AU597387B2 - A process for producing L-lysine - Google Patents

Description

COMMONWEALTH OF AUSTRA 9 7 FORM PATENTS ACT 1952 COMPLETE SPECIFICATION FOR OFFICE USE: Class Int.Class Application Number: Lodg~1' Complete Specification Lodged: Accepted: Published: Priority: :Related Art: Name of Applicant: KYOWA HAKKO KOGYO KABUSHIKI KAISHA Address of Applicant: NO. 6-1, OHTE-MACHI I-CHOME, CHIYODA-KU, TOKYO-TO, JAPAN *4 99 Actual Inventors: HIDEAKI YONEKURA, TOSHIHIKO HIRAO TOMOKI AZUMA and TOSHIHIDE NAKANISHI e "Address for Service: SHELSTON WATERS, 55 Clarence Street, Sydney Complete Specification for the Invention entitled: "A PROCESS FOR PRODUCING L-LYSINE" The following statement is a full description of this irvention, including the best method of performing it known to me/ds:- 1

(I

it% A PROCESS FOR PRODUCING L-LYSINE BACKGROUND OF THE INVENTION Field of the Art: The present invention relates to a process for producing

T

-lysine by fermentation.

Prior Art: L-lysine is one of the essential amino acids, which Ot is widely used for various purposes such as, for example, for the preparation of medicines, additives to feedstuffs and foodstuffs and the like. It is known to produce L-lysine by fermentation of mutant strains induced from certain microorganisms belonging to, for example, the genus o rynebacterium. Brevibacterium, Arthrobacter, fBailias, 1 etc. Such mutant strains include mutant strains tequiring, for example, homoserine or methionine and threonine, etc. for their growth and mutant strains resistant to various substances.

Among the known processes for producing L-lysine by fermentation, Japanese Patent Publication 4993/84 discloses a process for producing L-lysine, characterized by culturing an L-lysine-producing mutant strain of the geIus TBrevibacterium or Corynebacterium, in a culture medium to form and accumulate L-lysine, and reovering the resultant L-lysine from the medium, said mutant strain being re.sstant to lysine analogues as well afi to at least one compound having a quinone- or quinoline skeleton.

".J

Ii 4~j 7 In this prior art literature, the term "lysine analogues" denotes? for example, S- (2-aminoethyl )-L-cysteine, alpha -halogenocaprolactum, gamma -methylJ-.ysine and the like, having the following characteristics:- Its chemical struct ure is similar to the chemical structure of lysine; It i~s capable of inhibiting the growth of the micro- *:::organisms of the genera Brevibacterium, Corynebacterium and the like; and The above-mentioned inhibition is eliminated by *adding L-lysine to the culture medium.

The compounds having a quinone- or quonoline skeleton are exemplified by 8-hydroxyquinoline; 8-hydroxyquinoline- 15.q 5-sulfonic acid; 5,8-dioxy-6-arnino-7-chloroquinoline; 1,4naphthoquin )ne; 1 ,2-naphthoquinone; 2,3-dichloronaphthoquinone; 5-carboxy-3 0 ,thia,-6'-aza-2 cyclohexenonaphthalene-1 ,4qUinone; 6 '-(S)-4carboxy-3'-thia-7'-aza-2,3-cycloheptiononaphthalene-1, 4-quirione; Na N -bis (3 -chloro-1 ,4-naphtho- 2"-quinonyl)-L-lysine and granaticin, which exhibit the following characterisitcs: (1 They are capable, of inhibiting the growth of the microorganisms of the genera Brevibacterium and Corynebactairum; and.

Such an inhibit ion is at least partly eliminated by adding L-leucine, to the culture medium.

It has been constantly desired to provide a process for producing L-lysine at a cheaper cost on an industrial scale in view of the fact that L-lysine is very important for practical purposes.

The present inventin is based upon our discovery that L-lysine may be obtained with a higher yield by using a mutant strain of the genus Corynebacterium, Brevibacterium, Microbacterium or Arthrobacter, which has been made to be resistant *o to beta-naphthoquinoline.

1SUMMARY OF THE INVENTION ft*.

The present inventin is concerned with a process for 9 S Sproducing L-lysine by fermentation, which comprises culturing an L-lysine-producing mutant strain of the genus Corynebacterium, Brevibacterium, Microbacterium or Arthrobacter '19 in a culture medium to form and accumulate L-lysine in the S" cultured broth, and recovering the resultant L-lysine from ~the cultured broth.

The process of the present invention is characterized in that said mutant strain is resistant to beta-naphthoquinoline.

It has been found that the inhibition of the growth of the microorganism may not be restored 6 en by adding SL-leucine to the culture medium, on the contrary to the quinone- and quinoline type compounds disclosed in said Japanese Patent Publication 4993/84, from which it is apparent that beta-naphthoqUinoline is not equivalent to various quinones or quinolines of the known types disclosed in the -3- Japanese prior art literature in view of the inhibition of the growth of L-lysine-producing microorganisms.

Examples of the microorganisms belonging to the genus Corynebacterium, Brevibacterium, Microbactorium or Arthrobacter, which may be used as the parent microorganisms for inducing the mutant strain according to the present invention may be naturally-occuring or mutant strains, and include, for example, microorganisms which require various nutrients (for example, at least one member selected It from homoserine, methionine, threonine, histidine, proline, alanine, leucine, isoleucine, valine, serine, glutamic o acid, pantothenic aciC, nicotinic acid amide, acetic acid, adenine, hypoxanthine and inositol), microorganisms which are resistant to various amino acid analogues (for example, 15 at least one member selected from analogues of lysine, threonine, methionine, leucine, isoleucine, valine, aspartic acid, tryp ophan and histidine) and microorganisms which are resistant to various other substances (for example, at least one member selected from various '20 antibiotics, sulfa drugs, various organic acids, quinone compounds and quinoline compounds of the known types).

It is possible to obtain a mutant strain which may 0 1 be used for the purpose of the present invention by imparting beta-naphthoquinoline-resistance to the abovementioned parent strain capable of producing L-lysine.

Alternatively, it is possible to obtain the desired mutant strain capable of producing L-lysine according to the present invention by imparting the above-mentioned -4resistance to various nutrients, resistance to various amino acid analogues or resistance to various other substances to a beta-naphthoquinoline resistant microorganism.

Preferred examples of the microorganism which may be. used as the parent strain for the induction of the mutant strain of the present invention include Corynebacterium glutamicum ATCC 13032, Corynebacterium glutamicum H-3149 (FERM BP-158), Corynebacterium acetoacidophilum ATCC 13870, o Brevibacterium lactofermentum ATCC 13869 and Brevibacterium ~flavum ATCC 14067, all being capable of producing L-lysine 94, by fermentation.

00 The mutant strain which may be used for the purpose of the present invention may be induced in conventional .1.5 manner used for induction of mutant strains such as, for example, irradiation of ultraviolet rays, chemical o° treatment using, for example, N-methyl-N'-nitro-Nnitrosoguanidine and the like. The desired mutant strain Smay be separated from the microorganism subjected to mutation by colleting mutant strain grown on a suitable medium, for example, a minimum medium on an agar plate containing beta-naphthoquinoline at a concentration sufficient to inhibit the growth of the parent strain (for example, more than 40 pg/ml) with respect to the resistance to beta-naphthoquinoline and productivity of L-lysine.

The microbial mutant strain Corynebacterium glutamicum H-4412 resistant to beta-naphthoquinoline, induced by mutation of a naturally-occuring parent strain H-3149, was deposited with Bikoken (the Fermentation Research Institute, Agency of Industrial Science and Technology, the Japanese Government) and assigned with a deposition number of FERM-BP 1069 on 27th May 1986.

As the medium for carrying out the process according to the present invention, it is possible to use both synthetic and organic media containing suitable amounts of assimilable sources of carbon and nitrogen, inorganic e o o° substances and various other nutrients required for the S 0 j 3oo0 growth of the microorganism.

Preferred carbon sources include, for example, glucose, o fructose, sorbitol, glycerol, sucrose, starch, starch e0 4 o 0* Shydrolyzate, molasses, fruit jutice and various other carbohydrates; acetic acid, fumaric acid, lactic acid, maleic o* 4 *i acid and various other organic acids, although it is p possible, if desired, to use ethanol, methanol and other alcohols.

Preferred nitrogen sources include, for example, ammonia, ammonium chloride, ammonium sulfate, ammonium acetate, ammonium phosphate and various other ammonium salts of inorganic acids; urea, amines and other nitrogencontaining compounds; peptone, meat extract, yeast extract, corn steep liquor, casein hydrolyzate, soybean meal hydrolyzate, microbial cell bodies of fermented microorganisms and their hydrolyzate, etc.

Preferred inorganic substances can be exroiplified by potassium moncphosphate, potassium diphosphate, magnesium phosphate, magnesium sulfate, sodium chloride, ferrous

II,

sulfate, manganese sulfate, calcium carbonate, etc.

The medium may, if desired, contain suitable amount of nutrients specially required for the growth of the microorganism used. In some cases, these nutrients are plesent in the above-mentioned naturally-occuring nitrogen sources. Also, in some cases, the productivity of L-lysine can be improved by adding to the medium certain additives such as, for example, various antibiotics, alpha-aminolactic acid, cysteine, leucine, fermentation liquor of leucine, 00 ;0 aspartic acid, glutamic acid and the like.

*5* o Culturing may be carried out under aerobic conditions, Q 9 S, o for example, with shaking or by submerged culturing with 9 aeration and shaking, usually at a temperature within a range of from 20 to 40°C and at a pH of about 3 to 9 15 (preferably at around a neutral pH), although it is possible to carry out the culturing under other conditions so far as the microorganism can g7:ow. The pH14 of the medium may .9 4 be adjusted, for example, with addition of calcium carbonate, urea, solution of acid or alkali, pH regulator 44 '20 and the like. Usually, culturing is continued for 1 to S6 days which are sufficient to form and accumulate L-lysine lin the cultured broth.

After completion of culturing, it is preferred to remove the microbial cell bodies and other precipitates from the cultured broth. L-lykine may be recovered from the supernatant in conventional manner, for example, by treatment with ion exchange resin, concentration adsorption, salting-out and the like. If desired, it is -7possible to recover L-lysine from the microbial cell bodies in conventional manner.

Various experiments have revealed that an increase of the production yield of L-lysine of about 10-25 may be obtained by the process according to the present invention.

The following non-limiting examples illustrate the present invention.

EXAMPLE 1 140 An L-lysine-producing and beta-naphthoquinolineresistant mutant strain Corynebacterium glutamicum H- 4412 (FERM BP-1069) was obtained in the following manner.

9« Corynebacterium qlutamicum H-3149 (FERM BP-158; naturally-occuring microorganism; thialysine-resistant, 1.5 rifampicin-resistant, streptomycin-resistant and 6azauracil-resistant), which was used as the parent strain, was suspended in 0.1N tris-maleic acid-buffered solution (pH=6.0) at a concentration of 108 cells per ml, to which was added N-methyl-N'-nitro-N-nitrosoguanidine to a final 20 concentration of 0.2 mg per ml. The cell suspension was allowed to stand at room temperature for 30 minutes, followed by smearing onto an agar plate having a minimal medium of the following composition, which contained betanaphthoquinoline at a concentration sufficient to inhibit the growth of the parent strain (40 pg/ml).

A/ter culturing at 30 0 C for 3 days, the desired mutant strain was separated from the grown colonies with respect to the resistance to beta-naphthoquinoline, and was designated as Corynebacterium glutamicum 1-4412. This -I r was apparently different from the parent strain H-3149 with respect to the resistance to beta-naphthoquinoline and a higher productivity of L-lysine, as shown in~ the following Table 1.

CompositIon of the minimal medium (agar plate): Glucose 10 g/l; NH 4 Cl 1 g/l; urea 2 g/l; KH 2

PO

4 1 g/l;

K

2

HPO

4 3 g/l; MgSO 4 '7H 2 0 0.4 g/l; FeSO 4 .7H 2 0 10 mg/l; MnSO 4 '4H 2 O0 4 mg/l; biotin 50 pg/l; nicotinic acid 5 mg/l; ZnSO 4 '7H 2 0 mg/l; CuSO 4 *5H 2 0 mg/l; (NH 4 6 M 7 24 4H20 1 mg/l; aga.r 2 (pH TABLE 1 Stai -34 9 Be ptaon aphtho.meinoin (gml) ina30 30 40emee fak (Note. c=ullurgrowt +00CPor groth; wit shNokgrowth.) Cor' thebculture (2utml)cwas transferred to-a0690 wa Eremee fl0s putnoniaiseed medium (2(m)inaQ0 ml 7dnm 2e faskg teedhaiga composition of glucose (4 10 g/l acltda l,uroe ammonium sulfate (40 KH 2 PO4 (0.5 g/l),MgSO4.7H 2 0 g/l) and CaCO 3 (30 g/1) for culturing at 32 0 C for 4 days with shaking (220 After completion of culturing, L-lysine accumulated in the cultured broth was 52 g/l calculated as L-lysine hydrochloride (determined by the colour reaction method using acidic copper/ninhydrin reaction). For comparison, the parent strain H-3149 was cultured in a similar manner to that described above to obtain 44 g/l of L-lysine hydrochloride.

21< The resultant cultured broth (1 litre) was centrifuged to obtain a supernatant. After adjusting the pH to 1.5 with sulfuric acid, the supernatant was passed through a column packed with Diaion SK-1B (H form; strongly acidic ion exchange resin; commercial product of Mitsubishi Kasei Kogyo KK., 1 5 Tokyo) to adsorb L-lysine onto the resin. After washing the column with water, elution was effected by the use of 2N ammonia water to give L-lysine-containing fractions which were then collected, combined and concentrated. After adjusting to a pH of 2.0 with hydrochloric acid, the cncentrated solution was cooled whilst adding ethanol so that crystals of L-lysine (41 g) were obtained.

Claims (2)

1. A process for producing L-lysine by fermentation, which comprises culturing a microorganism selected from the genera Corynebacterium, Brevibacterium, Microbacterium and Arthrobacter in a culture medium to form and accumulate L-lysine in the cultured broth and recovering the resultant L-lysine from said cultured broth, said microorganism being capable of producing L-lysine and resistant to beta-naphthoqu,noline. A process for producing L-lysine by fermentation of a microorganism capable of producing L-lysine and selected from .IIP. the genera Corynebacterium, Brevibacterium, Microbacterium and *Arthrobacter, in a culture medium to form and accumulate L-lysine in the cultured broth and recovering the, resultant L-lysirne from the cultured broth, characterized in that said micro- organism is resistant to beta-.naphthoquinoline. '41j*. 3i A process accor.~ding to claim 1, In which the culturing is effected at a temperature of 20-40 C and at a pH of 3-~9 for 1 to 6 days,

4. A process accor~ding to c~i~1-2$ in which the ;14cro- organism is Corynebacterium glutamiqum "-4412. FORM 1OE9) K'ZOWA EIAKtO KOGYQ KABUSHIK~I XISflA Attorney: PETER IAEATHP,0TR Fllo nstitte 1) t~e s~Ait~i Of SHEL1Or' WATERS