KR100752928B1 - Fermentation for preparing l-lysine - Google Patents

Abstract

A fermentation method for preparing L-lysine is provided to activate Corynebacterium sp. producing L-lysine by preseed culturing and seed culturing of Corynebacterium sp. in a medium containing pyrrolidone carboxylic acid, so that fermentation yield is improved. The method for preparing L-lysine comprises the steps of: preseed culturing and seed culturing Corynebacterium glutamicum(KFCC 11043) producing L-lysine in a preseed and seed medium containing 0.1-5 g/l of pyrrolidone carboxylic acid supplied as a type of beet molasses; and culturing the activated Corynebacterium glutamicum(KFCC 11043) in growth medium at 20-45 deg.C for 10-160 hours, wherein the concentration of beet molasses in the medium is 5-100 g/l.

Description

L-lysine fermentation process {Fermentation for preparing L-lysine}

The present invention is to activate the strain by preseed and seed culture of Corynebacteria sp. Strains in a medium containing Pyrrolidone carboxylic acid (PCA) The method and a fermentation process for producing L-lysine comprising performing the main culture using the strain activated by the method.

L-lysine is one of essential amino acids and is used as feed additives, food additives, and pharmaceutical raw materials for livestock. In particular, the market size of L-lysine as a feed additive for livestock is a huge fermented product reaching about 860,000 tons in 2005.

Currently, L-lysine is a carbon source of raw sugar, glucose, molasses (sugar cane, sugar beet) and a nitrogen source such as corn steep liquor (CSL), soybean sulphate, ammonium sulfate, vitamins, minerals, etc. It is industrially produced by the direct fermentation method.

Looking at the prior art with regard to the production of L-lysine, Japanese Patent Application Laid-open No. Hei 4-66558 discloses sugar cane molasses and beet molasses diluted with water and then passed through a cation exchange resin. A method for improving L-lysine fermentation concentration and yield by adding an eluent to the fermentation medium is presented; In Japanese Patent Application Laid-Open No. 1982-026594, sugar cane molasses and beet molasses diluted with water are reacted with an invertase, followed by fermentation process using Corynebacterium to form L-lysine. Methods of making are described.

Accordingly, the present inventors have established a L-lysine fermentation process that achieves excellent fermentation efficiency through a method of activating the strain by seed culture and seed culture of Corynebacterium species strain in a medium containing pyrrolidone carboxylic acid. The present invention has been completed.

It is an object of the present invention to provide a method for activating Corynebacterium species used for L-lysine fermentation.

Another object of the present invention is to provide a fermentation process for producing L-lysine using Corynebacterium species activated by the above method.

The present invention provides a method of activating said strain by species culture and seed culture of a Corynebacterium species strain in a medium comprising pyrrolidone carboxylic acid.

In the present invention, the Corynebacterium spp. Strain is included in the Corynebacterium genus, and any one having L-lysine production ability is included. The four examples of Corey tumefaciens species include Corynebacterium glutamicum (Corynebacterium contain a strain selected from the glutamicum), Corynebacterium thermo amino to Ness (Corynebacterium thermoaminogenes), Corynebacterium Plastic boom (Corynebacterium flavum), and Corynebacterium, Lactobacillus Peer lactofermentum of species the group consisting of (Corynebacterium lactofermentum) Can be.

Corynebacterium sp. Strain of the present invention is preferably Corynebacterium glutamicum ( Corynebacteria glutamicum , Accession No .: KFCC 11043).

The present invention provides a method for activating the strain by culturing Corynebacterium species by adding pyrrolidone carboxylic acid or a substance containing the same to the seed culture medium and seed culture medium during the fermentation process for producing L-lysine. It is about. In this fermentation process carried out after the seed culture and seed culture does not significantly affect the fermentation efficiency even if pyrrolidone carboxylic acid or a substance containing the same is not added to the medium.

In general, the fermentation process is largely composed of seed culture, seed culture and main fermentation (main culture) process.

Species culture is carried out to maintain and maintain pure culture prior to seed culture for efficient production at the production site.

Seed culture carried out after the seed culture is an intermediate step before entering the main culture step to increase the cell mass and activity, and in order to increase the efficiency of the fermentation process, 5 ~ 30% of the initial volume of the fermentation is required. It is common for the seed culture to be carried out before the fermentation process. Seed culture is to culture the strain purely cultured in the species culture to have a cell mass and activity to the extent that can be used in the present culture. Seed cultures are used directly for this fermentation process.

In batch fermentation, purely isolated strains are cultured in a small volume of reactor, and when the cell mass increases to a certain level, they are transferred to a large volume of reactors. It is possible to obtain a culture medium in which the cell mass and activity is increased to such an extent that it can be used.

In the present invention, by culturing by adding a pyrrolidone carboxylic acid or a substance containing the same to the species culture and / or seed culture medium of Corynebacterium species carried out before the present fermentation process, The strain is activated and the productivity of L-lysine in the present fermentation process is also improved.

In the present invention, pyrrolidone carboxylic acid may be added alone or in the form of a substance containing it.

In the present invention, the pyrrolidone carboxylic acid is preferably added at a concentration of 0.1 to 5g / l to the seed culture medium and seed culture medium.

Substances containing pyrrolidone carboxylic acid include animal skin, aloe, beet, and the like, and preferably beet molasses. When beet molasses is used, the beet molasses is preferably added to the culture medium and seed culture medium at a concentration of 5 to 100 g / l. Beet molasses generally have a pyrrolidone carboxylic acid content of about 2-3%.

Seed culture medium, seed culture medium, and other fermentation medium components other than pyrrolidone carboxylic acid include various carbon sources, nitrogen sources and trace element components.

Examples of carbon sources that can be used include glucose, sucrose, fructose, maltose, carbohydrates such as starch, organic acids such as acetic acid and lactic acid. These carbon sources may be used alone or in combination. Examples of nitrogen sources that can be used include organic nitrogen sources such as peptone, yeast extract, gravy, malt extract, corn steep liquor, and soybean wheat and inorganic nitrogen sources such as urea, ammonium sulfate, ammonium chloride, ammonium phosphate and ammonium nitrate. These nitrogen sources may be used alone or in combination. The medium may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate and the corresponding sodium-containing salts as a person. It may also include metal salts such as magnesium sulfate or iron sulfate. In addition, amino acids, vitamins, and appropriate precursors may be included. These media or precursors may be added batchwise or continuously to the culture.

During the culture, compounds such as sodium hydroxide, potassium hydroxide, ammonia can be added to the culture in an appropriate manner to adjust the pH of the culture. In addition, during the culture, antifoaming agents such as fatty acid polyglycol esters can be used to suppress bubble generation. In addition, to maintain the aerobic state of the culture, oxygen or oxygen-containing gas (eg, air) is injected into the culture. The temperature of the culture is usually 20 to 45 ° C, preferably 25 to 40 ° C. The incubation period can continue until the desired amount of L-lysine production is obtained, preferably 10 to 160 hours.

Examples of the culturing method include batch, continuous and fed-batch cultivation. The fed-batch culture includes, but is not limited to, injection batches and repeated injection batch cultures.

The present invention also relates to a fermentation process for preparing L-lysine, comprising performing the main culture using the Corynebacterium sp. Strain activated by the above method.

Isolation of L-lysine from the culture of the present fermentation process can be separated by conventional methods known in the art. In such separation methods, methods such as centrifugation, membrane filtration, ion exchange chromatography, and crystallization may be used. For example, the culture may be centrifuged at low speed or the supernatant obtained by removing the biomass using a membrane may be separated by ion exchange chromatography.

Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.

Example 1

In this example, the effect of pyrrolidone carboxylic acid (PCA) on the increase of strain activity was examined.

After pure separation from lyophilized cells, Corynebacterium glutamicum (Accession No .: KFCC 11043), which was sufficiently identified for its titer, was incubated in LB (Luria Bertani) liquid medium for 20 hours, and then After washing several times, the PCA concentrations were inoculated into the minimum medium added with 0, 0.1, 0.25, 0.5 and 1.0 g / l, and then incubated at 30 ° C. and 220 rpm for 20 hours.

Composition of the minimum medium (pH 7.0, before sterilization) :

Glucose _{10g, (NH 4) 2 SO} 4 2g, elements _{2g, KH 2 PO 4 1.0g,} K 2 HPO 4 3.0g, MgSO 4 .7H 2 O 0.5g, FeSO 4 .7H 2 O 10mg, MnSO 4 .5H ₂ O 10 mg, biotin 100 μg, thiamine HCl 100 μg, niacinamide 1 mg, calcium pantothenate 1 mg, CaCl ₂ 2H ₂ O 0.1 g, Na ₂ B ₄ O ₇ 10H ₂ O 80 μg, (NH ₄ ) ₆ MoO and ₂₇ 4H ₂ O 40㎍, ZnSO ₄ and _{7H 2 O 10㎍, CuSO 4 .7H} 2 O 300㎍, MnCl 2 and 4H ₂ O 10㎍, FeCl ₃ and 6H ₂ O 1mg (in 1 liter of distilled water).

Then, the colony (colony) number was measured after diluting smear in LB solid medium, 2 days incubation at 30 to 32 ℃.

The number of viable cells was diluted with 0.85% sterile saline solution, and then diluted in LB (Luria Bertani) medium (peptone 10g / l, enzyme extract 5g / l, NaCl 5g / l, agar 20g /) and cultured. Measured. Equivalent weight was quantified using Bertrand method if necessary.

PCA addition amount (g / l) 0 0.1 0.25 0.5 1.0 Number of Cells per Unit Volume (Number / ml) 5X10 ⁶ 6X10 ⁸ 7X10 ⁸ 3X10 ⁸ 5X10 ⁸

From Table 1, it was confirmed that the activity of Corynebacterium glutamicum KFCC 11043 was improved by the addition of PCA. These results suggest that PCA increased the number of cells by physiologically affecting the cell membrane and enhancing resistance to the external environment (eg, pH, osmotic pressure change in medium).

Example 2: Fermentation in Flasks and Small Fermenters

In this example, the fermentation process was carried out in a flask and a small fermenter with L-lysine producing strain Corynebacterium glutamicum KFCC 11043.

(1) flask fermentation experiment

In this example, 25 ml of the culture medium was added to a 250 ml baffle flask, followed by shaking culture, and 50 ml of the fermentation medium was added to the 500 ml baffle flask, followed by shaking culture.

PCA was added to the culture medium and the fermentation broth at concentrations of 0, 0.5, 1.0 and 2.5 g / l, respectively. The medium composition other than PCA is as follows.

Seed culture composition (pH 7.0, before sterilization):

50 g / l of raw sugar, 10 g / l of soybean lysate, 1 g / l of yeast extract, 1.5 g / l of KH ₂ PO ₄ , MgSO ₄ 7H ₂ O 0.5 g / l, urea 5 g / l, biotin 50 μg / l.

Fermentation medium composition (pH 7.0, after sterilization):

100 g of raw sugar or glucose, 10 g of soybean acid hydrolysate, (NH ₄ ) ₂ SO ₄ 40 g, urea 4 g, KH ₂ PO ₄ 1 g, NaCl 2.5 g, MgSO ₄ 7H ₂ O 0.5 g, FeSO ₄ 7H ₂ O 10 mg, 10 mg of MnSO ₄ 5H ₂ O, 100 μg of biotin, 200 μg of thiamin-HCl, 40 g of CaCO ₄ (based on 1 liter of process water).

Each culture condition was incubated for 72 hours at 32 ℃, 220 rpm.

When the culture was completed, the culture was analyzed by High Performance Liquid Chromatography (HPLC) to determine the concentration of L-lysine and the OD of the culture, and the residue was calculated by the Berland method. Same as

PCA addition concentration L-lysine HCl concentration (g / L) L-Lysine HCl Yield (%) OD ^* (x100) Residual (%) No addition 52.1 52.1 0.35 0 0.5 g / l 54.8 54.8 0.29 0 1.0 g / l 56.3 56.3 0.32 0 2.5 g / l 56.0 56.0 0.33 0 ^* OD measurement at 562 nm using 0.1 N HCl as dilution water (100 times) after removal of remaining CaCO ₄

 (2) Small fermentation tank experiment

After culturing in a shaker incubator, seed cultured in a 5L small fermenter, the fermentation process was carried out by fed-batch method in a 30L fermenter.

The seed culture is first added with PCA to a concentration of 0 (no addition), 1.0 and 2.5 g / l in the following seed culture medium for 20 hours at 30 ° C. and 220 rpm in a medium volume (medium volume 25 ml / 250 ml baffle flask), or Shake culture was performed by adding beet molasses to a concentration of 10, 20 g / l.

Composition of the seed culture medium (pH 7.0, before sterilization) :

50 g / l of raw sugar, 10 g / l of soybean lysate, 1 g / l of yeast extract, 1.5 g / l of KH ₂ PO ₄ , MgSO ₄ 7H ₂ O 0.5 g / l, urea 5 g / l, biotin 50 μg / l.

After the seed culture was carried out, seed culture was performed in a 5L fermenter for 24 hours at a temperature of 30 ° C., pH 7.2 (adjusted with ammonia gas) and 600 rpm on the following seed culture medium. The aeration amount was 1 vvm, and the culture was carried out by adding PCA to the following seed culture medium with concentrations of 0 (no addition), 1.0 and 2.5 g / l, or beet molasses to a concentration of 10 and 20 g / l, respectively. Was performed.

Composition of seed culture medium:

70 g / l of raw sugar, 5 g / l of soybean acid hydrolysate, (NH ₄ ) ₂ SO ₄ 10 g / l, KH ₂ PO ₄ 1.0 g / l, MgSO ₄ ㆍ 7H ₂ O 0.5 g / l, biotin 300 µg / l, Thiamine / HCl 1,000 µg / l, Niacinamide 2,000 µg / l, Pantothenic Acid 500 µg / l, Defoamer 3 ml / l.

After the seed culture was carried out, the main fermentation reaction was carried out in a 30L fermenter for 50 to 80 hours at 600 rpm, temperature of 32 ℃, pH 7.0 (adjusted with ammonia gas) on the main fermentation medium. Aeration was 1vvm. The culture was performed by adding PCA to the fermentation broth at concentrations of 0 (no addition), 1.0, and 2.5 g / l, respectively.

This fermentation medium composition:

360g / l of raw sugar, molasses (as reducing sugar) 10g / l, soybean acid decomposition product 30g / l (additional sterilization after adjusting pH 2-3 using ammonia gas when preparing medium), (NH ₄ ) ₂ SO ₄ 55g / l, KH _{2 PO 4 2.0g / l, MgSO} 4 and _{7H 2 O 1.5g / l, FeSO} 4 and _{7H 2 O 50mg / l, MnSO} 4 and 5H ₂ O 50mg / l, biotin 300㎍ / l, thiamine HCl and 1,000㎍ / l, niacinamide 2,000 µg / l, pantothenic acid 500 µg / l, antifoam 3 ml / l.

When the culture was completed, the culture was analyzed by High Performance Liquid Chromatography (HPLC) to determine the concentration of L-lysine. The results are shown in Tables 3 and 4 below.

PCA concentration (g / l) L-lysine HCl concentration (g / l) Yield per L-lysine HCl (%) Fermentation productivity (g / l. Hours) Species / seed culture This fermentation No addition / no addition No addition 165 51.0 2.54 0.5 / 0.5 No addition 190 52.0 2.92 0.5 189 52.0 2.92 1.0 / 1.0 No addition 188 51.8 2.89 1.0 186 51.6 2.86 2.5 / 2.5 No addition 191 52.0 2.94 2.5 187 51.6 2.88

PCA / sugar beet molasses concentration (g / l) L-lysine HCl concentration (g / l) Yield per L-lysine HCl (%) Fermentation productivity (g / l. Hours) Species / seed culture This fermentation No addition / no addition No addition 163 51.0 2.50 PCA (0.5) / PCA (0.5) No addition 192 52.2 2.95 Beet molasses (10) / Beet molasses (10) / No addition 190 51.9 2.92 Beet molasses (20) / Beet molasses (20) No addition 188 51.8 2.89

According to Table 3 above, when 0.5, 1.0, and 2.5 g / l of PCA were added to the seed / seed culture medium, respectively, regardless of whether PCA was added to the fermentation reaction, the conventional method (without PCA) was used. In comparison, fermentation productivity increased by 15% and yield was increased by 2%.

On the other hand, similar effects were achieved when beet molasses, a substance containing pyrrolidone carboxylic acid, was used (see Table 4).

The production of L-lysine according to the fermentation process according to the present invention can achieve excellent productivity and yield per unit.

Claims (6)

By preseed and seed culturing Corynebacteria sp. Strain in a medium comprising pyrrolidone carboxylic acid and the pyrrolidone carboxylic acid is A method of activating the strain by feeding the medium in the form of beet molasses. The method of claim 1 wherein the concentration of pyrrolidone carboxylic acid in the medium is present at 0.1 to 5 g / l. delete The method of claim 1, wherein the concentration of beet molasses in the medium is 5 to 100 g / l. The method of claim 1, wherein the Corynebacterium sp. Strain is Corynebacteria glutamicum (Accession Number: KFCC 11043), characterized in that. A method for producing L-lysine, comprising culturing the activated Corynebacterium spp. Strain according to any one of claims 1, 2, 4 and 5.