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.

Isolation and Selection of Streptomyces Species from Semi-arid Agricultural Soils and Their Potential as Producers of Xylanases and Cellulases.

The Mezquital Valley (MV), Mexico, is a semi-arid region whose main economic activity is agriculture, this zone is characterized by the use of wastewater for crop irrigation. This condition has increased the amount nutrients in soils, organic carbon content and native microorganisms. The Streptomyces species are a group of saprophytic bacteria that represent between 20 and 60% of the total microbial population in soils, capable of producing metabolites of commercial importance. In this work, Streptomyces species were isolated from agricultural soils of the MV and was evaluated the production of endoglucanases (CMCase) and xylanases (Xyl) in Solid-State Cultivation (SSC). From soil samples, 73 possible strains of Streptomyces species were isolated for their ability to produce CMCase and Xyl in SSC. The study also included its characterization by morphological characteristics. Of the isolated microorganisms, 38 strains were selected as strong enzyme producers according to the measurement of the halo generated in plate and by growth on barley straw as only carbon source. Two different sizes of barley straw particle were tested, finding that the greatest enzymatic activity was observed in particle size 12. Three strains of Streptomyces species were chosen which presented the best catalytic capacities, a maximum of 100.69 AU Xyl/gram dry matter (gdm), 82 AU Xyl/gdm and 26.02 AU CMCase/gdm for strains 30, 28 and 12, respectively. The strains were identified by ribosomal gen16s sequence and identified as S. flavogriseus, S. virginiae and S. griseoaurantiacus. It is the first report of endogluconase and xylanolytic activity by S. virginiae isolated from a semi-arid soil.

Use of barley straw as a support for the production of conidiospores of Trichoderma harzianum.

In this work was to evaluate the conidiospores production of Trichoderma harzianum using barley straw as substrate. Four growth conditions were used; washed and unwashed barley straw and washed and unwashed barley straw supplemented with mineral salts. The highest spore production was observed when washed barley straw supplemented with mineral salts with 1.56 × 1010 conidiospores/gram of dry matter (gdm) at 216 h of cultivation was used. The effect of substrate moisture on spore production was studied, three initial moisture levels of the substrate were tested and it was observed that a humidity of 80 % of the substrate improves the production of conidiospores reaching a concentration of 2.35 × 1010 conidiospores/gdm at 136 h. Finally, conidiospores viability was evaluated for 12 months by keeping them on the conidia and substrate, and viability of 71 % of the conidiospores was observed, so this maintenance method is an excellent means of conserving the conidiospores viability.

Screening for thermotolerant ligninolytic fungi with laccase, lipase, and protease activity isolated in Mexico.

The State of Hidalgo (Mexico) has a large area of forests known as the Huasteca Hidalguense, with a large variety of microorganisms inhabiting it. They represent an important resource from the ecological and technological point of view because they can be used in a broad variety of industrial processes. Due to the climatic conditions of this region, fungi inhabiting it must be thermophile or, at least, thermotolerant, as temperatures can be higher than 45°C in the summer, declining to 20°C in the winter. Use of ligninolytic fungi relies on their capacity to produce enzymes of industrial interest, a topic that has been under continuous research by academic and industrial investigators. Among the most important enzymes are proteases that are widely used due to their biotechnological applications with a high economic impact. Other enzymes, laccases, peroxidases, and lipases are of interest for the industries of the state of Hidalgo, especially in the textile industry, specifically in effluent processing. Fungi (n=156) were collected in the Huasteca Hidalguense, of which 100 were isolated in potato-dextrose-agar covered plates and maintained in tilted tubes. Afterwards, enzymatic activity (laccase, protease and lipase) was determined in the plates. The purpose was to select those fungi with the highest potential for biotechnological applications. Fungi generally grew at either 30°C or 37°C, and for some isolates enzymatic activities were detected at this higher temperature. Results are presented as the relation between enzymatic activity and growth rate: 60 fungi presented laccase activity, 49 had lipase activity, and none had protease activity. In most cases, enzymatic activity was higher than the growth rate, indicating that the isolated fungi have a great biotechnological potential. Statistical analysis revealed that isolates 31 (Trametes) and 8.1 (unidentified) have a larger potential to be studied as laccase-producing fungi. On the other hand, isolates 144.2 (Fomes), 154 (Trametes), and 147.2 (Pycnoporus) are of interest as lipase activity producers, an activity scarcely studied in this type of microorganisms.