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3-Methyl-2-benzothiazolinone hydrazone and 3-dimethylamino benzoic acid as substrates for the development of polyphenoloxidase and phenoloxidase activity by zymograms

Authors
  • García-Esquivel, Y.1
  • Mercado-Flores, Y.1
  • Anducho-Reyes, M. A.1
  • Álvarez-Cervantes, J.1
  • Wobeser, E. Aguirre-von2
  • Marina-Ramírez, A. I.3
  • Téllez-Jurado, A.1
  • 1 Universidad Politécnica de Pachuca, Carretera Pachuca-Cd. Sahagún, km 20, ExHacienda de Santa Bárbara, Zempoala, Hidalgo, C.P. 43830, México , Zempoala (Mexico)
  • 2 Centro de Investigación en Agrobiotecnología Alimentaria, Cd. del Conocimiento, Boulevard Circuito La Concepción, San Agustín Tlaxiaca, Hidalgo, C.P. 42162, México , San Agustín Tlaxiaca (Mexico)
  • 3 Centro de Biología Molecular Severo Ochoa, Nicolás Cabrera No. 1, Madrid, C.P. 28049, Spain , Madrid (Spain)
Type
Published Article
Journal
3 Biotech
Publisher
Springer International Publishing
Publication Date
Jan 08, 2021
Volume
11
Issue
2
Identifiers
DOI: 10.1007/s13205-020-02622-6
Source
Springer Nature
Keywords
License
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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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