НОВИ ЕНЗИМИ ОТ ГРУПАТА НА СИАЛИДАЗИТЕ ПРИ ФИЛАМЕНТОЗНИ ГЪБИ (NEW ENZYMES OF THE SIALIDASE GROUP IN FILAMENTOUS FUNGI)

Abstract

This monograph presents the results of the research results of the author's team on the distribution of the sialidase enzyme in strains of filamentous fungi isolated from different habitats, the mechanisms of enzyme synthesis regulation, the preparation of a purified enzyme and the characterization of its properties. To our knowledge, this is the first study in this scientific field. The study includes strains isolated from various non-clinical substrates such as heavy metal-contaminated soils, decaying wood, bat guano murals from Magurata Cave, ancient tombs, mosses, lichens, and medicinal plants. Attention was also paid to fungi from habitats with extreme cold temperatures. The results revealed that more than 70% of the strains tested synthesized the enzyme sialidase. The sialidase-positive strains belong to 11 orders, 14 families, and 16 genera. The dependence of sialidase synthesis on the habitat of the isolated strains was demonstrated. The highest prevalence of this enzyme was found in isolates from ancient caves (100%), followed by those from extremely cold habitats such as Antarctica and Alaska (91%), endophytes (83%), and bat guano murals (62%). An efficient sialidase producer isolated from Terra Nova Bay, Antarctica, was selected. The strain was identified to species as Penicillium griseofulvum and deposited at the National Bank for Industrial Microorganisms and Cell Cultures, Bulgaria (NBIMCC 9106). Cultivation parameters were optimized and laboratory technology was developed to produce the enzyme at scale in 3 L bioreactors. Of interest is the identification of the sialidase gene in strains that synthesize the enzyme and in those that can be defined as sialidase-negative. A similar phenomenon is also reflected in the scientific literature, suggesting the involvement of regulation at the level of transcription or the manifestation of gene silencing. Sialidase synthesis by the Antarctic strain P. griseofulvum P29 is regulated by control mechanisms induction and catabolite repression. The present study is the first published in the literature for a fungal sialidase and, in particular, for a temperature-sensitive enzyme. Changes in enzyme synthesis in the presence of inducer and repressor are consistent with sialidase gene expression, defining transcription as the primary level of regulation. The authors are the first to demonstrate the involvement of the enzyme sialidase in the cellular response to oxidative stress in filamentous fungi. The 89 data illustrated the relationship between exposure to low temperature and biomarkers of oxidative stress. Increased sialidase activity was observed in parallel with the accelerated synthesis of the antioxidant enzymes SOD and CAT. An efficient laboratory procedure was developed for the isolation of pure P29 sialidase enzyme from P. griseofulvum strain. The purity of the enzyme was confirmed electrophoretically by SDS-PAGE and by mass spectrometric analysis. The exact molecular mass of the new sialidase P29 enzyme, determined by MALDI-TOF/MS analysis, is 39924.40 Da. Amino acid sequence analysis of 7 peptides extracted after trypsinolysis of the protein band at 40 kDa from 12.0% SDS-PAGE confirmed that the isolated enzyme is a sialidase with high homology to neuraminidases from P. griseofulvum, P. rubens Wisconsin 54-1255 and P. camemberti (with 98.3%, 90.9% and 91.8% identity, respectively). These results provide further evidence that the isolated enzyme belongs to the sialidase family. The new sialidase enzyme from P. griseofulvum shows a temperature optimum at 37C and high stability at low temperatures, characterizing it as a temperature-sensitive (cold-active) enzyme. It has a relatively narrow substrate specificity compared to similar bacterial sialidases. A 3D structural model of the new sialidase was constructed based on its primary structure and on the 3D structure of P. patulum sialidase (KXG51741.1 neuraminidase) using Swiss-Prot, BLAST, RasWin, and graphical processing with Swiss-Pdb Viewer. The spatial structure of P29 sialidase is necessary to explain the structure and dynamics in the conformational changes of the enzyme at different pH. The results of fluorescence studies show that the purified enzyme retains its conformational stability in aqueous solution at pH 6.5 to 8.5. Interestingly, the conformational stability of the enzyme was higher in alkaline than in acidic regions, although the maximum enzyme activity was found at pH 4.0. To date, few sialidase producers from non-pathogenic strains are known. At the same time, such sialidases are more suitable for food or medical purposes. The selection of a producer from the group of filamentous fungi isolated from non-clinical sources may meet the need for such an enzyme.