KR100649102B1 - Method for producing leucocin a - Google Patents

Abstract

 The present invention relates to a yeast production system of leucosin A. In particular, the present invention relates to a yeast production system that can mass-produce leucine a and can safely use the yeast itself as a food preservative without isolation of leucine a. The yeast transformed with a polynucleotide encoding leucine a protein It provides a yeast production system of leucine a comprising culturing in a medium containing 1 to 20 w / v% sucrose and 0.5 to 2.5 w / v% NaCl.

Lucosine A, antibacterial, yeast

Description

Production method of leucosin A {METHOD FOR PRODUCING LEUCOCIN A}

1 is a schematic diagram of preparation of a pAUR-LeucoA vector.

Figure 2 shows the results of leucosin A gene PCR on plasmid DNA of leucine a transformants.

3 is a growth curve of leucosin A transformants and controls ( Saccharomyces cerevisiae ATCC9763).

4a shows the cell growth per hour of leucine a transformant in terms of cell density (OD ₆₀₀ ) and cell dry weight.

Figure 4b shows the total secreted protein amount and protease activity per hour of leucine a transformant.

5 shows the antibacterial activity of Bacillus subtilis by the supernatant of the culture medium according to the incubation time of leucosin A transformant.

6A and 6B show the cell density (OD ₆₀₀ , 6a) and cell number (CFU / ml) of the Bacillus subtilis cultured in the medium to which the supernatant of each of the leucosin A transformants and the control yeast was added. It is represented by (6b).

Figures 7a and 7b confirmed the cell proliferation according to the fructose (△), glucose (□), glycerol (▲) and sucrose (■) carbon source of the leucosin A transformant, 7a is OD ₆₀₀ results and 7b is It is dry cell weight

Figure 8a shows the total amount of secreted protein according to the carbon source of leucine a transformant.

Figure 8b shows the total amount of secreted protease according to the carbon source of the leucine a transformant.

Figure 9 is a graph showing the inhibition of Bacillus subtilis proliferation in the medium containing the supernatant of the culture solution obtained for each incubation time of the leucosin A transformant or the control group.

10a and 10b confirm the cell proliferation according to the NaCl concentration (◇: 0%, ◆: 1%, ▲: 3%, △: 5%, ■: 7%) of the leucosin A transformant, 10a is OD ₆₀₀ result and 10b dry cell weight.

11a and 11b shows the total amount of secreted protein of leucine a transformant (11a) according to NaCl concentration conditions (◇: 0%, ◆: 1%, ▲: 3%, △: 5%, ■: 7%) And total secreted protease amount 11b.

Figure 12 shows the Bacillus subtilis antimicrobial activity by the supernatant of the culture broth obtained according to the culture time of each wild-type yeast and leucosin A transformant.

[TECHNICAL FIELD OF THE INVENTION]

The present invention relates to a yeast production system of leucosin A, more specifically, leucosin which can safely use the culture medium itself as a food preservative without separate separation of leucosin A by mass production of leucine A under optimized conditions. Provide A's yeast production system.

[Private Technology]

Humans have long tried various methods to prevent food corruption and deterioration and improve shelf life. At present, artificial additives used to increase the preservation of food are serious problems due to residual and toxicity in the human body.

Bacteriocin is an antimicrobial substance produced by many gram-positive and gram-negative bacteria, mainly composed of proteins or complexes of proteins and carbohydrates (Davis, DB, R. Dulbecco, NH Eisen, and SH Ginberg (1990), Microbilogy, 4th ed. Lippincott company, Philadelphia, PA). The antimicrobial range of bacteriocins is limited to those strains that are highly systematically related to the antimicrobial producing strains themselves and have similar characteristics to antibiotics. Since the discovery of colicin proteins that inhibit microbial growth in E. coli in 1925 (Riley, MA and JE Wertz (2002), Bacteriocin diversity: ecological and evolutionary perspectives, Biochimie 84, 357-364), bacteriocins A lot of research has been conducted and it is considered a natural nontoxic preservative. Bacteriocin is stable at high temperatures and a wide range of pH and is non-toxic, colorless, and odorless, and is expected to be used as a natural preservative and medicine that can be substituted for chemical synthetic preservatives (Reid, G. and J. Burton (2002). ) Microbes Infect. 4, 319-324). In addition, the application of bacteriocin to fish meat products that cannot be pasteurized (Kim, H.-J., N.-K. Lee, S.-M. Cho, K.-T. Kim, and H.-D. Paik (1999) Korean J. Food Sci.Technol. 31, 1035-1043) and resistance to carcinogens (Chen, J., DM Stevenson, and PJ Weimer (2004) Appl.Environ.Microbiol. 70, 3167-3170 ), And many other actions have recently been revealed.

Bacteriocin, the industry's most notable antibacterial substance, is mainly derived from lactic acid bacteria, a representative example of which is nisin (Delves-Broughton, J. (1990) Food Technol. 44, 100-117). Nisin, currently produced exclusively in the UK, is used in the cheese, fermented milk, fermented liquor and the canning industry.

Bacteriocin can be produced through a variety of microorganisms, but lactic acid bacteria have a disadvantage that can change the flavor of food by producing foreign substances other than bacteriocin, in the production using prokaryotes such as Escherichia coli, growth inhibition by bacteriocin may appear However, mass production of bacteriocin is difficult. Therefore, there is a demand for the development of strains useful for mass production of bacteriocin and the establishment of conditions for mass production.

In order to solve the problems of the prior art, an object of the present invention is to provide a yeast expression system that can produce a large amount of leucosin A in yeast.

It is another object of the present invention to provide a yeast expression system of leucine a which can be used as a food preservative or an antibacterial agent.

In order to achieve the above object, the present invention is a yeast production system of leucine a to produce leucine a protein from yeast transformed with a polynucleotide encoding a leucine a protein, the yeast is 5 to 20 w / v% It provides a system comprising culturing in a medium containing sucrose and 0.5 to 2.5 w / v% NaCl.

In another aspect, the present invention provides a method for producing a food preservative comprising lyophilizing or decompression concentration of the culture solution, cultured cells obtained from the culture medium, the culture solution obtained from the culture solution or lyophilized in the yeast production system of leucosin A.

In another aspect, the present invention provides a method for producing a food preservative comprising lyophilizing or decompression concentration of the culture solution, cultured cells obtained from the culture medium, the culture solution obtained from the culture solution or lyophilized in the yeast production system of leucosin A.

Hereinafter, the present invention will be described in detail.

The present invention relates to a yeast production system of leucosin A.

Lucocin A comprises the amino acid sequence of SEQ ID NO: 2, the gene of which comprises a polynucleotide encoding the amino acid sequence of SEQ ID NO: 2.

The yeast production system of leucine a includes leucine a transformed yeast and culture conditions. The leucine a transformed yeast may be prepared by transforming the yeast with a recombinant vector prepared to express the leucine a gene. The recombinant vector may be prepared by inserting a leucine a gene into a known yeast expression vector, and a vector capable of expressing a leucine a gene under an alcohol dehydrogenase promoter is particularly preferable. One example of the vector is pAUR123 shown in FIG.

Said culture conditions, the sucrose of 1 to 30 w / v% sucrose, preferably 5 to 20 w / v% sucrose and 0.1 to 5 w / v% NaCl, preferably Is cultured in a medium containing 0.3 to 3 w / v%, more preferably 0.5 to 2.5 w / v% NaCl, the medium is 0.5 to 2 w / v% yeast extract and 1 to 5 w / It may further include v% polypeptone and the balance of water. The culture temperature is preferably 25 to 40 ℃, it is preferable to culture for 0.3 to 2 days.

Sucrose used in the yeast expression system of the present invention is provided as a carbon source of the yeast, and differentiated from using glucose as a carbon source in the yeast medium YPD (1% glucose, 2% yeast extract, 2% polypepton) medium do. Sucrose improves about 13.5% of the antimicrobial activity of leucine a transgenic yeast over glucose. In addition, NaCl increases antimicrobial activity by 13.6% compared with no addition, and when it exceeds 2 w / v%, antibacterial activity is greatly decreased and protease secretion rate is significantly increased.

Yeast, culture medium or filtrate thereof prepared according to the yeast expression system of the present invention can be used as a food preservative or antimicrobial agent for inhibiting the growth of microorganisms in which leucine a shows antimicrobial activity. The culture filtrate refers to the supernatant from which the cells are removed from the yeast culture.

In another aspect, the present invention relates to a method for preparing a food preservative or antimicrobial agent comprising lyophilizing or depressurizing a culture solution cultured with a yeast production system of leucine A, the cells obtained from the culture solution or the culture filtrate obtained from the culture solution. . The lyophilization or reduced pressure concentration may be carried out in a conventional method and conditions to be powdered, and other excipients added to food preservatives or antibacterial agents may be further added. Cells, culture filtrate or dried product of the culture may be included in the food preservative or antimicrobial agent in 1 to 99% by weight.

Hereinafter, examples of the present invention will be described. The following examples are only for illustrating the present invention and the present invention is not limited to the following examples.

Example 1: Yeast Expression System

1-1. Reagents and Medium

Restriction enzymes, T4 polynucleotide kinase, DNA ligation kit ver. 2, Taq DNA polymerase was purchased from Takara Korea (Seoul, Korea).

E. coli DH5α was used as a host for gene cloning. Escherichia coli was cultured at 37 ° C. in LB medium (1% bacto tryptone, 0.5% yeast extract, 0.5% NaCl), and ampicillin was added to LB medium at a final concentration of 100 μg / ml for selection of transformants. Solid medium was prepared by adding agar to 1.5% in LB medium.

YPD medium (1% yeast extract, 2% polypeptone, 2% D-glucose) was used for the culture of yeast. The expression plasmid of yeast was used pAUR123 (Takara Korea, Seoul Korea), where expression is induced by an alcohol dehydrogenase promoter (ADH), and transformation was performed using a yeast transformation system (Clontech, USA). The lithium acetate method was used. For selection of transformed yeast, a medium in which aureobacidin A (Aureobacidin A, Takara Korea, Seoul Korea) was added to YPD medium at a final concentration of 0.2 μg / ml was used.

1-2. Recombinant vector production

After synthesizing a 117 bp long lucosine A gene (SEQ ID NO: 1) containing an initiation codon and an end codon, introducing a restriction enzyme Xho I site at the 5 'end and a restriction enzyme Xba I site at the 3' site, It was inserted into the Xho I site and the Xba I site of the pAUR123 vector (Fig. 1). The recombinant vector was named pAUR-LeucoA (7,099 bp), and transformed into E. coli (DH5α).

1-3. Preparation of leucosin A transformant

pAUR-LeucoA was transformed into Saccharomyces cerevisiae (ATCC 9763) by lithium acetate method, and inoculated in YPD medium to which aureobaccidin A (final concentration 0.2 μg / ml) was added and incubated at 30 ° C. for 3 days. By culturing, the cultured yeast was selected as leucosin A transformant.

Lucosin A transformants were inoculated in 4 ml of YPD medium containing aureobacsidin A (final concentration 0.2 μg / ml), incubated at 30 ° C. overnight, and then centrifuged (14,000 xg, 5 minutes). Was recovered. 200 μl of yeast lysis buffer (2% Triton X-100, 1% SDS, 10 mM tris-HCl (pH 8.0) buffer, 1 mM EDTA) was suspended, and the cells were suspended in acid-washed glass beads (425). 0.3 g of -600 μm, Sigma # G-8772) and 200 μl of phenol / CHCl ₃ solution were added and mixed for 2 minutes. The supernatant was collected by centrifugation (14,000 xg, 10 minutes), 1 µl of glycogen, 20 µl of 5 M LiCl solution and 500 µl of cold ethanol were added, followed by centrifugation (14,000 xg, 10 minutes) to precipitate plasmid DNA. DNA was washed with cold 70% ethanol, dried, and dissolved in 20 µl of ultrapure water. PCR was performed on the plasmid DNA using primers SEQ ID NOs: 2 and 3, which are leucinecin specific primers. The primer of SEQ ID NO: 1 and the primer of SEQ ID NO: 2 were used for 50 ng of the plasmid DNA, respectively, and 10 pmol of each was used. The PCR reaction product was electrophoresed on 2% agarose gel to confirm its position. Figure 2 shows the results of leucosin A gene PCR on the plasmid DNA of the leucosin A transformant, confirming a gene fragment of about 120 bp corresponding to the leucosin A gene.

1-4. Growth Pattern of Transformant

After culturing the transformant and the control group (Saccharomyces cerevisiae, ATCC9763), the culture medium was collected at regular time and then cell growth rate, protease activity and antimicrobial activity were measured.

The cultures collected at specific times were measured for optical density at OD ₆₀₀ , centrifuged at 6000 rpm for 5 minutes at 4 ° C., dried at 60 ° C., and then dried. In addition, protease activity analysis was carried out by modifying some known methods (12). That is, 200 μl of the sample was reacted with 200 μl of 1% casein hydrolysate (0.2 M Tris-HCl buffer, pH 5.8) at 30 ° C. for 2 hours, and then 600 μl of 0.4M trichloroacetic acid (TCA) was added to react the reaction. Stopped. After 5 minutes at 4 ° C, centrifugation (6000 rpm, 5 min) to remove the precipitated protein was measured OD ₂₈₀ of the supernatant.

Figure 3 shows the growth curve of the leucosin A transformant and the control group (Saccharomyces cerevisiae, ATCC9763), the growth pattern of the control group and the transformant is almost similar so that the expression of leucine a in the yeast growth It is confirmed that it does not have a great effect on.

In addition, the cell growth, total secreted protein amount and protease activity of the transformants were measured for each culture time. Figure 4a is a result of measuring the cell growth per hour of cell growth (OD ₆₀₀ ) and cell dry weight of leucosin A transformant, Figure 4b is the total amount of secreted protein and protease activity per hour of leucosin A transformant It is shown. In Figure 4a, the leucosin A transformant after the logarithmic growth step up to 12 hours incubation was confirmed to have a growth pattern to enter into the plateau and die after. In addition, the total amount of secreted protein in FIG. 4B was the same as that of FIG. 4A, and continued to increase until 12 hours of incubation, and then decreased. The difference between the maximum value and the initial value of total secreted protein amount was about 12.15 ml / L, and the increase was very large between 8 and 12 hours, which was different from the change of dry cell weight of 4a. The protease was shown to decrease up to 12 hours in contrast to the total secreted protein and then increase rapidly. Based on the above results, leucosin A transformants proliferate using nutrients in the medium until 12 hours of peak growth, after which the cells are destroyed by nutrient depletion and protease secretion is increased in living cells. It is confirmed.

1-5. Antimicrobial Activity of Lucosine A Transformants

Antimicrobial activity was measured in solid and liquid media. Experiments on solid media are important for the diffusion of bacteriocin into solid foods.

The culture solution obtained over time while liquid culture of leucosin A transformant was centrifuged, and a certain amount of the supernatant was dropped on a paper disk placed on a YPD solid medium plated with Bacillus subtilis and incubated for 24 hours. After culturing, the size (mm) of the transparent ring without culturing Bacillus was measured and shown in FIG. 5. The antimicrobial activity was increased with the incubation time and decreased after the logarithmic growth phase (12 hours). The antimicrobial activity was found to be greatest when the leucosin A transformant was incubated for 12 hours. The area showing antimicrobial activity was 2.52 times after 12 hours of culture and assuming that the degree of diffusion of the supernatant was the same, the volume was calculated to be 3.28 times. Inferring from the volume showing antimicrobial activity, it was found that after 12 hours of culture, the antimicrobial activity was three times higher than that after 1 hour of culture.

In addition, the culture broth obtained by time while liquid culture of leucosin A transformant was centrifuged, and 250 μl of the supernatant was liquid cultured at 250 rpm at 30 ° C. with 50 μl of Bacillus subtilis for 12 hours. After incubation, the growth rate of Bacillus subtilis was measured.

6A and 6B show the growth rate of Bacillus subtilis cultured in the supernatant of leucosin A transformant culture and the supernatant of control yeast culture as OD ₆₀₀ (6a) and cell number (CFU / ml) (6b). will be. The growth of Bacillus subtilis was not significantly different according to the sampling time in the culture of control yeast, but the culture of leucine a transformant showed a big difference in the growth of Bacillus subtilis according to the sampling time. . That is, the culture medium of leucosin A transformant showed an initial antibacterial activity of 9.23%, and showed an antimicrobial activity of 70.57% after 12 hours of culture.

Example 2 Establishment of leucosin A mass production condition of leucosin A transformant

2-1. Measurement of Proliferation Efficiency of Lucosine A Transformants According to Carbon Sources

Lucocin A transformants were cultivated in various carbon sources to select carbon sources advantageous for leucine a production. Thus, Lu a of Columbia A transformant fructose, glucose, glycerol and shoe were cultured to cross the inoculation in each of the liquid medium comprises 10 g / L, the cell proliferation to obtain a culture time culture medium to OD ₆₀₀ and dry weight Measured.

Figures 7a and 7b confirmed the cell proliferation according to the fructose (△), glucose (□), glycerol (▲) and sucrose (■) carbon source of the leucosin A transformant, 7a is OD ₆₀₀ results and 7b is Cell dry weight. As a result, the cell proliferation was slightly different at 7a and 7b, and when the cell density was measured, a higher cell proliferation rate was measured compared to the dry weight. In addition, in FIG. 7A, cell proliferation was found to be high in the order of sucrose>glycerol>glucose> fructose. Especially, when cultured in sucrose after 15 hours of cultivation, the cell density and dry weight were 18% compared to those cultured in glucose. And 7.4% higher.

2-2. Determination of Protein and Protease Secretion Rates of Lucosin A Transformants According to Carbon Sources

Total secreted protein amount and total secreted proteinase amount were measured, respectively.

Figure 8a shows the total amount of secreted protein according to the carbon source of the leucosin A transformant, the maximum amount of total secreted protein was measured at 15 hours of culturing in the carbon source except glycerol. Total secreted protein was highest in sucrose and lowest in fructose. The culture of sucrose increased the total amount of secreted protein by about 7.9% compared to that of glucose.

Figure 8b shows the total amount of secreted protease according to the carbon source of the leucine a transformant, the lowest total secreted protease amount was measured at all carbon sources at 15 hours of incubation, sucrose was the lowest throughout the culture time Protease secretion was shown.

Therefore, the leucine a transformant of the present invention increases the total secreted protein and simultaneously decreases protease secretion when sucrose is used as the carbon source.

2-3. Antimicrobial Activity of Lucosin A Transformants According to Carbon Sources

After incubating the leucosin A transformants in various carbon sources, the antimicrobial activity of the culture broth obtained at each incubation time was measured for Bacillus subtilis (30 ° C., 250 rpm, 24 h).

9 is a graph showing the inhibition of Bacillus subtilis proliferation in the medium containing the supernatant of the culture solution obtained by the incubation time of leucosin A transformant or the control, Bacillus subtler compared to the control group The proliferation was significantly suppressed, and the culture medium showed the highest antimicrobial activity for about 15 hours. In addition, 52 and 33% of the antimicrobial activity was observed when incubated in sucrose and glucose for 2 hours, respectively, and 78 and 68% of the antimicrobial activity was observed after 15 hours, respectively. It could be confirmed that the increase.

2-4. Determination of Lucosin A Expression Rate According to NaCl Concentration Conditions

Preparation of leucosin A transformants in a liquid medium containing 20 g / L polypeptone, 20 g / L sucrose, 10 g / L yeast extract and 0.2 μg / ml of aureobaccidin A as sucrose as a carbon source Lucocin A expression medium was used, and NaCl was added at 0, 1, 3, 5, and 7%, and the rate of leucosin A expression according to NaCl concentration was measured.

10a and 10b confirm the cell proliferation according to the NaCl concentration (◇: 0%, ◆: 1%, ▲: 3%, △: 5%, ■: 7%) of the leucosin A transformant, 10a is OD ₆₀₀ result and 10b dry cell weight. Under conditions other than 7% NaCl, leucosin A transformants showed a maximum growth rate at 15 hours of culture, and a maximum growth rate at 18 hours of culture under NaCl 7%. In conditions showing the maximal proliferation of leucine a transformants, an increase of 7.75% (FIG. 10A) and 4.9% (FIG. 10B) was observed as compared with no addition of 1% NaCl.

11a and 11b shows the total amount of secreted protein of leucine a transformant (11a) according to NaCl concentration conditions (◇: 0%, ◆: 1%, ▲: 3%, △: 5%, ■: 7%) And total secreted protease amount 11b. In NaCl 7% concentration, total secreted protein was produced at the highest level at about 18 hours of cultivation, and at other concentrations, total secreted protein was peaked at 12 hours. More than 3% showed a decrease. Comparing the total amount of secreted proteolytic enzymes, proteolytic activity of NaCl 3% <1% <7% <0% <5% was observed, and compared with the absence of NaCl at 15 hours of incubation at 3% NaCl. Protease activity of 55% was confirmed, and at the same time, about 85.7% of protease activity was confirmed in 1% NaCl condition, compared to no addition, and it was found that protease expression was reduced compared to no addition.

Figure 12 shows the Bacillus subtilis antimicrobial activity by the supernatant of the culture broth obtained according to the culture time of each wild-type yeast and leucosin A transformant, "T" is a leucosin A transformant, "C "Is wild type yeast. As shown in FIG. 12, wild-type yeast showed almost no antimicrobial activity regardless of NaCl concentration, and leucine a transformant showed various antimicrobial activities depending on NaCl concentration. The antimicrobial activity of leucosin A transformant was the best at 15 hours of culture, and the group incubated at 1% NaCl concentration showed the highest antimicrobial activity.

As described above, the yeast production system of leucine a of the present invention can mass produce leucine A and can safely use the yeast itself as a food preservative or an antimicrobial agent without separation of leucine a.

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Claims (6)

i) preparing a leucosin A transformant that is a yeast transformed with a polynucleotide encoding a leucosin A protein; ii) culturing the leucosin A transformant in a medium comprising 5-20 w / v% sucrose and 0.5-2.5 w / v% NaCl; And iii) a method for producing leucocin A using a transformant of leucosin A, comprising producing leucosin A using a leucosin A transformant. The method of claim 1, wherein the polynucleotide comprises a nucleotide sequence of SEQ ID NO: 1. The method of claim 1, wherein the transformed yeast expresses leucine a protein under the control of an alcohol dehydrogenase promoter. The method of claim 1, wherein the medium further comprises 0.5 to 2 w / v% yeast extract and 1 to 5 w / v% polypeptone and the balance of water. A food preservative comprising lyophilizing or depressurizing a culture solution, a cell obtained from the culture medium, or a culture filtrate obtained from the culture solution according to any one of claims 1 to 4 Manufacturing method. Preparation of an antimicrobial agent comprising lyophilizing or concentrating a culture solution obtained by the production method of leucine a according to any one of claims 1 to 4, the cells obtained from the culture solution or the culture filtrate obtained from the culture solution. Way.

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