RU2492231C2 - Method of bacteriocins isolation - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: culture fluid of producer strain is concentrated on hollow fibers truncating macromolecules with a molecular weight higher than 15 kDa. The cell concentrate is added to dry NaCl in a final concentration of 0.5 M. Mixed on a shaker for 20 min. The suspension is centrifuged for 15 min at 10000 g and the supernatant is separated, the supernatant pH is made up to 3.0 by four-molar solution of HCl. The suspension is centrifuged. The residue is added to water in a volume of 0.1% of the initial volume of the culture fluid, the residue is suspended and alcohol is added in amount of 0.1% of the initial volume of the culture fluid. Ethanol or propanol or isopropanol is used as alcohol. Exposed for 30 minutes at 0°C and centrifuged. The alcohol is removed from the solution by evaporation. The peptide fraction is purified. Water is added to 0.1% of the initial volume of the culture fluid, active carbon is added in an amount of 0.5% w/v (v - water volume). Centrifuged removing impurities adsorbed on the carbon. The aqueous solution is passed through the membrane truncating macromolecules with a molecular weight more than 10 kDa.

EFFECT: invention enables to accelerate preparation of bacteriocin and to increase the degree of purification of the resulting product in a yield of 90% of the total activity in the culture fluid.

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Description

The invention relates to biochemistry and biotechnology, and in particular to a method for purifying bacteriocins.

Bacteriocins are peptides with a molecular weight of several kilodaltons, secreted by some gram-positive and gram-negative bacteria, followed by interaction with the membranes of recipient microbial cells. Studies of a number of bacteriocins have shown their non-toxicity, non-reactogenicity and non-allergenicity [Miller KW. Isolation and characterization of pediocin AcH chimeric protein mutants with altered bactericidal activity. Appl. Environ. Microbiology. 1998; 64; 1997-2005].

Bacteriocins could be widely used in medicine and veterinary medicine (instead of antibiotics), as well as in the food industry (as preservatives). However, this is hindered by difficulties in obtaining purified bacteriocins.

It is known to use only one bacteriocin in industry - nisin secreted by a Streptococcus lactis culture. The preparation contains only 2.5% nisin [Mazzotta A.S., Crandall A.D., and Montville T.J. Nisin resistance in Clostridium botulinum spores and vegetative cells. Appl. Environ. Microbiol. 1997; 63: 2654-2659]. The use of high purity nisin is economically inefficient.

Currently, highly purified bacteriocins are obtained mainly for structural studies (for example, to determine the primary structure). Ammonium sulfate precipitation, ion exchange chromatography on cation and / or anion exchangers, hydrophobic chromatography, high performance liquid chromatography, gel filtration are usually used. If 1-2 purification methods are used, sometimes the yield of the target product up to 60-80% of the total activity in the culture fluid can be achieved, however, its purity is usually insufficient for use in the medical industry [Carolissen-Mackay V, Arendse G, Hastings JW. Purification of bacteriocins of lactic acid bacteria: problems and pointers. Int. J. Food Microbiol. 1997; 34: 1-16].

An increase in the number of cleaning methods leads to a sharp drop in yield, which can be percentages or even tenths of a percent [Mortvedt CI, Nissen-Meyer JM, Sletten K, Nes IF. Purification and amino acid sequence of lactocin S, a bacteriocin produced by Lactobacillus sakei L45. Appl. Environ. Microbiol. 1991; 57: 1829-1834]. Even after 3-4 x purification methods, the purity of the bacteriocins is often insufficient to determine the primary structure and one or two are needed to achieve electrophoretic purity. It is also significant that some of them, for example, gel filtration, are difficult to apply for a large-scale process.

Closest to the proposed is a method of sorption-desorption of bacteriocins from the surface of the cells of dried lactic acid bacteria (the authors call it "extraction") [Yang R, Johnson MC, Ray B. Novel method to extract large amounts of bacteriocins from lactic acid bacteria. Appl. Environ. Microbiol. 1992; 58: 3355-3359.]. It was shown that during fermentation at neutral pH values, the bacteriocins produced by four species of these bacteria are adsorbed on the cells, and at pH 1.5-2.0 with stirring for 1 hour in the presence of 0.1 M NaCl, added to prevent cell adhesion, desorbed from them. After removing the cells, the supernatant is dialyzed and frozen.

The disadvantages of this method include a strong variation in the most important indicators: yield and purity of the target products. The yields were from 44% to almost 100%, and the number of protein peaks according to electrophoresis ranged from one to several, which indicates contamination of the target product with ballast proteins. In addition, dialing will complicate the isolation and purification during scaling and can be a critical operation. The authors dialyzed 1 L of the supernatant, with pores of the dialysis bags cutting off molecules> 1 kDa. Such dialysis should go very slowly and for volumes of the order of 5 l will be completely unacceptable.

The objective of the invention is to accelerate the purification of bacteriocins, increase the yield and purity of the target products from producers of various species and genera that fully preserve the overall activity of the culture fluid after precipitation with ammonium sulfate and subsequent dialysis of the precipitate and suitable for a large-scale process.

The problem is solved in that the proposed method includes the cultivation of a producer strain, followed by isolation and purification of the peptide fraction. First, the culture fluid is concentrated on hollow fibers, cutting off macromolecules> 15 kDa, and dry NaCl at a final concentration of 0.5 M is added to the cell concentrate. It is stirred on a rocking chair for 20 minutes, the suspension is centrifuged and the supernatant is separated. In the supernatant, the pH was adjusted to 3.0 with a four molar HCl solution. The resulting suspension is centrifuged, and the precipitate is added water in a volume of 0.1% of the initial volume of the culture fluid. The precipitate was suspended and the same volume of alcohol dissolved in water in any ratio was added, held for 30 minutes at 0 ° C and centrifuged, the alcohol was removed from the solution by evaporation, water was added to the initial volume of the alcohol solution, activated carbon was added in an amount of 0.5% w / v (v-volume of water), centrifuged, removing impurities adsorbed on coal. An aqueous solution is passed through a membrane cutting off macromolecules> 10 kDa. All centrifugation steps are carried out at 10000g, 15 min. The eluate is collected and analyzed.

A distinctive feature of the proposed method is the addition of alcohol (ethanol, propanol, isopropanol) to the fragments of cells on the surface of which there is bacteriocin. Under the action of alcohol, bacteriocin is removed from surface cell fragments and the proposed purification method can be called "hydrophobic chromatography on surface fragments of producer cells". In the overwhelming majority of works, it was believed that bacteriocins in a soluble state were purified and methods that were commonly used for the purification of soluble high molecular weight proteins were used. This led either to a low yield if several cleaning methods were used, or to a higher yield, but a lower degree of purification if one or two methods were used.

The proposed method is based on the general properties of bacteriocins and the general properties of the cell surface of producers of various species and genera that completely preserve the overall activity of the culture fluid after precipitation with ammonium sulfate and subsequent dialysis of the precipitate, therefore, it is suitable for cleaning not one, but many or even all bacteriocins. All bacteriocins are low molecular weight peptides (molecular weight - 2-7 kDa). These are hydrophilic-hydrophobic thermostable substances. Hydrophilicity (and good solubility in aqueous solution) are necessary for the delivery of bacteriocin to the recipient cell, and hydrophobicity is necessary for binding to membranes. A common property of the surface of gram-negative and gram-positive producer cells (peptidoglycan and membranes) is their hydrophobicity.

The proposed method for the purification of bacteriocins is as follows:

- the culture fluid is concentrated on hollow fibers, cutting off substances with a molecular mass of more than 15 kDa;

- dry NaCl with a final concentration of 0.5 M is added to the culture fluid concentrate and stirred on a rocking chair at a speed of 100 cpm for 20 minutes;

- centrifuged cells treated with 0.5 M NaCl, separate them from the supernatant;

- adjust the pH of the supernatant of 4 M HCl to a value of 3.0 and obtain a suspension of cell fragments;

- centrifuged the resulting suspension, remove the supernatant;

then the stage: add water to the sediment of cell fragments in a volume of 0.1% of the initial volume of the culture fluid, suspend fragments, add isopropanol in a volume of 0.1% of the initial volume of the culture fluid, hold the suspension for 30 min at 0 ° C, centrifuge, separate the precipitate from alcohol solution;

- remove alcohol from the solution on a rotary evaporator at 60 ° C;

- add water to the initial volume of the alcohol solution and activated carbon in an amount of 0.5% w / v,

- incubated for 15 minutes, centrifuged, remove activated charcoal with impurities adsorbed on it;

- an aqueous solution is passed through a membrane cutting off macromolecules> 10 kDa.

In all cases, centrifugation is carried out at 10000 g, 15 minutes

The activity of bacteriocins is determined in arbitrary units of the "agar test". For this test, an indicator strain is applied to a Petri dish with agar mixed with nutrient medium so that a continuous crop lawn grows overnight at 37 ° C. As an indicator strain sensitive to the action of bacteriocin, a Lister ea monocytogenes culture is used in all examples.

5 mm diameter wells are made in agar. Test samples of the supernatant, concentrate or eluate are added to these wells in a volume of 10 μl, titrated in double step, pre-incubated for 3 hours at room temperature, and then overnight at 37 ° C. After growth is complete, inhibition zones become visible. The dilution value at which the sample further does not exhibit bactericidal activity is the dilution value, after which the inhibition zones are not visible (final dilution). The maximum dilution of the sample is taken as the unit of activity, in which lysis of the cells of the indicator strain is still observed, related to 1 μl of the solution or suspension in which bacteriocin is present.

An agar test is also used to determine if a substance causing cell death of an indicator strain is inactivated in the presence of an enzyme, proteinase K, i.e. whether it is a polypeptide. For this, a solution of the enzyme with a concentration of 4-5 mg / ml in 0.05 M phosphate buffer, pH 7.0 and a volume of 20 μl is added to the supernatant of the culture fluid in a volume of 1 ml and a pH value of 7.0-7.2. Incubated for 1 hour at 37 ° C. 10 μl was collected and placed in a well with agar mixed with culture medium. In parallel, 10 μl of enzyme-free supernatant is added to the well, both samples are titrated in double step and the presence or absence of growth inhibition zones is compared.

Examples of the method of purification of bacteriocins.

The following microorganisms from the State collection of pathogenic microorganisms and cell cultures “GKPM-Obolensk” were used as producers of bacteriocins: Bacillus subtilis, registration number B-6799; Streptococcus raffinolactis registration number S-4515; Streptococcus raffinolactis registration number S-4516; Bacillus polymixis, registration number B-5831. As a test culture used Listeria monocytogenes, registration number L-5949.

Example 1

The culture of Bacillus subtilis B-6799 is grown in a bioreactor with a working volume of 8.2 l in saline medium of the following composition per 1 liter: K ₂ HPO ₄ - 10.5 g; KH ₂ PO ₄ - 4.5 g; (NH ₄ ) ₂ SO ₄ - 1.0 g; citrate Na · H ₂ O - 0.5 g; MgSO ₄ · 7H ₂ O (20 g / 100 ml) - 1 ml / l; 20% glucose - 10 ml. Growing conditions: pH 7.2, temperature 37 ° C, stirrer speed 200-250 rpm, air purge: 6 l / min, fermentation time 10 hours until stationary phase is reached. The culture fluid is concentrated 8 times on hollow fibers that cut off macromolecules with a molecular weight of more than 15 kDa. Dry NaCl was added to the concentrate to a final concentration of 0.5 M in small portions with constant stirring on a rocking chair at a speed of 100 cpm. Stirred for 20 minutes. Then centrifuged at 10000g, 15 min. The pH of the supernatant was adjusted with 4M HCl to 3.0, left at 0 ° C for 30 minutes to sediment cell fragments and centrifuged at 10,000 g, 15 minutes. 6 ml of water was added to the precipitate and the precipitate was suspended, 6 ml of isopropanol was added, the mixture was kept at 0 ° С for 30 min, then centrifuged at 10000 g, 15 min. Isopropanol is evaporated on a rotary evaporator at 60 ° C and water is added to the previous volume of the alcohol suspension. Activated carbon is added in an amount of 0.5% w / v. Centrifuged at 10,000 g, 15 min, remove the coal and impurities adsorbed on it. An aqueous solution is passed through a membrane cutting off macromolecules> 10 kDa. The yield is 94% of the initial total activity in the culture fluid; get electrophoretically pure product. The results are presented in table 1.

Table 1 Sample Volume μl Final dilution Number of conventional units 10 ⁶ Exit, % Culture fluid 8.2 × 10 ⁶ 2 ⁷ 1050 one hundred Suspension at pH 3.0 10 ⁶ 2 ¹⁰ 1024 98 Supernatant after Isopropanol Added 12.0 × 10 ³ 2 ¹³ 983 94

Thus, as can be seen from the results of the table, the proposed method allows to obtain bacteriocin with a yield of more than 90%.

Example 2

The Streptococcus raffmolactis S-4515 culture was grown in rocking flasks at a temperature of 37 ° C, 200-250 cpm / min, cultivation time 12 h, pH 7.0, the volume of solution in the flask was 250 ml. The composition of the nutrient medium (g / l): enzymatic meat hydrolyzate - 10.0; casein enzymatic hydrolyzate - 10.0; yeast extract - 2.0; dextrose - 1.0; sodium chloride - 5.0; sodium bisulfite - 0.1. The culture fluid in a volume of 1.5 l is concentrated 10 times on hollow fibers, cutting off macromolecules with a molecular weight of more than 15 kDa and centrifuged at 10000 g, 15 minutes Dry NaCl was added to the resulting supernatant to a final concentration of 0.5 M with constant stirring on a shaker at a speed of 100 cpm / min and stirred for 20 minutes. Centrifuged at 10000g, 15 min. The supernatant pH was adjusted with 4M HCl to 3, and left at 0 ° C for 30 minutes to sediment cell fragments. The suspension is centrifuged at 10000 g, 15 min, the supernatant is removed, and water in a volume of 1.5 ml is added to the precipitate, the precipitate is suspended and 1.5 ml of propanol is added and the mixture is kept for 30 min at 0 ° C and centrifuged at 10000 g for 15 min. Propanol is evaporated on a rotary evaporator at 60 ° С, water is added to the previous volume, then activated carbon (0.5% w / v) is added and centrifuged at 10000g, 15 min, removing adsorbed impurities, the aqueous solution is passed through a membrane cutting off the macromolecules> 10 kDa. The yield of bacteriocin: 95% of the total activity in the initial culture fluid, get electrophoretically pure product.

Example 3

The Streptococcus raffinolactis S-4516 culture is grown in rocking flasks at a temperature of 37 ° C, 200-250 cpm / min, cultivation time is 12 hours, pH 7.0, the volume of solution in the flask is 250 ml. The composition of the nutrient medium (g / l): enzymatic meat hydrolyzate - 10.0; casein enzymatic hydrolyzate - 10.0; yeast extract - 2.0; dextrose - 1.0; sodium chloride - 5.0; sodium bisulfite - 0.1. The culture fluid in a volume of 1.5 l is concentrated 10 times on hollow fibers that cut off macromolecules with a molecular weight of more than 15 kDa. Dry NaCl was added to the concentrate to a final concentration of 0.5 M, stirred for 20 minutes and centrifuged at 10000 g, 15 minutes to obtain a supernatant. The pH of the supernatant was adjusted with 4M HCl to 3.0 and left in the refrigerator at 0 ° C for 30 minutes to sediment cell fragments. The supernatant is centrifuged at 10000 g, 15 min, the supernatant is removed and 1.5 ml of water is added to the precipitate. Suspended in water, add 1.5 ml of propanol. After 30 min, centrifuged at 10000g, 15 min. The alcohol is removed by evaporation, water is added to the previous volume. Activated carbon is then added in an amount of 0.5% w / v and centrifuged at 10,000 g for 15 minutes, carbon and adsorbed impurities are removed. An aqueous solution is passed through a membrane cutting off macromolecules> 10 kDa.

The yield of bacteriocin: 95% of the total activity in the initial culture fluid, the product is electrophoretically pure.

Example 4

The culture of Bacillus polymixis B-5831 is grown in rocking flasks at a temperature of 37 ° C, 200-250 cpm / min, the cultivation time is 12 hours, pH 7.0, the volume of solution in the flask is 250 ml. The composition of the nutrient medium (g / l): meat extract - 5.0; peptone - 4.0; yeast extract - 5.0; dextrose - 1.0; sodium chloride - 2.5; glucose - 8.0-10.0 ml; The culture fluid in a volume of 1.5 l is concentrated 10 times on hollow fibers that cut off macromolecules with a molecular weight of more than 15 kDa. Dry NaCl was added to the concentrate to a final concentration of 0.5 M with stirring on a rocking chair for 20 minutes, centrifuged at 10,000 g, 15 minutes to obtain a supernatant. The pH value was adjusted with 4M HCl to 3.0 and left at 0 ° C for 30 minutes to sediment cell fragments. It is centrifuged at 10000 g, 15 min, the supernatant is removed, and 1.5 ml of water are added to the precipitate, suspended and 1.5 ml of propanol are added. Stand for 30 min at 0 ° C, centrifuged at 10000g, 15 min. Propanol is evaporated on a rotary evaporator at 60 ° C, water is added to the previous volume of the alcohol suspension, then activated carbon is added at a concentration of 0.5 w / v. It is centrifuged at 10000 g, 15 min, the coal and its adsorbed impurities are removed, and the aqueous solution is passed through a membrane cutting off macromolecules> 10 kDa. The yield of bacteriocin: 93% of the total activity in the initial culture fluid, the product is electrophoretically pure.

To determine the molecular weight and purity of the target product using SDS-PAG electrophoresis in 16% polyacrylamide gel (standard protocol from Bio-Rad) with a set of protein markers from 10 to 170 kDa (Fermentas). 5 μl of a solution of bacteriocin diluted to a concentration of ~ 0.5 mg / ml, mixed with 5 μl of buffer for protein SDS-PAG electrophoresis and heated at 60 ° C. The movement of proteins occurs at a current strength of 10 mA in a separation gel for 30 minutes; in a concentrating gel of 14 mA - 60 min. The gel is fixed in a solution of 10% acetic acid and 20% isopropanol for one hour, washed with distilled water for 2 hours and stained in a solution of Coomassie G-250.

Figure 1 shows the electrophoregram purification of bacteriocin (example 2).

1 - sample after the action of propanol on the fragments

2 - a sample on a nitrocellulose membrane

3 - bacteriocin passing through the membrane, cutting off proteins> 10 kDa

4 - protein markers

Thus, the proposed method of purification by removing bacteriocin from surface cell fragments under the influence of alcohol can be called "hydrophobic chromatography on surface fragments of producer cells". The method is applicable for large-scale production of bacteriocins secreted by producers of various genera and species, there are no operations requiring a long time (for example, dialysis), and the result is an electrophoretically pure product with a yield of more than 90% of the total activity in the culture fluid.

Claims (2)

1. The method of isolation of bacteriocins from the culture fluid of the producer strain, followed by purification of the peptide fraction, characterized in that the culture fluid is concentrated on hollow fibers that cut off macromolecules with a molecular mass of more than 15 kDa, and dry NaCl is added to the cell concentrate at a final concentration of 0, 5 M, stirred on a rocking chair for 20 minutes, the suspension is centrifuged at 10000 g for 15 minutes and the supernatant is separated, the pH of the supernatant is adjusted to 3.0 with a four-molar HCl solution, the resulting suspension is centrifuged, to adka add water in a volume of 0.1% of the initial volume of the culture fluid, then the precipitate is suspended and alcohol is added in a volume of 0.1% of the initial volume of the culture fluid, incubated for 30 min at 0 ° C and centrifuged, the alcohol is removed from the solution by evaporation, add water to 0.1% of the initial volume of the culture fluid, add activated carbon in an amount of 0.5% w / v (v-volume of water), centrifuged to remove impurities adsorbed on coal, the aqueous solution is passed through a membrane that cuts off macromolecules with a molecular weight more 10 kDa. 2. The method according to claim 1, characterized in that ethanol or propanol or isopropanol is used as the alcohol.

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