project administration, and supervision.
Corresponding author
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest in the publication.
Ethical approval
This paper does not contain any studies with human participants or animals performed by any of the authors.
Rights and permissions
About this article
Cite this article
García-Esquivel, Y., Mercado-Flores, Y., Anducho-Reyes, M.A. et al. 3-Methyl-2-benzothiazolinone hydrazone and 3-dimethylamino benzoic acid as substrates for the development of polyphenoloxidase and phenoloxidase activity by zymograms. 3 Biotech 11, 39 (2021). https://doi.org/10.1007/s13205-020-02622-6
Received:
Accepted:
Published:
DOI: https://doi.org/10.1007/s13205-020-02622-6