Action of Extracellular Proteases of Aspergillus flavus and Aspergillus ochraceus Micromycetes on Plasma Hemostasis Proteins.

In this study, we investigated the properties of proteolytic enzymes of two species of Aspergillus, Aspergillus flavus 1 (with a high degree of pathogenicity) and Aspergillus ochraceus L-1 (a conditional pathogen), and their effects on various components of the hemostasis system (in vitro) in the case of their penetration into the bloodstream. We showed that micromycete proteases were highly active in cleaving both globular (albuminolysis) and fibrillar (fibrin) proteins, and, to varying degrees, they could coagulate the plasma of humans and animals (due to proteolysis of factors of the blood coagulation cascade) but were not able to coagulate fibrinogen. The proteases of both Aspergillus fully hydrolyzed thrombi in 120-180 min. Micromycetes did not show hemolytic activity but were able to break down hemoglobin.

Vermiculite as a new carrier for extracellular protease production by Aspergillus spp. under solid-state fermentation.

A new method has been developed to increase the productivity of aspergilli - producers of extracellular proteinases based on their cultivation on vermiculite under solid-state fermentation conditions. The productivity of the mycelium Aspergillus ochraceus L-1 and Aspergillus ustus 1 was 3-18 times higher not only in comparison with submerged cultivation, but also in comparison with growth on other carriers studied under solid-state fermentation conditions. Vermiculite can be considered as a new promising carrier for solid-state fermentation of micromycetes.

Combined microbiological approach to screening of producers of proteases with hemostasis system proteins activity among micromycetes.

A scheme for screening of micromycetes - producers of proteases with the activity of hemostasis system proteins, based on their enzymatic indices determination and the activity towards chromogenic peptide substrates for proteins of the hemostasis system was developed. Depending on the ability of proteases producers to cleave such substrates, an enzymatic reaction in conditions containing human plasma is suggested, which makes it possible to identify the potentiality of the target plasma hemostasis proenzymes activation.

Structural basis for the fast maturation of Arthropoda green fluorescent protein.

Since the cloning of Aequorea victoria green fluorescent protein (GFP) in 1992, a family of known GFP-like proteins has been growing rapidly. Today, it includes more than a hundred proteins with different spectral characteristics cloned from Cnidaria species. For some of these proteins, crystal structures have been solved, showing diversity in chromophore modifications and conformational states. However, we are still far from a complete understanding of the origin, functions and evolution of the GFP family. Novel proteins of the family were recently cloned from evolutionarily distant marine Copepoda species, phylum Arthropoda, demonstrating an extremely rapid generation of fluorescent signal. Here, we have generated a non-aggregating mutant of Copepoda fluorescent protein and solved its high-resolution crystal structure. It was found that the protein beta-barrel contains a pore, leading to the chromophore. Using site-directed mutagenesis, we showed that this feature is critical for the fast maturation of the chromophore.