JPS59169497A - Production of l-lysine - Google Patents

Abstract

PURPOSE:A strain in Corynebacterium, which is capable of producing L-lysine and resistant to the antibiotics produced by Bacillus, is cultured to effect low- cost production of L-lysine which is used as a medicine or food additive, on a practical scale. CONSTITUTION:A strain in Corynebacterium or Brevibacterium, which is resistant to antibiotics produced by Bacillus such as thermolysin or subtilisin, and capable of producing L-lysine, such as Corynebacterium glutamicum H-3330 (FERM P-6819) is cultured in an enriched medium to produce L-lysine, which is collected from the culture mixture.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing L-lysine by a fermentation method, and more particularly, the present invention relates to a method for producing L-lysine by a fermentation method, and more specifically, a method for producing L-lysine that belongs to the genus Corynebacterium or Brevibacterium and has the ability to produce L-lysine, and that belongs to the genus Bacillus. A production method characterized by culturing a bacterial strain resistant to antibacterial substances produced by bacteria in a nutrient medium, producing this in the culture, and collecting L-lysine produced and accumulated from the culture. This invention relates to a method for producing lysine.

The purpose is to provide a method for industrially producing lysine, which is in great demand as an additive to pharmaceuticals, feeds, and foods, at low cost.

The antibacterial substances produced by bacteria belonging to the genus Bacillus in the present invention include batidrazine, thiocillin, licheniformin, basilicin, colistatin, porcillin, serexin, gramicidin, and tyrocidin.

Chee cook chin. Colistin, prevolin, circulin or antibacterial enzyme.

The six antibacterial substances include thermolysin, subtilisin, phospholipase A, and phospholipase C.

Examples include phospholipase D and the like.

It has been known that bacteria of the genus Corynebacterium and Brevibacterium produce L-lysine. Additionally, methods using antibiotic-resistant bacteria are also known (U,
S, P3687810).

Bacterial contamination is a major problem in lysine production, and there is a need for a solution to the reduction in yield due to contamination, especially in continuous fermentation. As a result of examining the cause, it was found that the majority of the problems were due to the contamination of Bacillus bacteria with heat-resistant spores, which inhibited the growth of lysine-producing bacteria. As a countermeasure against this problem, we have found that the damage can be significantly reduced by using mutant strains that are resistant to antibacterial substances produced by Bacillus.

The present invention will be explained in detail below.

In the method of the present invention, any lysine-producing strain that belongs to the genus Corynebacterium or Brevibacterium and is resistant to antibacterial substances produced by bacteria of the genus Bacillus can be used. .

Such a mutant strain can be obtained by imparting the above properties to the L-lysine production line, or conversely, by imparting lysine productivity to a bacterium having the above properties. In addition to the above properties, the strain used in the present invention may have any properties that contribute to L-lysine production.

Examples of strains used in the present invention are shown below.

Corynebacterium glutamicum H-3330FE
RM P-6819H-3311FERM P-682
0 H-3386FERM P-6822 Brevibacterium lactofermentum H-3331FERM P
-6821 Among the five, H-3330, H-3371, H-3
The 386 strain was prepared by culturing Corynebacterium glutamicum FERM-BP-158 (resistant to thialidin, streftomycin, rifampicin, and 6-azauracil) cells capable of producing lysine in 0.1N Tris-maleate buffer ( pH 6,0) for 10 days Ce1ls/
N-methyl-N'-nitro-N-nitronguanidine was suspended at a final concentration of 0.2 mg/ml.
After adding it so that it becomes like this and leaving it for 30 minutes at room temperature.

A minimal agar plate medium having the following composition containing batidrazine (BCT) 5 μg/ml or gramicidin (GRM) 50 μg/ml, and thermolysin (TL)
, 500 μg/ml on a similar agar plate medium, and were selected from the colonies that grew, each having BCT, GRM, or TL resistance.

As shown in Table 1, it can be clearly distinguished from the parent strain.

In addition, the strain H-3331 is Brevibacterium lactofermentum FERM-P, which has lysine-producing ability.
6209 (resistant to thialidine, streptomycin, and rifampicin, can be distinguished by leucine).

Table 1 Composition of the above minimal agar plate medium Nigelcose 10g/β,
Ammonium chloride 4 g/1, K H2P
OaIg/p, 2HPO43g/β, MgSO47H
200.4g/I2. FeSO47H200,01'g
/ j! , MnSO4・4H200,01g/β, urea 2g/It, biotin 50μg/C agar 20g/C
L-Leucine 200μg/ml (pH 7,2) The medium composition used in the present invention may be a synthetic medium or a natural medium as long as it contains appropriate amounts of carbon, nitrogen, inorganic substances, and other necessary nutrients that can be utilized by the strain used. Either can be used. In other words, carbon sources include glucose, fructose, and sorbitol.

Various carbohydrates such as glycerol, sucrose, starch, starch hydrolyzate, molasses, fruit juice, acetic acid, fumaric acid.

Organic acids such as lactic acid and succinic acid, as well as ethanol.

Alcohols such as methanol can also be used. Nitrogen sources include ammonia and ammonium chloride.

Ammonium salts of various inorganic acids such as ammonium sulfate, ammonium acetate, ammonium phosphate, urea, amino acid, other nitrogen-containing compounds, peptone, meat extract,
Yeast extract, corn steep liquor, casein hydrolyzate, soybean meal acid hydrolyzate, various fermented microbial cells and digested products thereof, etc. can be used. Furthermore, as inorganic substances, potassium phosphate, dipotassium phosphate/magnesium phosphate, magnesium sulfate, sodium chloride, ferrous sulfate, manganese sulfate, calcium carbonate, etc. are used.

If the microorganisms used in the present invention require specific nutrients for growth, the nutrients must be present in appropriate amounts in the culture medium.

These substances may be added as a nitrogen source by being included in the natural bamboo. In addition, various additives 2 such as various antibiotics and α-aminobutyric acid are added to the medium.

By adding cysteine, leucine, leucine fermentation liquid or aspartic acid, glutamic acid, etc., L-
May increase lysine production. Cultivation is performed under aerobic conditions such as shaking culture or deep aeration agitation culture. The culture temperature is usually in the range of 20 to 40°C, and the pH of the medium is in the range of 3 to 9.

It is preferable to maintain it near neutrality.

It can be carried out under other conditions if the strain used grows. The pH of the medium is adjusted using calcium carbonate, acid or alkaline solutions, pH buffers, and the like. Usually, L-lysine is produced and accumulated in the culture solution in 1 to 6 days.

After culturing, precipitates such as bacterial cells are removed from the culture solution using known ion exchange treatment methods, concentration methods, and adsorption methods.

L-lysine can be recovered from the culture solution by using a salting-out method or the like.

It goes without saying that the effect of implementing the present invention is that damage in the event of bacterial contamination can be significantly reduced, and that conventional excessive sterilization to prevent bacterial contamination can be minimized. , steam, and energy can also be significantly saved.

Hereinafter, the present invention will be specifically illustrated by giving examples.

Corynebacterium glutamicum H-33 as an example inoculum
30 (BCTR) is used.

Glucose 40g/β, HR3g/l, KH
2'PO41.5g/j! , 2HPO40,! 5g
/β, Mg5o4·lH2O0,5g/β.

Biotin 50 μg/C peptone 20 g/L Yeast extract 5 g/L seed medium (p)I7.2) Containing 20 ml of 3 (10 ml triangular) “Waco” was sterilized by autoclaving at 120°C for 30 minutes, then inoculated at 30°C. Incubate for 24 hours.

2ml of this culture solution was mixed with 90g/β of blackstrap molasses (in terms of glucose) and 10g/j of soybean meal acid hydrolyzate! (converted to soybean meal weight), ammonium sulfate 20 g/It, urea 3 g/ρ.

MgSO4・7 H'20 0.5 g/j2,
K2HP O4Q, 7 g/A, Ca
CO3' 30 g/j! A fermentation medium consisting of (
pH 7.2) 20n+] was inoculated into a 300 ml Erlenmeyer flask (sterilized by autoclaving at 120°C for 30 minutes) and cultured with shaking at 30°C for 3 days. 41.5g/j of L-lysine (as hydrochloride, hereinafter the same) in the culture solution! Generate and accumulate. The same procedure as above is carried out except that FERM-BP-158 is used as a control to obtain 41 g/β lysine.

On the other hand, Bacillus subtilis ATCC 14593, which has the ability to produce batidrazine, was similarly cultured in the above seed medium.
00 times diluted, 1 ml of the same was inoculated at the same time as the above-mentioned lysine-producing bacteria and cultured in the same manner, and 33 g/Il of L-lysine was accumulated in the culture solution.

When FERM-B P158 is cultured, 13.5 g/β
Met.

After completion of the culture, the supernatant liquid obtained by centrifuging the culture solution 1β contaminated with H-3330 was adjusted to pH 1 to 5 with sulfuric acid, and then Diaion 5K-IB (H+ type) was obtained.

L-lysine was adsorbed by passing it through a column chromatograph using a strongly acidic ion exchange resin (trade name, manufactured by Mitsubishi Kasei Corporation), and after washing the column with water, it was eluted with dilute aqueous ammonia and both fractions containing L-lysine were collected. and concentrate. After adjusting the concentrated solution to pH 2 with hydrochloric acid, L-
Crystallize lysine, L ~ Crystals of lysine hydrochloride 26.5
I got g.

Example 2 The strains shown in Table 2 were each inoculated into the seed medium shown in Example 1, cultured at 30°C for 24 hours, and 2 ml of this culture solution was mixed with 90 g of glucose/20 g of soybean meal acid hydrolyzate/7!
(converted to soybean meal weight), ammonium sulfate 20g/j! , 3 g of urea
/ 7! , M g S Oa ・7 H200,5g
/β, K, H2P Oa 1 g/ I! ,
Biotin 50, ljg/l), “jIamine・HCn
200pg/ml-, F6SO4・7H2010m
g/ll, Mn5Oa ・4H2010■/12. C
Fermentation medium (pH 7.2) consisting of aCO220g/it
3'00 ml Erlenmeyer flask containing 20 ml (120
℃, autoclave sterilization for 20 minutes) and cultured with shaking at 30°C for 3 days to obtain the results shown in Table 2. Table 2 shows the results when Bacillus plevis ATCC 8185, which was cultured in the same manner as in Example 1 and has the ability to produce gramicidin, was inoculated at a ratio of 1/1000 to live JI bacteria.

Table 2 Example 3 The culture was carried out in the same manner as in Example 1, except that the strains shown in Table 3 were used, and the results shown in Table 3 were obtained. Table 3 also shows the results when the production medium was autoclaved at 100'C for 20 minutes and cultured in the same manner.

Table 3 Example 4 H, -3330 and FERMBP-15 as inoculum
2 containing 100 ml of the nine types of culture medium shown in Example 1.
The mixture was inoculated into a 1-volume Erlenmeyer flask with a pamphlet and cultured with shaking at 30°C for 24 hours. Add 100ml of this culture solution to 100ml of blackstrap molasses.
g/A! (Glucose equivalent) Corn steep liquor 10g/L Ammonium sulfate 20g/il! , to 2 HPO4,
1g/j! , Mg5Oa ・7H200, Sg/l, F
Fermentation medium consisting of e5Oa・7H205■/1 ('pH
6, 8) In a 2 A' jar fermenter containing 600 rl, at 32°C, stirring number 90 rpm, ventilation t 1! l
/min.

During cultivation, the pH was controlled to 6.8 with sterilized ammonia water. After consuming the initial sugar, the feed liquid with the following composition (
Blackstrap molasses 200g/7! <Glucose conversion>.

Ammonium sulfate 30g/β, KH2PO40.5g/Cl2O℃2
sterilization) at a rate of 30 ml/hr. When the liquid volume in the fermenter exceeds 1200 ml, the liquid is continuously drained aseptically at a rate of 30 ml/hr to keep the liquid volume in the tank constant. No matter which strain was used, the amount of mustard-lysine accumulated 50 hours after the start of culture was 80 g/7!
reach. If sterility can be maintained, the amount of L-lysine accumulated is 80
g/n or higher for more than 360 hours.

However, when the feed solution was sterilized at 100°C for 20 minutes, the FERM BP-158 strain was already 10 g/1. The amount of lysine accumulated is as follows, H-
For strain 3330, 70 g/β can be maintained until the 150th hour.

On the other hand, the feed liquid was sterilized at 120°C for 20 minutes.
At 100 hours of culture, Bacillus subtilis ATCC14593 cultured in the same manner as in Example 1 was incubated at 0. When L+++1 was added, FERM BP, 158 strain was 70 g/j at 120 hours! It will be as follows.

H-3330 strain can maintain 70 g/β or more for up to 190 hours.

Patent applicant (102) Kyowa Hakko Kogyo Co., Ltd. Representative Ikube Kinoshita

Claims (1)

[Claims] fi+ A lysine-producing bacterium belonging to Corynebacterium or Brevibacterium and resistant to antibacterial substances produced by Bacillus bacteria is cultured in a nutrient medium, and L-lysine is added to the culture medium. Generate. Method for producing perilla-lysine characterized by collecting perilla-lysine from a culture solution (2) The antibacterial substance is v! (3) The antibacterial enzyme is thermolysin or subtilisin. Phospholipase A, phospholipase C or phospholipase D'? 'Production method according to claim 2