KR950014462B1 - Preparation method of l-leucine by using microorganism - Google Patents

Abstract

The L-leucine fermentation method using Corynebacterium MS 3382 (KCTC 8509P) (I) yields 47 g/l at pH 6.8-7.5 and 1.0 vvm aeration on the medium based on 12% glucose and isoleucine 500 mg/l and histidine 300-500 mg/l. (I) is an isoleucine requiring mutant from histidine requiring Corynebacterium MS 3178 (KFCC 10011) by uv.

Description

Method for producing L-leucine by microorganisms

The present invention relates to a method for producing L-leucine by fermentation using a mutant strain that requires L-isoleucine and simultaneously requires L-histidine.

L-Leucine is an essential amino acid, which is used in pharmaceuticals, food additives, etc., and is recently used as a raw material and additive of liquid crystals or waste oil decomposition agents.

The present invention selects the mutant strains that are required for the growth of L-leucine and L-isoleucine and L-histidine when producing L-leucine by microbial fermentation, and then aerobicly in a medium containing sugar as a main ingredient. It relates to a method for producing L- leucine by culturing.

The production method of commercial L-leucine known so far is mainly extracted from hydrolysates of various plant proteins of wheat gluten, but "fermentative production using microorganisms" is the L-isoleucine requirement strain of Cerachia malceseus. Method (Japanese Patent Digestion 47-26313), Pseudomonas genus, Agrobacterium genus, Escherichia coli, Bacillus genus, etc. A method of culturing by mixing one or two strains (Japanese Patent Digestion 52-64486), L -Method using new mutant strains requiring isoleucine, L-threonine, and L-methionine as growth factors (US Patent 149640), S- (β-aminoethyl) -L-cysteine resistant bacteria in a medium containing acetic acid Method of using strain (Japanese Patent Digestion 51-37347), method of accumulating L-Leucine in culture medium by regulating oxygen supply in culture of L-leucine producing strain (Japanese Patent Digestion 57-798), sugar in medium It is known and a method of producing L- leucine using a semi-continuous fermentation method while maintaining the degree of extension of fermentation time (Republic of Korea Patent Publication No. 83-2485 No.).

Isolation method of the strain used in the present invention is a conventional mutant treatment method using the parent strain of Corynebacterium dongsae MS-03178 (KFCC-10011), the present inventors have been used for the production of L- leucine. Mutant strain MS-03382 (KCTC-8509P), which requires L-histidine as a growth factor, was isolated from the strains obtained by cultivation, and cultured in a fermentation medium containing leucine and L-histidine at a certain concentration. Discovering the fact that leucine accumulates, the present invention has been completed.

That is, the parent strain Corynebacterium Dong-a MS-03178 (KFCC-10011) strain of the present invention is appropriately diluted in sterile distilled water, treated with UV light for a predetermined time, and cultured by plating on complete medium, and then growing colonies at least. Replica culture was performed on top plate medium (L-isoleucine addition) and the minimum plate medium to which various amino acids were added.

After 2-3 days of incubation, L-histidine-required mutants were isolated by selecting strains which were not grown on at least top plate medium but not at least top plate medium to which L-histidine was added.

TABLE 1

Growth of Parental (KFCC-10011) and Mutant (KTCT-8509P)

Note (1) Add 100 μg / m1 L-isoleucine to the minimum medium.

Note (2) L-histidon; 100 μg / ml addition

Note (3) +++; Growth, ++; Normal growth, +; Low growth,-; Inability to grow

Isolated L-histidine-required mutants were cultured in fermentation broth supplemented with L-isoleucine and L-histidine, resulting in higher amounts of L-leucine than the parent strain Corynebacterium Dong-a MS-03178 (KFCC-10011). Has been shown to accumulate in the culture.

Among these mutant strains, the mutant strains (KCTC-8509P) showing clear L-histidine requirements as described in Table 1) were selected and separated.

As a result of comparing and analyzing the mutant strain (KCTC-8509P) of the present invention selected and separated in this manner and the growth of L-histidine addition concentration and the accumulation amount of L-leucine, are shown in Table 2) below.

Table 2) Comparison of growth and accumulation of L-leucine by strains by L-histidine addition by concentration (L-isoleucine was added to both strains to 500μg / m1 in fermentation medium)

TABLE 2

Note (1) The amount of L-histidine added to the fermentation broth (μg / ml)

Note (2) Growth rate; Measure absorbance at 610 nm by diluting the culture broth cultured in fermentation broth for 72 hours

Note (3) L-leucine accumulation in the culture medium incubated for 72 hours in L-leucine and fermentation broth (mg / ml)

As shown in Table 2), the optimal participation concentration of L-histidine of the mutant strain KCTC-8509P was 300 µg / ml.

The detailed mechanism of L-histidine, which is a requirement of the mutant strain (KCTC-8509P) used in the present invention, promotes the biosynthesis of L-leucine is not known, but as shown in Fig. 1, the target product using glucose in the culture medium is Biosynthesis of L-leucine blocks the synthesis of L-histidine, which is a precursor of 5-phosphoribosyl-1-pyrophosphoric acid (PRPP) and ATP, thus leading to an overall L-leucine biosynthetic pathway. It is judged to be due to.

(Figure 1) Biosynthetic pathway of L-leucine

PRPP is 5-phosphoribosyl-1-pyrophosphate

(5-phosphoribosyl-1-pyrophosphate)

ATP is Adenosine 5'-triphosphate.

That is, it can be presumed that the metabolic pathway to L-histidine, which is not an amino acid directly involved in the same pathway, is blocked or the flow is weakened, but the accumulation of L-leucine is promoted.

The medium composition of L-leucine can be used as a carbon source, such as glucose, raw sugar, starch hydrolyzate, etc. As a nitrogen source, ammonia gas, ammonia water, ammonium chloride, ammonium acetate, yuan, urea, ammonium phosphate, corn steep liquor can be used. And a medium in which its natural nutrient source and inorganic metal salt were mixed.

As a cultivation method, it is incubated for 48-72 hours under an aeration of 0.5-1.0vvm while maintaining a pH of 6.5-7.5 at a temperature of 30 ± 1 ° C.

L-leucine accumulated in the culture broth was adsorbed and separated from the ion exchange resin according to a conventional method to obtain an eluent, concentrated and decolorized, and then treated with ethanol to obtain crude crystals of L-leucine.

It will be described in detail in the following Examples.

Example 1

Used strain; Corynebacterium Donga MS-03382 (KCTC-8509P)

ph7.2 (before sterilization)

ph7.0 (before sterilization)

(Cultivation method)

The seed medium having the composition as described above is sterilized and cooled by dispensing 50m1 in 500m1 shake-flask, and then cultured by shaking the platinum strain inoculated with the strain MS-03382 (KCTC-8509P) of the present invention as a culturing culture.

The fermentation broth 300m1 was dispensed into a 1 L triangular flask and inoculated with 12 ml of the seed culture medium prepared by sterilization and cooling, and then cultured at 30 ° C. at a speed of 120 revolutions per minute for 72 hours.

The accumulation amount of L- leucine at the end of the culture was 38g / L.

Example 2

In the fermentation broth of Example 1, 2L of all medium except calcium carbonate was added to a 5L small fermentation tank, and sterilized and cooled. Here, inoculated with the pre-prepared seed culture medium 80m1 and incubated for 48 hours at 30 ℃ under aeration conditions of 600rpm, 0.5-1vvm.

At this time, the fermentation medium pH was automatically adjusted to 6.8-7.5.

The accumulation amount of L-leucine at the end of fermentation was 47 g / L.

Claims (5)

Fermentation medium mainly containing saccharides from Corynebacterium sinus MS-03382 (KCTC-8509P), which requires both L-isoleucine and L-histidine for growth in the production of L-leucine using nutrient-forming mutants. Method of producing L- leucine by trophic microbial fermentation, characterized in that the aerobic culture in a large amount to accumulate a large amount of L- leucine in the culture medium. The method according to claim 1, wherein the fermentation broth is cultured by adding 500 μg / m1 of L-isoleucine and 300-500 μg / m1 of L-histidine. The method of claim 1, wherein the pH of the culture medium of the fermentation broth is maintained at 6.8-7.5. The method according to claim 1, wherein the culture is carried out while maintaining the aeration amount at 0.5-1.0vvm. The method according to claim 1, wherein L-isoleucine is 500 μg / m1, L-histidine is 300-500 μg / ml, the pH of the culture medium of the fermentation broth is 6.8-7.5, and the aeration amount is 1.0vvm. Method for preparing L-leucine.