KR100868330B1 - A method for preparing L-threonine - Google Patents

Abstract

The present invention relates to a method for producing L-threonine, which is generated during sterilization by adjusting the pH before sterilization of a seed culture and seed culture to 2-4 with sulfuric acid, phosphoric acid, hydrochloric acid, and the like. Inhibiting the formation of complex salts and activating the Esherichia coli strain has a very good effect of providing a method for producing L-threonine with higher fermentation efficiency, ie, high yield and productivity. have.

Threonine, L-Threonine, Escherichia coli, Fermentation efficiency, Per yield

Description

A method for preparing L-threonine

The present invention relates to a method for producing L-threonine, and by suppressing the formation of complex salts generated during sterilization by adjusting the pH of the subsidiary material before sterilization to 2-4 with sulfuric acid, phosphoric acid, hydrochloric acid, etc. Esherichia during preseed and seed cultures coli ) relates to a method for producing L-threonine with improved yield and productivity characterized by activating a strain.

L-threonine is one of the essential amino acids and is used as feed additives, food additives, and pharmaceutical raw materials for livestock. In particular, the market size of L-threonine as a feed additive for livestock reached about 90,000 tons ($ 2.7 billion / year) in 2006, which is the second largest fermentation product after L-lysine.

Currently, L-threonine is a carbon source such as glucose (corn / tapioca syrup), raw sugar (enzyme digest), and a nitrogen source such as corn steep liquor (CSL), soybean sulphate, ammonium sulfate, vitamins, minerals, etc. It is industrially produced by a direct fermentation method using as a medium raw material.

As a conventional method for producing L-threonine, Korean Patent Application Publication No. 2003-33039 (US Pat. No. 6,562,601) discloses a method for producing L-threonine-producing microorganisms of the Enterobacteriaceae subfamily by a method of feeding oil. The method of culturing and remaining part of the fermentation broth in the fermentation tank to fill a new medium to further culture. It is suggested that the yield can be further increased. US Pat. No. 4,427,774 discloses a method of fermenting L-threonine using ethanol as a main carbon source, while US Pat. No. 5,474,918 provides resistance to purine analog compounds. The method for producing L-threonine by artificially mutant strains, US Pat. No. 5,376,538, is an artificially mutant strain that is resistant to L-phenylalanine or L-leucine. L-Threonine production method, US Patent No. 3,616,217 is a method of producing L- threonine by an artificial mutant strain that methionine and valine, or leucine nutritional requirements, US Patent No. 5,188,949 is a mycophenolic acid A method for producing L-threonine by the genus Brevibacterium or Corynebacterium that has given the resistance to (mycophenolic acid) is disclosed in US Pat. No. 7,074,602. -The method for producing threonine, Korean Patent Publication No. 1999-0016872 discloses L-threonine by culturing in a medium a microorganism belonging to the genus Eshrichia and having resistance to purine analogs and the ability to produce L-threonine. Method of recovering the method, Korean Patent No. 0134840 discloses a novel strain as a method for producing a large amount of L- threonine by aerobic culturing a strain of L- threonine producing strains of the genus Serratia. La by the method of writing a large amount of L- using thiazole in SW9410 Leo non-accumulated in the culture medium for producing L- threonine, Republic of Korea Patent No. 10-0478468 discloses have the metJ gene inactivated chromosomal used L- A novel Escherichia coli strain having the property of producing leonin and a method of producing L-threonine using the same are described.

Among the L-threonine production methods, L-threonine production methods using Escherichia coli strains have a disadvantage of inactivating Escherichia coli strains in culture due to the formation of a double salt during sterilization.

Accordingly, the present inventors suppressed the formation of complex salts generated during sterilization by adjusting the pre-sterilization pH of preseed culture and seed culture medium to 2-4 with sulfuric acid, phosphoric acid, hydrochloric acid, and the like. Esherichia coli ) to complete the present invention by establishing the L-threonine fermentation process to achieve a higher fermentation efficiency by activating the strain.

Accordingly, an object of the present invention is to provide a method for activating Escherichia coli species used in L-threonine fermentation to produce L-threonine with high yield and productivity.

The present invention suppresses the formation of complex salts generated during sterilization by adjusting the pre-sterilization pH of the seed culture and seed culture medium to 2-4 with sulfuric acid, phosphoric acid, hydrochloric acid and the like. teeth Collie (Esherichia coli ) to provide a method for producing L-threonine to achieve higher fermentation efficiency by activating the strain.

In the present invention, tapioca starch sugar (glucose) may be used as a carbon source, and potassium dihydrogen phosphate, dipotassium hydrogen phosphate, and corresponding sodium-containing salts may be included as a subsidiary material. Also, complex salts such as magnesium, iron, phosphorus, etc. may be prepared by adjusting the pH of the seed medium and the seed medium containing metal salts such as magnesium sulfate or iron sulfate to 2-4 with sulfuric acid, hydrochloric acid, phosphoric acid, etc. before sterilization. By inhibiting production), thereby providing a method of activating said strain during species culture and seed culture.

In the present invention, strains used for L-threonine fermentation belong to Escherichia spp., And any one having L-threonine production ability is included.

The Esherichia sp. Strain of the present invention is preferably Esherichia coli (Accession No .: KCCM-10392).
The Escherichia coli KCCM-10392 was issued in June 2002 in connection with the Korean Patent Application No. 10-2003-62423, filed on September 6, 2003, entitled "Method for Producing L-Threonine". It was deposited with the Korea Microbial Conservation Center on 25th.

The present invention relates to a method of activating the strain by sterilizing the seed culture medium and seed culture medium in the fermentation process for producing L-threonine by adjusting the pH to low before sterilization and then adjusting the culture pH again.

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, and is a step for increasing the cell mass and activity. Generally, in order to increase the efficiency of the fermentation process, 10-30% 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, the strain is activated by changing and culturing the seed culture medium and / or seed culture medium of Escherichia coli, which is carried out before the present fermentation process, and as a result, the L-settle in the present fermentation process is activated. The yield and productivity of leonin are also improved.

Examples of carbon sources that may be used during fermentation in the present invention include glucose, sucrose, fructose, organic acids such as carbohydrates such as crude sugar enzyme digests, lactic acid and citric acid. These carbon sources may be used alone or in combination.

Examples of nitrogen sources that can be used in the present invention 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 Included. These nitrogen sources may be used alone or in combination.

Phosphorus sources in the present invention may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate and the corresponding sodium-containing salts. 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 and 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 may continue until the desired amount of L-threonine production is obtained, preferably 30 to 120 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 producing L-threonine, comprising performing the main culture using an Escherichia coli species strain activated by the above method.

Isolation of L-threonine 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: Investigate the effect of medium sterilization conditions on strain growth

Esherichia isolated purely from lyophilized cells and confirmed its titer coli , Accession No .: KCCM-10392) cells cultured in LB (Luria Bertani) liquid medium for 5 hours, sterilized the seed culture medium under the same sterilization conditions as in Table 1, and then adjusted to the culture pH 7.0 before inoculation again. After phagocytosis in a predetermined amount, shaking culture for 5 hours at 35 ℃, 220rpm.

Then, after diluting with 10-fold distilled water, absorbance (Optical Densiyt) was measured at 562 nm, and the equivalent was quantified using Bertrand method if necessary.

Seed composition:

Ingredients: 20g of glucose (separate sterilization),

Sub material: yeast extract _{(Yeast extract) 5g, KH 2} PO 4 3.0g, K 2 HPO 4 3.0g, MgSO 4 .7H 2 O 0.5g, NaCl 0.5g, NH 4 Cl 1g, L-met 0.1g, L- _{ile 0.1g, FeSO 4 .7H 2 O} 10mg, MnSO 4 .5H 2 O 10mg, CaCl 2 and _{2H 2 O 0.1g, Na 2 B} 4 O 7 and _{10H 2 O 80㎍, (NH 4} ) 6 MoO 27 and 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 ₂ and H ₂ O 1mg (process water 1 liters basis).

PH adjustment before subsidiary media sterilization PH 3.0 adjustment with diluted sulfuric acid before subsidiary media sterilization Absorbance (Opical Density 562nm, * 10-fold dilution) 0.05 0.45

From Table 1, it was confirmed that when the subsidiary medium was adjusted to pH 3.0 with diluted sulfuric acid before sterilization, the activity of Escherichia coli was improved.

These results indicate that under low pH sterilization conditions, magnesium, phosphorus, iron, and ammonia exist in free ions, and enough E- herichia coli ( Esherichia ) is a L-threonine producing strain. coli , Accession No .: KCCM-10392).

Example  2: in small fermentation tank Seed culture

In this example, a culture process in a small fermentation tank of Escherichia coli (Accession No .: KCCM-10392), an L-threonine producing strain, was performed.

Stage 1: First Small Fermenter Seed culture test

In this example, 50 ml of the above culture medium (to pH 3.0 with sulfuric acid diluted before sterilization and pH 7.0 after sterilization) was added to a 500 ml baffle flask, followed by shaking culture for 5 hours.

Thereafter, seed culture was performed in a 5L fermenter for 15-30 hours at a temperature of 33-37 ° C., pH 6.5-7.0 (adjusted with ammonia gas) and 600 rpm on the following seed medium. Aeration was 1vvm and was carried out under the test conditions as shown in Table 2.

Seed culture medium

Raw materials: 40g of glucose (separate sterilization)

Sub material: yeast extract _{(Yeast extract) 3.0g, KH 2} PO 4 3.0g, MgSO 4 .7H 2 O 0.5g, NaCl 0.5g, NH 4 Cl 1g, L-met 0.1g, 200㎍ biotin, thiamine and HCl 500 Μg, niacinamide 10 mg, calcium pantothenate 10 mg, FeSO ₄ 7H ₂ O 10 mg, MnSO ₄ 5H ₂ O 10 mg, CaCl ₂ 2H ₂ O 0.1 g, Na ₂ B ₄ O ₇ 10H ₂ O 80 μg, (NH _{4) 6} MoO ₂₇ and 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 ₂ and H ₂ O 1mg (step 1 Liters).

When the culture was completed, the culture was analyzed by High Performance Liquid Chromatography (HPLC) to measure the concentration of L-threonine. The results were as shown in Table 2 below.

PH adjustment before subsidiary media sterilization PH 3.0 adjustment with sulfuric acid before subsidiary media sterilization Absorbance (Opical Density 562nm, * 100-fold dilution) 0.2 0.4 Incubation time (hrs) 25 18 L-Threonine Concentration (g / l) 12 20 Residual (%) 0.5 0

Table 2 shows that when the pH of the subsidiary medium was adjusted with sulfuric acid before sterilization, it had a remarkable effect in terms of cell volume, L-threonine fermentation yield, and culture time.

Stage 2: 2nd Small Fermenter Seed culture test

Sulfuric acid was used to lower the pre-sterilization pH of the subsidiary medium to 2-4 in the first stage experiment, but sulfuric acid has a significant risk of being applied to large production fermenters in handling.

Therefore, the following experiments were carried out in a small fermenter using safer acid, phosphoric acid. Medium and culture conditions were adjusted in the same manner as in the first step.

PH adjustment before subsidiary media sterilization PH 3.0 adjustment with sulfuric acid before subsidiary media sterilization PH 3.0 adjustment with phosphoric acid before subsidiary media sterilization Absorbance (Opical Density 562nm, * 100-fold dilution) 0.22 0.45 0.42 Incubation time (hrs) 28 19 20 L-Threonine Concentration (g / l) 11.7 18 19 Residual (%) 0.5 0 0

As a result, when the pH was adjusted using phosphoric acid, it was found that there was no difference in cell volume, incubation time, fermentation yield, and the like when using sulfuric acid.

Step 3: fermentation test of small fermenter

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

First, the seed culture was carried out by shaking culture at 35 ° C. and 220 rpm for 5 hours in a medium volume (50 ml / 500 ml baffle flask).

After the seed culture, seed culture was performed in a 5L fermenter for 20-25 hours at a temperature of 33-37 ° C., pH 6.5-7.0 (adjusted with ammonia gas) and 600 rpm on the following seed culture medium.

In seed culture, the subsidiary medium was adjusted to pH 3.0 before sterilization using phosphoric acid, followed by incubation.

After the seed culture was carried out, the main fermentation reaction was carried out in the following main fermentation medium at a temperature of 33 to 37 ° C., pH 6.5 to 7.0 (adjusted with ammonia gas), 45 to 60 hours at 600 rpm, and aeration amount of 1 to 1.5 vvm 30L fermenter. Was performed.

This fermentation medium ingredients:

Ingredients: Glucose 40g

Sub material: yeast extract _{(Yeast extract) 1g, KH 2} PO 4 2.0g, MgSO 4 .7H 2 O 0.5g, NaCl 0.5g, NH 4 Cl 1g, DL-met 0.1g, 200㎍ biotin, thiamine and HCl 500㎍ , 10mg niacinamide, calcium pantothenate _{10mg, FeSO 4 .7H 2 O 10mg} , MnSO 4 .5H 2 O 10mg, CaCl 2 and _{2H 2 O 0.1g, Na 2 B} 4 O 7 and 10H ₂ O 80㎍, (NH ₄ ) ₆ MoO ₂₇ and 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 ₂ and H ₂ O 1mg, anti-foaming agents 3㎖ / l. (Based on 1 liter of process water).

Additional medium medium components: glucose _{50 ~ 55%, KH 2 PO} 4 2.0g, MgSO 4 .7H 2 O 0.5g, DL-met 0.1g, 200㎍ biotin, thiamine and HCl 500㎍, niacinamide 10mg, 10mg calcium pantothenate _{, FeSO 4 .7H 2 O 10mg,} MnSO 4 .5H 2 O 10mg, CaCl 2 and _{2H 2 O 0.1g, Na 2 B} 4 O 7 and _{10H 2 O 80㎍, (NH 4} ) 6 MoO 27 4H ₂ O and 40㎍, ZnSO ₄ and _{7H 2 O 10㎍, CuSO 4 .7H} 2 O 300㎍, MnCl 2 and 4H ₂ O 10㎍, FeCl ₂ and H ₂ O 1mg, anti-foaming agents 3㎖ / l.

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

PH-free adjustment before seed sterilization medium Adjusting pH 3.0 with phosphoric acid before seed material medium sterilization L-Threonine Concentration (g / l) 105 120 Fermentation yield (%) 47 52 Fermentation Productivity (g / hrs) 2.2 3.0

According to Table 4, when the seed culture subsidiary medium was adjusted to 3.0 with phosphoric acid before sterilization, the yield was increased by 10% and the fermentation productivity was increased by 30% or more.

As described above, the present invention forms complex salts generated during sterilization by adjusting the pH before sterilizing the seed culture and seed culture medium to 2-4 with sulfuric acid, phosphoric acid, hydrochloric acid, and the like. Inhibiting Esherichia coli ) is a very useful invention in the food industry, livestock feed industry and pharmaceutical industry because it has a very excellent effect to activate the strain to provide a method for producing L- threonine with higher fermentation efficiency, that is, excellent yield and productivity. .

Claims (4)

In the method of producing L-threonine by escalating, seeding and main culture of Escherichia coli strain, the culture medium is adjusted to 2-4 before sterilization and sterilized again. Process for producing L-threonine, characterized in that readjusted to the appropriate pH. In the method of producing L-threonine by seed culture, seed culture and main culture of Escherichia coli strain, the pH of the medium is adjusted to 2-4 prior to the seed culture, followed by sterilization Process for producing L-threonine, characterized in that readjusted to the appropriate pH. The method for preparing L-threonine according to claim 1 or 2, wherein the pH adjustment is performed by adding sulfuric acid, hydrochloric acid or phosphoric acid. The method of claim 1 or 2, wherein the Escherichia coli ( Esherichia) coli strain is Esherichia coli ) (Accession No .: KCCM-10392), a method for producing L-threonine.