Naujo bakteriocino, produkuojamo termofilinės bakterijos, identifikavimas, klonavimas ir heterologinė raiška / ; Identification, cloning and heterologous expression of novel bacteriocin produced by thermophilic bacterium.

Bacteriocins are ribosomally-synthesized antimicrobial peptides which are active against closely related or non-related to producer species. Bacteriocins are very heterogenous group of compounds. Bacteriocins activity against food spoiling and pathogenic bacteria could be widely used in food industry, agriculture, and medicine. Bacteriocins have been studied for a while now but knowledge about bacteriocins from thermophilic bacteria are still scarce. Thermophilic bacteria mostly produce thermostable proteins therefore bacteriocins produced by these bacteria could be also thermostable, possibly even more than bacteriocins produced by mesophilic bacteria. In this research thermophilic Geobacillus stearothermophilus 15 strain producing several bacteriocins was used. One of them is 19 kDa molecular weight geobacillin 19. This research is continuation of work with geobacilin 19. During this research we optimized growth medium and liquid chromatography purification strategy. Purified geobacillin 19 was analyzed by mass spectrometry and using bioinformatic tools we determined his amino acid sequence. The sequences of geobacilin 19 and proteins coded near geo19 were compared to protein sequences in NCBI database. We discovered that there is a similarity between geo19ABCD and sdpABC gene clusters. Geobacilin 19 share features typical to class II bacteriocins, for this reason Geo19 was assign to this class. To determine the functions of proteins coded in geo19ABCD gene cluster geo19A, geo19B, geo19C and geo19D genes using different ligation methods were cloned into pCDFDuet-1, pETDuet-1 and pRSFDuet-1 expression vectors. Constructs were moved to E. coli BL21 (DE3) and Rosetta (DE3) expression strains to determine individual geo19ABCD gene cluster genes functions in production of active Geo19.