DEVELOPMENT OF RECOMBINANT PEPTIDE TECHNOLOGY WITH ANTIMICROBIAL PROPERTIES OF A BROAD ACTION SPECTRUM

Abstract

Bacteriocins are antibacterial, mainly complex, substances of protein nature. The promising strains producing bacteriocins used in the food industry are lactic acid microorganisms. This study examines the development of a technology for the production of a recombinant peptide with broad-spectrum antimicrobial properties. An important step is the isolation and purification of the recombinant peptide. It has been proved that the highest antimicrobial activity is manifested by a recombinant peptide isolated by a method based on salting out with ammonium sulfate. During the purification of the recombinant bacteriocin preparation, three kinds of columns were used. In the purification process, the volume of bacteriocin produced decreases 3-fold, while the RU/mL increases 3-fold, and RU/mg increases 6-fold. Purification allows the use of a smaller amount of recombinant bacteriocin in technologies with greater efficacy. Based on the results of determining the molecular weight and purity of the recombinant bacteriocin, it was found that the molecular weight of the recombinant bacteriocin having the amino acid sequence: KYYGNGVTCCKHSCSVDXGKASSCIINNGAMAXATGGH GGNHCCGMSRYIQGIPDFLRGYLHGISSANKHKKGRL, is 13 kDa. A technology for the preparation of a broad-action antimicrobial spectrum peptide has been developed. The process of production of antimicrobial peptide includes such stages as: cultivation of the recombinant strain of Escherichia coli BL21DE3; separation of biomass from the nutrient medium; precipitation of bacteriocins by ammonium sulfate; centrifugation; washing the precipitate; centrifugation at 4200 rpm and separation of the preparation; purification of bacteriocins by HPLC method; packing in bags of polymeric and combined materials; storage at a temperature of 18±2°C for 12 months.