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

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.

Biological decolorization of Amaranth dye with Trametes polyzona in an airlift reactor under three airflow regimes.

In the present work, a strain of the basidiomycete fungus Trametes polyzona was used to decolorize the Amaranth dye. The decolorization was carried out in an Airlift reactor with three flow regimes: 1, 2, and 3 vvm. The results showed that the decolorization was a function of the flow regime. The decolorization times for the regimes of 1, 2, and 3 vvm were 30, 25, and 19 days, respectively. The COD (Chemical Oxygen Demand) decreased from 1600 to 72 mg COD/L. The enzymatic activity kinetics of laccase (Lcc), lignin peroxidase (LiP), and manganese peroxidase (MnP) were determined. In all the treatments, the enzyme LiP was expressed during the first 6 days, at which point 80% decolorization was observed, whereas Lcc and MnP enzymes were produced from day 6 until the end of the decolorization process. The effluent generated showed no inhibitory effects on the growth of the algae Nannochloropsis salina. T. polyzona showed great versatility in the decolorization of synthetic effluents containing the Amaranth dye, and the fungus was able to use this dye as its only carbon source starting at the beginning of the process. LiP was the enzyme that contributed the most to the decolorization process, and on average, 95% decreases in color and the COD were observed.