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MOLECULAR DESIGN AND SYNTHESIS OF ANTIMICROBIAL PEPTIDES OF WIDE RANGE
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MOLECULAR DESIGN AND SYNTHESIS OF ANTIMICROBIAL PEPTIDES OF WIDE RANGE
Ivanova Svetlana 1
Astakhova Lidiia 2
Piskaeva Anastasia 3
Dyshlyuk Lyubov 4
Mercè Pallàs 5
Authors:
1.  Kemerovo Institute of Food Science and Technology (University)

Russian Federation
2.  Immanuel Kant Baltic Federal University

Kaliningrad, Kalinigrad, Russian Federation
3.  Kemerovo Institute of Food Science and Technology (University)

Russian Federation
4.  Kemerovo Institute of Food Science and Technology (University)

Russian Federation
5.  Universitat de Barcelona

Russian Federation
Type:
Article
DOI:
https://doi.org/10.21603/2500-1418-2017-2-1-60-67
Pages:
from 60 to 67
Status:
Published
Received:
27.06.2017
Accepted:
27.06.2017
Published:
27.06.2017
Subject area:
GRNTI 29.01 Общие вопросы физики
GRNTI 27.01 Общие вопросы математики
GRNTI 31.01 Общие вопросы химии
GRNTI 34.01 Общие вопросы биологии
Language:
English
Keywords:
Lactic acid bacteria, bacteriocins, antimicrobial peptides, milk products, E. coli, enzymatic hydrolysates
Abstract and keywords
Abstract (English):
Lactic acid bacteria form a wide range of bacteriocins, the compounds that inhibit ineligible food product microflora. New producers are vigourously searched and bacteriocin properties are actively studied. Creation of new antimicrobial peptides consisting of C- and N-parts of various bacteriocins is of great concern. Based on co-use of nisin, lactocin and enterocin, their antimicrobial effect is proved to increase with the well matching combination of bacteriocins of different origin. Either, development of genetically altered strains of fungi and bacteria is promising that are capable of producing one or more enterococcus bacteriocins and other LAB. This work describes procedures to integrate structures producing Lactobacillus paracasei and Pediococcus acidilactici strain bacteriocins isolated from fermented milk products used by people residing in the south of the Western Siberia into E. coli BL21DE3 competent cells using the commercial vector Mud5005-13. The strains obtained were analyzed for successful integration by induction of pathogenic bacteria by lysates and it was shown that, irrespective of the induction time, the recombinant strain effectively synthesizes the bacteriocin construct integrated. Best conditions were developed for the strain culturing to achieve the highest yield of the recombinant peptide into the culture medium comprising of: medium № 6 containing the following per 1 liter: tripton - 12 g, sucrose - 12 g, sodium acetate 0.5 g, magnesium sulfate - 0.2 g, ammonium sulfate - 0.5 g, sodium chloride - 6 g, calcium chloride - 2 g; culturing temperature - 40°C; agitation speed - 200 rpm; aeration value is 1 l/l*min.

Keywords:
Lactic acid bacteria, bacteriocins, antimicrobial peptides, milk products, E. coli, enzymatic hydrolysates
Text
Science Evolution, 2017, vol. 2, no. 165Fig. 3. Dynamics of recombinant bacteriocin accumulation in the culture medium depending on the medium composition.Based on the data in Figure 3, we may conclude that the best composition for recombinant strain cultivation is the medium No. 6 that allows the maximum release of the recombinant peptide in the culture medium.The temperature impact was studied using the fermentation system with the optimized medium No. 6. Figure 4 shows summary data on the release of recombinant peptide in the medium at different temperatures. It was shown that the maximum yield of the recombinant peptide (2.8 g/l) was achieved at 40°C. The enzyme yield factor in the studied temperature range differed by more than a factor of 2.5.Fig. 4. Dependence of the recombinant peptide yield on the strain incubation temperature.The frequency of the effective mix of the culture liquid with microorganisms was determined at three velocity values, amounting to 150, 200 and 250 rpm at 40°C in the medium No. 6. Figure 5 shows the data on dependence of the enzyme activity, the biomass gain and the degree of foaming on the rate of mixing. Fig. 5. Dynamics of the recombinant peptide yield in processes with different agitation intensity in the fermentation system.Based on the data obtained, we may conclude that at the mixing rate of 200 rpm, the enzyme yield is most active, while the biomass gain is uniform steady in the total volume of the nutrient medium. Parameters of aeration effect on the yield of the enzyme were studied at 40°C in the medium No. 6 at the mixing rate of 200 rpm in the fermenter. As it is seen in Fig. 6, the aeration value of 1 l/l*min is the effective for the peak enzyme containment in the culture liquid. Under this regime, the productivity was 4.4 g/l, which is 1.2-1.6 times higher as compared with other aeration conditions to study.Fig. 6. Dynamics of the recombinant peptide yield in processes with different aeration parameters.Thus, the following results were obtained:1) Pediococcus acidilactici, Lactococcus lactis subsp. lactis, Lactobacillus paracasei strains isolated from dairy products from the south of the Western Siberia were sequenced. Sequences encoding bacteriocins were selected based on BLAST, NCBI, UniProt KB, Bagel2, and Bactibase databases. In line with nding for antimicrobial activity of isolated bacteriocins, it is rational to use pediocin and lactocin from Lactobacillus paracasei and Pediococcus acidilactici bacteria having the most prominent antimicrobial characteristics. The coordinates of gene ends are dened as coordinates of stop codons (ends of assumed reading frames) located at the small distance from the alignment ends of the best ndings or coinciding with them. Resulting from ped1 and lac1 gene cloning, total of twelve clones was obtained containing 5.0-kb fragments.2) A commercial vector Mud5005-13 was used to create a system for gene expression encoding antimicrobial peptides (bacteriocins), where constructs were inserted containing sequences of bacteriocins. Further, the vector containing bacteriocins was integrated into competent cells of E. coli BL21DE3 by electroporation. To verify the proper insertion, the strain culture was exposed to the induction effect factor such as the mixture of lysates Bacillus subtilis B-1325 and Bacillus fastidiosus B-5651 at 1 μg/ml. The lysates analysis data of transformed cells showed mobilization of the recombinant bacteriocin expression, and its amount slightly depended on the time of expression induction. 3) Best conditions were developed for the recombinant strain incubation to achieve the peak yield of the recombinant peptide into the culture medium. The 8 10 12 14 16 18 20Time, h3 2.521.510.50Protein release, g/l 25 30 35 40 45 50Temperature, °C3 2.521.510.50Protein release, g/l 8 10 12 14 16 18 20 22 24 26 28 30Inoculation time, h4 3.532.521.510.50Protein release, g/l 8 10 12 14 16 18 20 22 24 26 28 30Temperature, °C4 3.532.521.510.50Protein release, g/l Science Evolution, 2017, vol. 2, no. 166medium No. 6 was the most effective containing the following per 1 liter: 12 g of tryptone, 12 g of sucrose, 0.5 g of sodium acetate, 0.2 g of magnesium sulfate, 0.5 g of ammonium sulfate, 6 g of sodium chloride, and 2 g of calcium chloride. When studying the effect of various parameters on the yield of the recombinant peptide, it was revealed that the peak yield of the recombinant peptide (2.8 g/l) reached at 40°C at the mixing rate of 200 rpm, aeration value of 1 l/l*min. The enzyme yield factor in the studied temperature range differed by the factor more than 2.5. ACKNOWLEDGEMENTSThe work was carried out as part of the grant 15-08-02003 A of the Russian Foundation for Basic Research (RFBR)
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