In silico Design of Laccase Thermostable Mutants From Lacc 6 of Pleurotus Ostreatus

Fungal laccase enzymes have a great biotechnological potential for bioremediation processes due to their ability to degrade compounds such as ρ-diphenol, aminophenols, polyphenols, polyamines, and aryldiamines. These enzymes have activity at different pH and temperature values, however, high temperatures can cause partial or total loss of enzymatic activity, so it is appropriate to do research to modify their secondary and/or tertiary structure to make them more resistant to extreme temperature conditions. In silico, a structure of the Lacc 6 enzyme of Pleurotus ostreatus was constructed using a laccase of Trametes versicolor as a template. From this structure, 16 mutants with possible resistance at high temperature due to ionic interactions, salt bridges and disulfide bonds were also obtained in silico. It was determined that 12 mutants called 4-DB, 3-DB, D233C-T310C, F468P, 3-SB, L132T, N79D, N372D, P203C, P203V, T147E, and W85F, presented the lowest thermodynamic energy. Based on the previous criterion and determining the least flexibility in the protein structures, three mutants (4-DB, 3-DB, and P203C) were selected, which may present high stability at high temperatures without affecting their active site. The obtained results allow the understanding of the molecular base that increase the structural stability of the enzyme Lacc 6 of Pleurotus ostreatus, achieving the in silico generation of mutants, which could have activity at high temperatures

Isolation of Bacteria with Antifungal Activity against the Phytopathogenic Fungi Stenocarpella maydis and Stenocarpella macrospora

Stenocarpella maydis and Stenocarpella macrospora are the causal agents of ear rot in corn, which is one of the most destructive diseases in this crop worldwide. These fungi are important mycotoxin producers that cause different pathologies in farmed animals and represent an important risk for humans. In this work, 160 strains were isolated from soil of corn crops of which 10 showed antifungal activity against these phytopathogens, which, were identified as: Bacillus subtilis, Pseudomonas spp., Pseudomonas fluorescens, and Pantoea agglomerans by sequencing of 16S rRNA gene and the phylogenetic analysis. From cultures of each strain, extracellular filtrates were obtained and assayed to determine antifungal activity. The best filtrates were obtained in the stationary phase of B. subtilis cultures that were stable to the temperature and extreme pH values; in addition they did not show a cytotoxicity effect against brine shrimp and inhibited germination of conidia. The bacteria described in this work have the potential to be used in the control of white ear rot disease

β-Xylosidase SRBX1 Activity from <i>Sporisorium reilianum</i> and Its Synergism with Xylanase SRXL1 in Xylose Release from Corn Hemicellulose

Sposisorium reilianum is the causal agent of corn ear smut disease. Eleven genes have been identified in its genome that code for enzymes that could constitute its hemicellulosic system, three of which have been associated with two Endo-β-1,4-xylanases and one with α-L-arabinofuranosidase activity. In this study, the native protein extracellular with β-xylosidase activity, called SRBX1, produced by this basidiomycete was analyzed by performing production kinetics and its subsequent purification by gel filtration. The enzyme was characterized biochemically and sequenced. Finally, its synergism with Xylanase SRXL1 was determined. Its activity was higher in a medium with corn hemicellulose and glucose as carbon sources. The purified protein was a monomer associated with the sr16700 gene, with a molecular weight of 117 kDa and optimal activity at 60 °C in a pH range of 4–7, which had the ability to hydrolyze the ρ-nitrophenyl β-D-xylanopyranoside and ρ-Nitrophenyl α-L-arabinofuranoside substrates. Its activity was strongly inhibited by silver ions and presented Km and Vmax values of 2.5 mM and 0.2 μmol/min/mg, respectively, using ρ-nitrophenyl β-D-xylanopyranoside as a substrate. The enzyme degrades corn hemicellulose and birch xylan in combination and in sequential synergism with the xylanase SRXL1