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.

在本研究中，通过酶谱技术设计了多酚氧化酶和酚氧化酶的连续染色过程。第一步，在自然条件下进行电泳，然后，使用 3-甲基-2-苯并噻唑啉腙 (MBTH) - 3-二甲氨基苯甲酸 (DMAB) 的显色溶液进行第一次染色，以实现可视化多酚氧化酶，然后使用相同的凝胶，我们继续进行酚氧化酶的差异染色，添加H ₂ O ₂溶液。该技术使用漆酶（T. versicolor）和辣根的商业酶进行标准化。该技术用于鉴定从辐射松上的担子菌Lentinus strigosus生长获得的粗提物中的多酚氧化酶（漆酶）和酚氧化酶（木质素过氧化物酶） 。该技术在检测同一凝胶中的不同酶活性（最小 1.56 活性单位/mL）方面表现出极高的灵敏度，且酶和所用溶液之间不会产生干扰。另一方面，将该技术的效率与常用于检测此类活性的底物（例如 2,2'-偶氮基双（3-乙基苯并噻唑啉-6-磺酸）二铵盐）进行了比较。 ABTS）和愈创木酚，观察到更高的灵敏度和最小的干扰，因此本方法将允许在同一凝胶中同时可视化多酚氧化酶和酚氧化酶活性，从而促进表达研究。