KR810001746B1 - Process for the production of l-lysine by fermentation - Google Patents

Abstract

Corynebacterium glutamicum FERM-P 3633, NRRL-B-8182 was cultured for 24 hrs. at 30≰C or 7 ml medium (pH 7.2) contg. glucose 4, urea 0.3, KH2PO4 0.15, K2HPO4 0.05, MgSO47H2O 0.05, biotin 50, peptone 2 and yeast ext. 0.5 g/dl. The seed cultrue (2ml) was inoculated into 20ml medium (pH 7.2) contg. molasses 8.5, soybean acidic hydrolyzate 2, (NH4)2SO4 0.5, Urea 3, MgSO47H2O 0.05, KH2PO4 0.07 and CaCO3 3 g/dl and cultured with shaking for 3 days at 30≰C to produce 30 mg/ml L-lysine.

Description

Method for preparing L-lysine by fermentation method

The present invention relates to a method for producing L-lysine by fermentation.

More specifically, the present invention is a strain belonging to the genus Corynebacterium and resistant to at least one strain selected from the group consisting of L-lysine producing ability, aspartic acid analogs and sulfases in a nutrient medium. It relates to a method for producing L-lysine by the fermentation method, characterized in that the cultivation, and recovered and deposited L-lysine from the culture solution.

L-lysine is a type of essential amino acid well known in the art, and is in demand as an additive for medicine, animal feed and food.

Until now, the following methods are known as a method for producing L-lysine by the fermentation method. That is, methods using homoserine (or methionine and threonine) requiring mutant strains (US Pat. No. 2,979,439) or nutritional conditions for threonine, methionine, arginine, histidine, leucine, isoleucine, phenylalanine, cystine, or cysteine Methods of using homoserine (or methionine and threonine) that require mutant strains (US Pat. No. 3,700,557) using mutant strains resistant to lysine analogs (US Pat. No. 3,707,441), L-lysine production capacity and Nutritional conditions and lysine for methods of using mutants with resistance to vaccicin, penicillin G or polymyxin (US Pat. No. 3,687,810) and homoserine, threonine, threonine and methionine, leucine, isoleucine or mixtures thereof Methods of using mutant strains resistant to threonine, isoleucine or analogs thereof (US Pat. No. 3,70) 8,395), methods of using mutants with resistance to lysine analogs and nutritional conditions for serine, proline, arylene, nicotinamide, nicotinic acid, pantothenic acid, thiamine, guanine, adenine, hypoxanthine, vitamin B ₁₂ (US Patent No. 3,825,472).

In light of the recently validated demand for L-lysine, a number of studies have been conducted to obtain strains with improved L-lysine production ability.The present invention belongs to the genus Corynebacterium which is resistant to at least one selected from aspartic acid analogs and sulfases. The present invention was completed by discovering that L-lysine-producible strains have significantly improved L-lysine production.

In the production of L-lysine by fermentation, it was first discovered by the present inventors to improve the productivity of L-lysine by using a strain resistant to at least one selected from the group consisting of aspartic acid analogs and sulfases.

Hereinafter, the method of the present invention will be described in detail.

In the method of the present invention, certain strains belong to Corynebacterium, are resistant to L-lysine production and at least one selected from aspartic acid analogs and sulfases, and any may be used. That is, in the present invention, the strain belongs to Corynebacterium and has the ability to produce L-lysine, and has a resistance to at least one selected from aspartic acid analogs and sulfases, or among the Corynebacterium and aspartic acid analogs and sulfases. Any of the strains having L-lysine production ability as the resistance to at least one selected can be used. Strains belonging to Corynebacterium and having L-lysine-producing ability, such as nutrients (eg, homoserine, methionine, threonine, histidine, proline, alanine, leucine, isoleucine, valine, serine, pantothenic acid, nicotinic acid, nicotinic acid amide) , Requirements for thiamin, adenine, hipoxanthin and blends thereof, resistance to various amino acid analogs (eg, lysine analogs, threonine, methionine, leucine, isoleucine, valine, or histidine and blends thereof) And strains capable of producing L-lysine having one or more of a combination of resistance to other drugs (eg, various antibiotics such as penicillin G, polymyxin baccitracin, and formulations thereof). Thus, the strains used in the present invention can be obtained by imparting resistance to at least one selected from aspartic acid analogs and sulfases to the above-described L-lysine-producing strains, or belonging to Corynebacterium and among aspartic acid analogs and sulfases. L-lysine-producible strains obtained by imparting the above-mentioned various nutritional conditions, resistance to various amino acid analogues or resistance to various drugs to the selected strains resistant to at least one kind can also be used in the present invention. In addition, the strain used in the present invention may have other properties necessary for enhancing L-lysine production capacity in addition to the properties described above.

Examples of aspartic acid analogues are aspartic acid hydroxitrate, α-methylasparonic acid, β-methylaspartic acid cysteinesulfinic acid, difluorosuccinic acid, hadacidine and the like. Examples of sulfases include sulfaguanidine, sulfadiazine, sulfamethazine, sulfamerazine, sulfamethazole, sulfamethimidine, sulfamethoxypyridazine, sulfatiazole, homosulfamine, sulfadimethoxine, solfamethoxazole, Sulfisoxazoles and the like.

Among the strains used in the present invention, Corynebacterium glutamicum FERMP 3633 (NRRL-B-8182) can be said to be an example of a strain having resistance to aspartic acid analogs, Corynebacterium glutamicum Qum FERM-P 3634 (NRRL-B-8183) is a type of strain that is resistant to sulfase. FERM-P 3633 strain was subjected to Corynebacterium glutamicum ATCC 21543 (homoserine requirement, leucine requirement and S- (β-aminomethyl) -cysteine with L-lysine production in 0.1 N trismaleic acid buffer (pH 6.0). Resistant to cells (see Canadian Patent No. 939,518) and suspended at a concentration of 10 ⁸ cells / ml, to which N-methyl-N'-nitro-N-nitro soguanidine has a final concentration of 0.2 mg / ml. After the addition, the suspension was allowed to stand at room temperature for 30 minutes, and then the suspension was applied on an agar plate of a vegetable medium having the following composition containing 1 mg / ml of aspartic acid hydroxamic acid salt, which is a type of aspartic acid analogue. It is a mutant strain obtained by screening the strain among the strains, and it is clearly distinguished from the parent strain ATCC 21543 in the resistance to aspartic acid hydroxitrate. In addition, the FERM-P 3634 strain is a mutant strain selected in the same manner as described above, except that agar plate of minimal medium containing 1 mg / ml of sulfamethazine, which is a kind of sulfase, is used instead of aspartic acid hydroxitrate. It is distinguished from the parent strain in resistance to sulfamethazine and sulfadiazine.

Microbiological formulations of Corynebacterium glutamicum are described in Monoreactive Microbiology, Vol. 18, pp. 279-301 (1967).

Any synthetic and natural medium may be used as the medium for the present invention as long as the carbon source, nitrogen source, inorganic substance and other necessary nutrients are properly contained. Examples of carbon sources include glucose, fructose, sorbitol, mannitol, glycerol, starch, starch hydrolyzate, molasses, hydrocarbons such as blackstrap molasses, hydrocarbons such as n-paraffin and kerosene, acetic acid, fumaric acid, lactic acid, Organic acids such as fibric acid and succinic acid Alcohols such as methanol and ethanol can be used. As a nitrogen source, ammonia can be used, and inorganic and organic ammonium salts such as ammonium chloride, ammonium sulfate, ammonium phosphate, ammonium acetate, urea, amine, other nitrogen compounds, peptone, gravy, yeast extract, corn steep liquor, casein hydrolyzate Fish meal, fish meal immersion liquid, skim soybean, skim soybean immersion liquid, soybean protein hydrolyzate, various microbial cells, various microbial cell immersion liquid and the like can be used. As the inorganic substance, potassium hydrogen phosphate, dipotassium hydrogen phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium carbonate and the like can be used. When the microorganism to be used in the present invention requires a special growth nutrient, of course, an appropriate amount of nutrient is added to the medium. In some cases, these and nutrients are added with components of natural substances foreseen as a nitrogen source.

In the present invention, the productivity of L-lysine by the present microorganism can be further enhanced by adding leucine fermentation broth to the medium as shown in Example 3. In this case, the lysine fermentation broth may be added in an amount ranging from 0.2 to 15% by volume of the medium volume.

In addition, the productivity of L-lysine by the present microorganism can also be enhanced by addition of various other additives, such as various antibiotics, α-aminobutyric acid, cysteine, norleucine, leucine, aspartic acid, glutamic acid, and the like.

The culture is carried out under aerobic conditions, for example, by shaking culture, shaking low culture. The incubation temperature is generally in the range of 20-40 ° C., pH 3 to 9 of the medium, and is preferably maintained near neutral, but can be cultured under conditions outside this range as long as the microorganisms used can grow. The pH of the medium is adjusted with calcium carbonate, organic or inorganic acid or ammonia, alkali hydroxides, pH buffers and the like. Usually, after 1 to 7 days of incubation, L-lysine is produced and precipitated in the final culture solution.

After the incubation is completed, the precipitate such as cells and the like can be removed from the culture solution, and L-lysine can be recovered using known methods such as ion exchange resin treatment, concentration, adsorption, salting, and the like.

When the present invention is described in detail as an embodiment as follows.

Example 1

As seed strains, Corynebacterium glutamicum FERM-P 3633 and NRRL-B-8182 (having resistance to aspartic acid hydroxate) were used as seed strains. This seed strain contains glucose 4g / dl, urea 0.3g / dl, KH ₂ PO ₄ 0.15g / dl, K ₂ HOP ₄ 0.05g / dl, MgSO ₄ 7H ₂ 0 0.05g / dl Biotin 50g / dl, peptone 2g / 50 ml (190 mm x 20 mm) of large in vitro containing 7 ml (pH 7.2) of seed medium consisting of dl, and yeast extract, 0.5 g / dl, were inoculated and incubated at 30 ° C for 24 hours. 2 ml of the resulting seed medium were blackstrap molasses (as glucose) 8.5 g / dl, soy cake acid hydrolyzate (as soy cake) 2 g / dl, ammonium sulfate 0.5 g / dl, urea 0.3 g / dl, MgSO ₄ 7H ₂ O was inoculated into 300 ml triangle triangle containing 20 ml (ph 7.2) fermentation medium consisting of 0.05 g / dl, 0.07 g / dl KH ₂ PO ₄ and ₃ g / dl calcium carbonate, and incubated with shaking at 30 ° C. for 3 days. . As a result, 35 mg / ml of L-lysine (as monohydrochloride to be used later) was formed and deposited in the culture. The amount of L-lysine by the parent strain ATCC 21453, which was a control strain cultured under the same conditions at the same time, was 25 mg / ml.

After incubation, 1 L of this strain culture was centrifuged to remove cells and other precipitates. The supernatant was passed through a column of Diaion SK-1 (H ⁺ type, Mitsubishi Chemical Industries, Ltd., a strong acidic ion exchange resin) to adsorb L-lysine. After washing the column, the column was eluted with dilute aqueous ammonia solution, and then the fractions containing L-lysine were collected and concentrated. The pH of the concentrate was adjusted to 2 using hydrochloric acid, and the concentrate was cooled while methanol was added thereto to precipitate L-lysine. As a result, 26.5 g of crystals of L-lysine hydrochloride were obtained.

Example 2

Culture was carried out in the same manner as in Example 1 except for using Corynebacterium glutamicum FERM-P 3634, NRRL-B-8183 (resistance to sulfamethazine) as the seed strain. , 33 mg / ml of L-lysine was produced and deposited in the culture. As a control bacterium, 25 mg / ml of L-lysine was obtained when cultured in the same manner as described above using the parent strain ATCC 21543.

Example 3

Corynebacterium glutamicum FERM-P 3633 used in Example 1 was used as the seed strain. This seed strain was inoculated into a 300 ml Erlenmeyer flask containing 20 ml of the seed medium used in Example 1 and incubated at 28 DEG C for 24 hours with stirring. 1 liter of the seed medium was black strap molasses (as glucose) 14 g / dl, MgSO ₄ .7H ₂ O 0.03 g / dl, KH ₂ PO ₄ 0.07 g / dl urea 0.3 g / dl, soy cake acid hydrolyzate (as soy cake) ) Inoculated into a 30-liter self fermenter containing 10 g (pH 7.2) of fermentation medium consisting of 1.8 g / dl and 1.4% (volume) of lysine fermentation broth prepared in advance as described below. The pH was adjusted to 6.8 and incubated at 28 ° C. for 48 hours at an air supply of 10 L / min, stirring speed, and 400 r.pm. As a result, 58 mg / ml of L-lysine was produced and deposited in the culture solution. L-lysine 43 mg / ml was obtained when the mother strain ATCC 21543 was cultured in the same manner as described above.

The leucine fermentation broth used in the fermentation broth is prepared by the following method. Corynebacterium glutamicum 21885, a leucine-producing strain, was prepared using glucose 5g / dl, peptone 1g / d1, yeast extract 1g / d1, corn steep liquor 0.5g / d1, NaCl 0.25g / d1, urea 0.3g / d1, and biotin 50μg. It was inoculated into a 5 L autogenous fermenter containing 3 L of a medium medium (pH 7.2) composed of / L, followed by incubation for 17 hours at 30 DEG C with an air supply of 3 L / min and a stirring speed of 600 r.pm. The heterogeneous medium 1ℓ ammonium acetate _{0.5g / d1, KH 2 PO 4} 0.2g / d1, MgSO 4 · 7H 2 O 0.55g / d1, FeSO 4 · 7H 2 O 0.01g / d1, MnSO 4 · nH 2 O 0.001g / d1, inoculated into a 30-liter porcelain incubator containing 10 (pH 6.8) fermentation broth consisting of 50 μg / l biotin and 100 μg / l thiamine hydrochloride and inoculated at 10 ° C. with an air supply of 10 L / min and a stirring rate of 400 r.pm. Time incubated with air stirring. During the incubation, a mixed solution containing 7% ammonium acetate and 38% acetic acid was continuously supplied to the culture medium to secure a carbon source, and the pH 6.8 was also adjusted. As a result, 15.3 mg / ml L-leucine was obtained and used as part of the fermentation medium for L-lysine.

Claims (1)

A strain belonging to Corynebacterium, having L-lysine production ability, and resistant to a species selected from the group consisting of aspartic acid analogs or sulfases was cultured in a nutrient medium, and L-lysine in the culture medium. After producing and depositing, L-lysine by the fermentation method characterized in that for recovering L-lysine.