KR890003681B1 - Process for preparing l-phenyl-alanine by microorganism - Google Patents

Abstract

The method for preparing L-phenylalanine by the mutant of Escherichia coli is presented. Thus, MWEC101-b strain is precultured in the medium containing 5% of glucose, 1 tryptone, 1 yeast extract and 0.1 of sodium chloride. The precultured strain is cultured in fermentation medium containing 6% of glucose, 0.04 potassium sulfate, 2 ammonium sulfate and 0.05 sodium citrate etc. at 31≰C for 16 hrs. The fermented broth is separated by ionexchange resin.

Description

Method for preparing L-phenylalanine by microorganism

1 is a graph showing the growth rate by the addition of nicotinic acid.

The present invention is aerobic culture of a special strain of Escherichia coli in a medium containing a carbon source, a nitrogen source, and other organic and inorganic nutrients useful for microorganisms, so L-phenylalanine accumulates at a high concentration in the medium L-phenylalanine It relates to an industrial production method of. L-phenylalanine is a substance used as a pharmaceutical ingredient of essential amino acids or a synthetic raw material of aspartame, a low-calorie sweetener.

Conventional methods for producing L-phenylalanine by microorganisms include tyrosine-constituting phenylalanine of Escherichia coli (Silicon Publication 37-6345, 60-160890) by Tyrosine Regimen of Brevibacterium or Corynebacterium. Tryptophan analog-resistant mutant strains (Japanese Patent Application Publication 55-165797) and the like are known.

L-phenylalanine has a complicated biosynthetic pathway of microorganisms, and the biocidal yield having an aromatic ring in its structural formula is generally very low. In addition, there are many disadvantages such as adding expensive L-tyrosine to the medium or delaying fermentation due to poor growth.

로 본 발명을 완성하게 되었다.The inventors of the present invention have solved these shortcomings in the past, and have been studying to invent a strain having excellent productivity of L-phenylalanine.

The present invention has been completed.

The strains of the present invention are based on L-tyrosine, L-tryptophan co-requirement and MWEC-101 strains resistant to L-phenylalanine analogues from E. coli K-12 strains. L-phenylalanine is a strain having a special physiological properties improved productivity.

The inventors of the present invention, MWEC-101 strain of the main strain of the present strain requires L-tyrosine and L-tryptophan to grow a large amount of these expensive substances added to the production medium, poor growth and require a long fermentation time point In order to solve the shortcomings such as growth inhibition by L-phenylalanine produced in the fermentation broth and lower productivity, MWEC-101 strains of the mother strain do not require L-tyrosine and L-tryptophan. L-Phenylalanine is produced by screening strains with higher L-phenylalanine productivity among strains with enhanced resistance to L-phenylalanine, L-tyrosine and L-tryptophan analogues Better strains were invented as strains.

Specific physiological properties of strain E. coli MWEC 101-6 strain of the present invention are as follows.

1. An unrequired nutritional reversion variant that does not require L-tyrosine and L-trypan in growth.

2. L-varin resistant main point.

3. High concentration of resistance to analogues of L-phenylalanine, L-tyrosine and L-tryptophan.

4. It is sensitive to the culture temperature and the growth of nicotinic acid is significantly promoted.

The mutant treatment method used by the present inventors for strain improvement was performed by suspending the cultured cells of MWEC 101 strain at 37 ° C. in a medium of medium 1 for 12 hours in 5 ml of 0.05 M phosphate buffer (pH 7.0), and then 30 cm distance with 15 W ultraviolet lamp. After irradiating for 15 minutes at and then inoculated in the medium of medium 2 and cultured shaking at 37 ℃ 7 days. The culture medium was plated again in medium 3 medium and cultured at 37 ° C. for 24 hours, only the best bacterial colonies were grown, followed by shaking culture at 37 ° C. for 50 hours in medium 4 medium, followed by analysis of L-phenylalanine concentration in the culture medium. After MWEC 101-1 strain, which had the highest productivity, was isolated and subjected to mutation treatment again.

The cells of MWEC 101-1 strain were well suspended in 2 ml of 0.05 M phosphate buffer (pH 7.0), and then 2 ml of 50 g / ml N-methyl-N'-nitro-N-nitroso-guanidine solution was added at room temperature. After shaking for 30 minutes, cells and mutants were separated by centrifugation and washing, plate was plated in medium 5, and cultured for 48 hours at 37 ° C. MWEC 101-5 had the highest productivity of L-phenylalanine. Weeks were selected.

MWEC 101-5 strains were further mutated by the above-described UV treatment to use a medium of medium 6 as a selection medium. Each strain of the mutated cells was inoculated into a medium of medium 6, and then shaken at 37 ° C. for 24 hours, and then grown at 610 mm to examine the absorbance at 610 mm to select only the strains with the highest relative growth, and again to produce L-phenylalanine. After comparison, the strain MWEC 101-6 (KCTC 8235 P) of the present invention having the highest production capacity was finally selected.

Badge 1:

Yeast Extract 0.5%

Peptone 1%

Juicy 1%

Sodium chloride 0.25%

Agar 2%

pH 6.8 120 ℃ × 20 minutes sterilization

Badge 2:

10 g of glucose

Ammonium Sulfate 4g

1 g of potassium monophosphate

Potassium Diphosphate 3g

Magnesium sulfate 0.5g

20 mg of iron chloride

Manganese Chloride 10mg

Zinc sulfate 10mg

Thiamine Hydrochloride 1mg

0.5 g of fumaric acid

Yeast Extract 0.2g

1l of distilled water

pH 7.4 minutes 120 ℃ × 20 minutes sterilization

Badge 3:

Add 4 g / l L-phenylalanine and 20 g / l agar to Medium 2

Badge 4:

60 g of glucose

Yeast Extract 0.5g

Potassium Diphosphate 0.8g

0.4 g of potassium sulfate

20 g ammonium sulfate

0.8 g magnesium chloride

Cobalt Chloride 0.1mg

20 mg of iron chloride

Manganese Chloride 10mg

0.5 g citric acid

0.5 g of fumaric acid

Calcium Carbonate 35g (Added after Sterilization)

1l of distilled water

40ml aliquots in 500ml flasks

PH 7.5 adjusted with 1N potassium hydroxide and sterilized for 20 minutes at 120 ℃

Badge 5:

Add 20 g / l of agar to the medium 2 excluding 0.2 g of yeast extract.

P-Fluorine-Diel-phenylalanine, 5-Methyl-Del-Tryptophan, and 3-Amino-L-Tyrosine were each added at a concentration of 100 μ / ml

Badge 6

Add 100 mg / l of L-varin to the medium of medium 2 excluding 0.2 g of yeast extract

The culture and physiological characteristics of the strain MWEC 101-6 of the present strain were as shown in the following table.

Table 1

Nutritional Components Survey

-: No growth, +, ++, +++: Growth degree display

The irradiated amino acid was added to each 200mg / l concentration basal medium was used as the medium 2 except for yeast extract.

[Table 2]

Comparison of Resistance to L-Phenylalanine, L-Tyrosine and L-Tryptophan Analogue Materials

^* 1. -DL- fluoro-phenylalanine as m-

2. β-2-thienyl-DL-alanine

3. p-amino-DL-phenylalanine

4. 5-Methyl-DL-Tryptophan

5. 3-amino-L-tyrosine

50 ml of nicotinic acid was added to a 500 ml flask in a medium containing 100 mg / l of nicotinic acid in the medium 2 except for the yeast extract, and the relative growth was also compared by absorbance irradiation at 610 nm for 10-fold dilutions of the culture medium after shaking for 24 hours at 37 ° C.

MWEC 101 strain added L-tyrosine and L-tryptophan at 200 mg / l concentration in medium, respectively.

Table 3

Effect of L-Barine Addition

Growth test was carried out in accordance with the method of Figure 1 attached (influence of nicotinic acid).

Table 4

Comparison of L-phenylalanine Production Capacity by Culture Temperature

The culture method was investigated the effect of each temperature by the method of Example 1 and in the case of MWEC 101 was added to L-tyrosine and L- tryptophan concentration of 600 mg / l, respectively.

As can be seen in Tables 1 and 4, the strain E. coli MWEC 101-6 of the present invention is significantly promoted growth by the addition of nicotinic acid, and there is no nutritional composition for special substances such as amino acids, compared to conventional strains. Cheap fermentation medium is available and fermentation management is very easy. In addition, resistance to L-phenylalanine, L-tyrosine, and L-tryptopin analogues was markedly enhanced compared to the parent strain as shown in Table 2, and the nutritional investigation of the addition of L-varin in Table 3 revealed that The strains of the present invention can be regarded as L-varin resistant strains because they show clearly different growth rates from the parent strains of MWEC 101 strains and MWEC 101-5 strains.

In addition, cell growth as well as L-phenylalanine productivity showed a lot of difference according to the orientation temperature. MWEC 101 strain, the parent strain, showed the optimum growth and production at 37 ℃, but the strain showed the optimal growth and production capacity at 31 ℃. This sensitivity to incubation temperature was altered by the separation of L-varin resistant strains. These various physiological changes are believed to be the cause of high production of L-phenylalanine.

When L-phenylalanine is produced using the strain of the present invention, it is cultured in an initial condition in a medium containing a carbon source, a nitrogen source, an inorganic salt, and an organic nutrient source in accordance with the physiological and culture characteristics of the strain of the present strain. The fermentation broth in which L-phenylalanine is accumulated is separated and decolorized using an ion exchange resin or the like according to a conventional method, and then concentrated to recover L-phenylalanine.

Although the detailed method of this invention is described in the Example, it is limited to the implementation of this application.

Example 1

Use strain MWEC 101-6

Species medium: glucose 5, tryptone 1%, yeast extract 1%, sodium chloride 0.1%

Fermentation medium: glucose 6%, potassium sulfate 0.04%, yuan 2%, sodium citrate 0.05%, fumaric acid 0.05%, magnesium chloride 0.08%, potassium monophosphate 0.1%, dibasic potassium phosphate 0.1%, yeast extract 0.1%, glutamic acid 0.05%, cobalt chloride 0.1 mg / l, zinc sulfate 1 mg / l, manganese chloride 2 mg / l, calcium chloride 5 mg / l, thiamine hydrochloride 5 mg / l, nicotinic acid 10 mg / l.

Culture method: Dispense 50ml of the seed medium into a 500ml shake flask and autoclave it according to a conventional method, inoculate strain MWEC 101-6 of the present invention, and incubate at 31 ° C for 16 hours to make a seed culture.

After fermentation broth 201 injection sterilization in 501 fermenter was inoculated with 1l of the seed culture solution was incubated for 48 hours at 31 ℃ under 400rpm, 0.75VVM conditions. During incubation, ammonia water was adjusted to pH 7.0-7.2 and 60% glucose solution adjusted to pH 3.0 with phosphoric acid was added twice when the residue was 1%. The total equivalent used for fermentation was 140 g / l with L-phenylalanine accumulated 23.2 g / l. After adsorbing 1 l of the culture solution to the ion exchange resin according to a conventional method, the eluate was concentrated to obtain 18 g / l of crude phenylalanine.

Example 2

After inserting 3 l into a 5 l small fermentation tank by the method of Example 1, autoclaved at 120 ℃ for 15 minutes, inoculated with 200ml of the seed culture solution and incubated at 31 ℃ under 600rpm, 0.75VVM conditions for 24 hours.

The pH in the culture was adjusted to 7.0 using ammonia water. The accumulation amount of L-phenylalanine in the culture solution was 11.4 g / l.

Example 3

In the fermentation broth of Example 1, sugar was used instead of glucose after enzymatic digestion into fructose and glucose, and the total amount of sugar used was 130 g / l.

The culture medium had a L-phenylalanine accumulation of 24.8 g / l.

Claims (1)

L-tyrosine and L-tryptophan are required for growth, resistant to L-varin, high concentration resistance to analogues of L-phenylalanine, L-tyrosine, L-tryptophan and markedly promoted by nicotinic acid. Method of preparing L-phenylalanine by culturing the Ashericia coli MWEC 101-6 (KAIST, KCTC 8235 P).

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