KR810001114B1 - Process for preparation of l-alginine by fermentation - Google Patents

Abstract

L-Arginine was prepd. by fermn. Thus, Breribactrium. flavum MS-05760 (KFCC 10018) was inoculated in culture media which contained molasses 5%, NH2Cl 2.5%, peptone 0.1%, yeast ext. 0.1%, K2HPO4 1.0%, MgSO4 0.05%, FeSO4 0.001%, corn steep liquor 1.5%, MnSO4 0.001%, CaCO3 2%, and fermented at 30oC in 65hr. with shaking condition. L-Arginine productivity is 20.8 mg/m in broth.

Description

Preparation of L-arginine by fermentation

The present invention relates to a method for producing L-arginine by the fermentation method. L-arginine is a kind of natural amino acid, which is very important for nutrition and medicine, and at the same time, it is an important amino acid used for seasoning and feed.

In the past, the industrial production of L-arginine was a method of extracting and separating from hydrolysates of natural proteins. However, the extraction separation structure was very complicated, and thus L-arginine was inexpensive.

In recent years, however, many methods for producing L-arginine have been reported using fermentation methods, which are mainly resistant to L-arginine antagonists, that is, mutants whose control mechanisms are released by L-arginine.

(Japanese Patent 48-3391 Agr. Biol Chem 36, 1675-1684 (1972))

The microorganism used for the L-arginine fermentation of the present invention is resistant to L-arginine metabolic antagonists and does not release the metabolic regulation by L-arginine, but the microorganism itself deletes arginine decarboxylase and arginine decarboxylase simultaneously. By activating the arginine biosynthesis system, the production capacity of arginine is remarkably improved.

In other words, the present inventors have conceived that arginine decarboxylase and arginine decarboxylase are involved in the metabolic regulation of arginine biosynthesis, and thus mutagenesis of alginine decarboxylase and arginine decarboxylase by simultaneously inducing mutations in alginine decarboxylase. The present invention has been completed by discovering that the enzyme inhibition of the arginine biosynthesis system by oxidase and arginine decarboxylase can be released significantly in the nutrient medium containing a common carbon source.

The microorganism used in the present invention is a mutant Brevibactetilumfura boom MS-05760 (L-arginine-producing brevy bacterium fura boom ATCC 14067, which simultaneously depleted the mutated arginine decarboxylase and arginine decarboxylase). KFCC 10018).

This deficiency strain is a conventional mutation-inducing operation treated with, for example, X-ray irradiation, ultraviolet irradiation, or chemical variant organic matter (for example, N-methyl-N'-N nitrosoguanidine, diesulfide, ethylamine, etc.). Then, the bacteria suspension was incubated for 2-3 days in a plate medium (for example, Sbittzen basal medium) by adding arginine (1.5 mg / ml) and a very small amount of oil (2-10 µg / ml) as a medium nitrogen source. After the colony was purely separated and selected as a good strain.

When the physiological characteristics of the arginine decarboxylase and the arginine decarboxylase co-defective strain MS-05760 (KFCC 10018) of the present invention were compared with the parent strain, it was found that the parent strain and the sugar fermentability were completely different.

Comparing the sugar fermentation of parental and mutant strains,

1.A: acid production

-: Does not generate acid or gas.

2. Fermentation broth: Peptone broth containing sugar (1.0% peptone, 0.5% sodium chloride, 0.5-2.0% sugar).

Microorganism experiment published by Bergy's Manual of Determinative Bacteriology and American Bacterial Association of Brevibacterium puraboom MS-05760 (KFCC 10018), which is a co-deleted variant of arginine decarboxylase and arginine decarboxylase of the present invention According to the Manual of Microbiological Method, the results were as follows.

end. Morphological properties

(1) Form: Simple bacilli, 0.7 × 1.0μ ~ 1.5 × 3.0μ

(2) Mobility: None

(3) Gram dyeing: positive

I. Physiological Properties

(1) Optimal growth temperature: 28-32 ℃

(2) Optimal growth pH: 6.8-7.2

(3) Oxygen Requirements: Aerobic

(4) hydrogen sulfide production: negative

(5) Gelatin liquefaction power: None, grow on the surface.

(6) urease: positive

(7) Starch Liquefaction: Negative

(8) catalase: positive

All. Culture properties

1. Agar Slope Cultivation (Juicy Agar Medium 28 ℃ × 30 hours)

(1) Growth: Normal

(2) Shape: Butter

(3) Gloss: Weak Gloss

(4) Cell color: light yellow

2. Agar plate culture (Juicy agar plate 28 ℃ × 30 hours)

(1) Growth: Normal

(2) Shape: Round

(3) Shape: Semi-lens type

The fermentation medium for the mutant strains used in the present invention contains a common carbon source, nitrogen source, inorganic salts, and organic nutrients. As the carbon source, sugars such as glucose, molasses, and starch hydrolyzate are used.

As a nitrogen source, 2-5% of inorganic ammonium such as ammonium chloride and lactic acid ammonium was used. As inorganic salts, small amounts of potassium phosphate, magnesium lactate, and iron lactate were added. As organic nutrients, peptone and yeast ekis , Cone tip rica and the like were used at 0.01-2.0%.

The fermentation method was shaken for 2-7 days in aerobic conditions while maintaining the temperature of 24-35 ℃, pH 6.0-8.0.

Collected L-arginine was combined with a conventional ion exchange resin and other known methods.

The production of amino acids other than L-arginine was extremely small.

L-arginine was identified by paper chromatography, and L-arginine was quantified by bioassay.

In addition, the deficiency of arginine decarboxylase and arginine decarboxylase was measured by CO ₂ .

(EF Gale, Biochem. J. 34, 392 (1940), ES Taylor, EF Gale, Biochem. J. 39, 52 (1945))

Example 1

Molasses 5%, ammonium chloride 2.5%, peptone 0.1%, yeast ekis 0.1%, dibasic phosphate 1.0%, magnesium lactic acid 0.05%, ferrous lactic acid 0.001%, corn steep rika 1.5%, manganese 0.001 30 ml of medium (pH 7.3) containing% and 2% calcium carbonate was added to a 500 ml shaken flask and sterilized under pressure at 115 ° C. for 15 minutes. At this time, molasses and calcium carbonate are added by sterilization separately. This medium was inoculated with Brevibacterium puraboom MS-05760 (arginine decarboxylase and arginine decarboxylase) (KFCC 10018) after inoculation with platinum and incubated for 65 hours at 30 ° C.

The amount of L-arginine produced in the culture was 20.8 mg / mL and 2.46 mg / mL in the case of parent strain.

Example 2

Platinum is inoculated with Brevibacteriumfuraboom MS-05760 (KFCC 10018), which is a strain of Example 1, by adding 2.5% aspartic acid to the same medium composition as in Example 1.

As a result of treatment in the same manner as in Example 1, the amount of L-arginine produced in the culture medium was 21.3 g, and in the case of the parent strain of the same method, 2.57 mg / ml.

Example 3

L-glutamic acid 2.0% was added to the same medium composition as in Example 1, and the strain Brevibacteriumfuraboom MS-05760 (arginine decarboxylase, arginine decarboxylase) was simultaneously platinum. After inoculation, incubate at 30 ° C. for 72 hours. (130 rpm) 1 L of the fermentation broth is heated and filtered. This solution is passed through an Amberlite IR-120B (type H) column, followed by washing with water to elute L-arginine with 5% ammonia. The resulting L-arginine was concentrated under reduced pressure to precipitate and determine the residue with methanol and hydrochloric acid.

This crystal is recrystallized from hydrated methanol.

At this time, 23.6 g of L-arginine hydrochloride was obtained, and 2.45 g of the parent strain was obtained in the same method.

Example 4

Medium containing glucose 3.0%, urea 1.0%, lactic acid ammonium 0.5%, corn steep lyca 0.7%, dibasic phosphate 0.2%, magnesium lactate 0.01%, ferrous lactate 0.001%, calcium carbonate 1.5% pH 7.3) 30 ml is put into a 500 ml shake and the flask is autoclaved at 120 ° C. for 10 minutes. At this time, calcium carbonate is added by sterilization separately.

This medium was inoculated with Brevibacterium puraboom MS-05760 (KFCC 10018) by platinum and shaken at 30 ° C for 72 hours.

The amount of L-arginine produced in the culture was 20.8 mg / ml, and the parent strain of the method was 2.16 mg / ml.

Claims (1)

L-arginine, characterized by culturing Brevibacterium furaboom MS-05760 (KFCC 10018) (arginine decarboxylase, arginine decarboxylase co-deleted strain) and collecting and accumulating large amounts of L-arginine in the medium. Recipe.