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Starch catabolism leading to high glucose level in blood is highly problematic in chronic metabolic diseases, such as type II diabetes and obesity. α-Amylase catalyzes the hydrolysis of starch, increasing blood sugar concentration. Its inhibition represents a promising therapeutic approach to control hyperglycaemia. However, only few drug-like molecule inhibitors without sugar moieties have been discovered so far, and little information on the enzymatic mechanism is available. This work aims at the discovery of novel small α-amylase binders using a systematic in silico methodology. 3D-pharmacophore-based high throughput virtual screening of small compounds libraries was performed to identify compounds with high α-amylase affinity. Twenty-seven compounds were selected and biologically tested, revealing IC50 values in the micromolar range and ligand efficiency higher than the one of the bound form of acarbose, which is used as a reference for α-amylase inhibition.

Dispersin B (DspB) from Aggregatibacter actinomycetemcomitans is a β-hexosaminidase exhibiting biofilm detachment activity. A series of β-(1→6)-linked N-acetyl-D-glucosamine thiophenyl glycosides with degree of polymerisation (DP) of 2, 3, 4 and 5 were synthesized, and substrate specificity of DspB was studied on the obtained oligosaccharides. For oligomer synthesis a 1+2, 2+2, 1+4 coupling strategy was applied, using bromo-sugars as glycosyl donors. The formation of 1,2-trans interglycosidic bond has been ensured by 2-phtalimido protecting group; chloroacetyl group was installed to mask temporarily the 6-hydroxyl and acetate esters were applied as permanent protecting groups. Enzymatic studies revealed that DP of the GlcNAc oligomers strongly affected the hydrolysis rate, and the hydrolytic activity of DspB on the tetramer and pentamer have been found to be approximately 10-fold higher than that of the dimer. This fact indicates that four units are required for a strong binding at the active centre of DspB. The role of aromatic amino acids W237, Y187 and Y278 in substrate specificity and catalysis was also examined using mutant enzymes.

Iron is an essential element for all microorganisms. Bacteria and fungi produce versatile siderophores for binding and storing this essential transition metal when its availability is limited in the environment. The aim of the study was to optimize the fermentation medium of Aspergillus fumigatus for siderophore production. Triacetyl-fusarinine C and ferricrocin yields were dependent on glucose and glycine supplementations as well as the initial pH of the culture media. The optimal fermentation medium for triacetylfusarinine C production contained 8% glucose, 0.4% glycine and the initial pH was set to 5.9. Meanwhile, maximal ferricrocin yields were recorded in the presence of 10% glucose, 0.5% glycine and at an initial pH of 7.4. Under optimized fermentation conditions, the yields for triacetylfusarinine C and ferricrocin increased up to 2.9 g/l culture medium and 18.9 mg/g mycelium, respectively.