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.
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This work was carried out thanks to the support of the National Council of Science and Technology (CONACyT) of Mexico.
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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.
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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
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DOI: https://doi.org/10.1007/s13205-020-02622-6