JPS5816691A - Preparation of l-lysine - Google Patents

Abstract

PURPOSE:To recover the titled substance accumulated in a medium, by cultivating aerobically under a specific temperature condition a bacterium belonging to the genus Nocardia, capable of producing lysine, subjecting to growth inhibition by DL-methionine. CONSTITUTION:A strain such as Nocardia alkanoglutinousa 7040(FERM-P 6046), Nocardia alkanoglutinousa 7360, etc. belonging to the genus Nocardia, capable of producing L-lysine, observed to have growth inhibition in a medium containing methionine only, is cultivated in a medium containing a carbon source such as glucose, lactic acid, etc., nitrogen source such as glutamic acid, peptone, etc., vitamin, etc. The cultivation is carried out conventionally in a pH of 6-9 at 30-45 deg.C aerobically, subjected to an ion exchange resin, etc., to isolate L-lysine.

Description

DETAILED DESCRIPTION OF THE INVENTION The present invention provides that in lysine fermentation using bacteria of the genus Nocardia, growth is inhibited by DL-methionine.
This invention relates to a method for producing lysine by carrying out lysine fermentation using lysine-producing bacteria, accumulating a significant amount of lysine in a medium, and recovering lysine from the medium.

A method using bacteria of the genus Nocardia for lysine fermentation using ethyl alcohol as the main carbon source, a method using ethyl alcohol as the main carbon source. How to do that, USP 412332
There is a method using n-paraffin of No. 9 as the main carbon source, etc.
spNrx4123329 has been reported to produce 52.5 f/l of lysine (as hydrochloride) from 10% n-paraffin; however, when using carbohydrates as raw materials for lysine production using Nocardia bacteria, There are no reports that any amount of lysine was produced.

The present inventors used bacteria of the genus Nocardia and found that when carbohydrates are used as a carbon source in lysine fermentation, growth is inhibited by DL-methionine alone. discovered that it can be produced in the same way, and completed the present invention.

The microorganism used in the present invention belongs to the genus Nocardia, and its growth is inhibited in a medium supplemented with DL-methionine alone (hereinafter referred to as "susceptible"), and a significant amount of L-lysine is produced and accumulated in the medium. It is a strain that has the ability to

Therefore, any strain having such susceptibility is included within the scope of the microorganisms used in the present invention, even if it also has properties such as sensitivity or resistance to other drugs and auxotrophy. Nocardia alcanoglutinosa (
Nocardia alkanoglutinousa)
7040, Microtechnical Research Institute Deposit No. 6046, Nocardia arcanoglutinosa 7360, etc. These resistant bacteria may be isolated from nature or may be induced by conventional mutation methods. Also, amino acid analogs such as lysine analogs such as S-2 aminoethyl-L-cysteine, cysteic acid, α-methylaspartic acid, DL-threoβ
- Aspartic acid analogs such as hydroxyaspartic acid, deaminosuccinic acid, aspartophenone, β-methylaspartic acid, α-aminolevulinic acid, β-aspartic acid hydrazide, α-methylglutamic acid, methionine sulfoxide, γ-glutamyl ethyl Glutamic acid analogs such as amide, S-carnozumylcysteine, D-carbamylserine, D-canolebasilserine, methionine analogs such as ethionine, crotonylalanine, crotoninoreglycin, methionine sulfoxime, methkinine, β-hydroxy Noruno Sunrin,
β-hydroxynorleucine, threonine analogs such as serine, methionine, cysteine, threonine,
It may be a strain that grows in a medium containing amino acids such as lysine and valine alone or in combination. or,
This sensitive bacteria contains threonine, homoserici, methionine,
Leucine, alanine, inleucine, palin, cysteine, serine, glycine, arginine, etc. may be added singly or in combination. Alternatively, it may be obtained from bacteria that have both the above-mentioned sensitivity and requirements.

Next, the DL-methionine sensitive Nocardia alkanoglutinosa 7040 used in the present invention and the non-sensitive Nocardia alkanoglutinosa 22B-5.
An example of an experiment is shown in which changes in the growth rate of Nx8260 deposited at the National Institute of Fine Arts and Science were investigated in response to combinations of DL-methionine, L-lysine, and L-threonine.

Experimental Example 1 Nocardia alcanoglutinosa 7040 and Nocardia alcanoglutinosa 223-59 were mixed with 1% peptone S (Daigo Nutrition K.K.), 1% meat extract, 0.5% yeast extract, and chloride. Sodium 0.3%, agar 2%, P
After culturing on a H7.2 (neutralized with caustic soda) plate medium (hereinafter abbreviated as Peshoton S broth agar medium) at 33°C for 1 day, one platinum loop was inoculated into the following preculture medium and incubated at 33°C for 29 hours. After culturing, the following minimal medium was added with DL-methionine, L-lysine, and L-threonine as shown in Table 1.
After inoculating one strain at a time and culturing for 33° C4B hours, the bacteria grown in the medium was diluted 10 times with a 1:80 (concentrated hydrochloric acid:water) hydrochloric acid solution, and the absorbance at 610 nm was measured using a spectrophotometer.

Preculture medium; glucose 20f1 fructose 20g,
K2HPO40.5 (lXKH2PO40.5 da, Na
2HPO4・12H201. Q4 da, NaH2P04・
2H200.589, MgS04・7H201. Og,
K.C.I.O. 5f, NaC60.5fXFeSO4・7H
2020mg, znS04・7H2010qXMnSO
4.4H2010q1 peptone 85g, pro extract 2g
(Tomen K.K.), cacg2-2H2o1.93
f, CaC0330f, ammonium sulfate 17.5!9 (separate sterilization added), pH 7.4 (neutralized with caustic soda), final liquid volume 1 liter, dispense 5 zl into a large test tube, and boil at 120°C for 15 minutes in minimal medium; glucose 20 g, Fructose 2Of, K2H
PO40.5f, KH2PO40.5f, Na2/HP
O4'12H201.049, NaH2PO4"2H2
00.58y, MgSO4・7H201. OfXKCI
O. 5f, Na(J!0.59, FeSO4”7H20
20q, ZnSO4'7H2010W, MnSO4"4
H201qSCaC4・2Hp1.93f/, CaCO
330f, ammonium sulfate 17.5fI (separate sterilization added), PH7.
4 (caustic soda neutralization), final liquid volume: 667 ml Dispense 2 mt each into 71 large test tubes and boil at 120°C for 15 minutes. Separately, sterilize the amino acid solution and add 1yttt to make the concentration shown in Table 1.

As is clear from the results in Table 1, DL-methionine 0,
In the presence of 3 to 1 f/l, the growth of Nocardia alcanoglutinosa 223-59 was hardly inhibited, whereas the growth of the susceptible strain Nocardia alcanoglutinosa 70
Growth of No. 40 was significantly inhibited under these conditions. This also applies to the other susceptible strain Nocardia arcanoglutinosa 7360.

Based on the above results, the microorganisms that are said to be sensitive to DL-methionine in the present invention, to give one example, have a growth rate that is lower than that in a minimal medium in which DL-methionine is added to a minimal medium at a concentration of lf/l. 50% or less.

Carbon sources used in the culture medium include monosaccharides such as glucose, fructose, starch or molasses hydrolysates, acetic acid,
Organic acids such as lactic acid, fumaric acid and citric acid, ethanol and n-alkanes are used. As a nitrogen source, amino acids such as glutamic acid and methionine, thawed plant proteins such as soybean protein, peptone, meat extract, yeast extract, ammonium sulfate, aqueous ammonia, ammonium chloride, etc. can be used. Further, vitamins may be added as necessary.

As inorganic salts, phosphates, magnesium salts, calcium salts, potassium salts, sodium salts, iron salts, manganese salts, zinc salts, and other trace metals are used as necessary.

When culturing, the pH is 6 to 9, preferably 6 to 8, the temperature is 20 to 45°C, preferably 33 to 39°C, and shaking culture or aeration stirring culture is carried out.

L-lysine can be isolated by conventional methods.

That is, Amberlight IR-120 or Amberlight I
It is obtained by adsorbing on an ion exchange resin such as RC-84, washing with water, eluting with aqueous ammonia, concentrating, neutralizing with concentrated hydrochloric acid, and further concentrating and cooling to crystallize.

L-IJ gin was measured by a piezoassay method using a mutant strain of E. coli.

The present invention will be explained below with reference to Examples.

Example 1 Peptone S of Nocardia alcanoglutinosa 223-59, Nocardia alcanoglutinosa 7040 and Nocardia alcanoglutinosa 7360
One platinum loop of bacterial cells was taken from broth agar slant culture (33°Cl days), inoculated into a large test tube containing 5 tttl of the above-mentioned preculture medium, and cultured for 33°Cl days. 8 txt of this seed seed was inoculated into a 500-capacity Sakaguchi flask containing 24 ml of the following main culture medium, and at the same time, 3 ml of separately sterilized 38% (w/v) ammonium sulfate was added and cultured with shaking at 36°C. The lysine produced in the solution was as follows as a hydrochloride.

Cylysine concentration at 72 hours
g/1 node ν 1ia anole b node retino-470
Glucose 5
0g1 fructose 50F/, K2HPO40.25!
, KH2PO40.25f, Na2HPO4・12H2
00.52g, NaH2P04・2H200.29f,
MgSO4-7H200.6f, NaClO. 39.K
CI0.3f, FeS04・7H2020m+1i'X
ZnS04'7H20101#j, peptone 85g, proextract 2g, sodium glutamate monohydrate 1f.
．． CaC4・2H200.97f-, CaCO3401
-, PH7.4 (neutralized with caustic soda), total liquid i80
0tst Example 2 The carbon source of the main culture medium of Example 1 was 80 g of glucose,
Shaking culture was carried out in the same manner as in Example 1, except that the amount of fructose was 20 g. The results are as follows.
1f/1 no calvia ano tr noku λi F no sa 70
40423Fl/1 Nopu ν Anorekanoku) Retino-4736048.49/1 Example 3 Nocardia arcanoglutinosa 7040 and Nocardia arcanoglutinosa 7360 were carried out except that the main culture temperature was 38°C Shaking culture was carried out in the same manner as in Example 1, and the following results were obtained.

72nd hour Lysine concentration

Claims (1)

[Claims] [+] A lysine-producing bacterium belonging to the genus Nocardia whose growth is inhibited by DL-methionine is aerobically cultured in a medium containing an assimilable carbon source and a nitrogen source, and in the medium! 4L-
! A method for producing lysine, which is characterized by accumulating J-sine and recovering it. (2) The strain is Nocardia alkalineglutinosa 70
40. The method for producing lysine according to claim 1. (3) The method for producing Shi-IJ gin according to claim 1, wherein the culture temperature is in the range of 30 to 45°C.