JPS6192586A - Production of l-lysine - Google Patents

Abstract

PURPOSE:To produce L-lysine in improved formation and accumulation ability, by cultivating a specific variant belonging to the genus Brevibacterium or Corynebacterium, and collecting L-lysine. CONSTITUTION:A variant belonging to the genus Brevibacterium or Coryne bacterium, having resistance to L-alpha-amino-epsilon-caprolactam, capable of forming and accumulating L-lysine, is cultivated. L-Lysine accumulated in the medium is collected by a means such as ultraviolet light radiation, treatment with N- methyl-N'-nitro-N-nitromesoguanidine, etc. A variant (FERM P-7693 and 7694) obtained by providing Brevibacterium lactofermentum, or Corynebacterium glutamicum with resistance to L-alpha-amino-epsilon-caprolactam may be cited as the bacterium to be applied.

Description

[Detailed description of the invention] [Industrial application fields] The present invention relates to a method for producing L-lysine.

[Prior Art] Conventionally, as a method for producing lysine by fermentation, 5-(2-aminoethyl)-L-cysteine (AE), which belongs to Brevibacterium m.
C) There is a known method using bacteria resistant to resistant or halo-substituted ε-caprolactam (Japanese Patent Publication No. 4).
8-28078, Japanese Patent Publication No. 53-45391).

Although this method is also a method with considerable industrial potential, it is still effective in producing and storing lysine! There was room for improvement in terms of improving performance.

[Problem to be Solved by the Invention 1] Therefore, the present inventors conducted intensive studies with the aim of improving the above-mentioned problem, and found that if a newly discovered specific mutant strain is used, the % product of lysine production can be reduced. (I discovered the fact that work improves performance and completed the present invention.)

[Means for Solving the Problems] The above-mentioned object of the present invention is to obtain Brevibacterium
acterium ) genus and IC are Corynebacterium (
Corynebacterium ) belongs to the genus L-
-C can be achieved by culturing a mutant strain that is resistant to α-amino-ε-caprolactam and has the ability to produce and accumulate lysine, and then collecting L-lysine accumulated in its rear.

The composition and effects of the present invention will be explained in detail below.

The microorganism used in the present invention is Brevibacterium (3r
It is a microorganism belonging to either the genus Corynebacterium or the genus Corynebacterium. Even microorganisms belonging to these classes are limited to those that are resistant to -α-amino-ε-caprolactam. These microorganisms are Brevibacterium lactofermentum (Bre
vibacterium lactoferment
un+) (A T C.

C-13869 (hereinafter referred to as TM-1118) and Corynebacterium glutamicum (Corynebacterium glutamicum).
erium glutamicuIIIA T CO
-13286 hereinafter referred to as TM-1117) to L-α~
It is a mutant strain that has been conferred resistance to lumino-ε-caprolactam (hereinafter referred to as LAG), and has already been registered as FIKEN Deposit No. 7693 and No. 7694, respectively.

In addition, the non-optically active α-amino-ε-caprolactam referred to in the present invention refers to one in which an amino group is stereoselectively introduced onto the asymmetric carbon at the α position, and has an optical rotation of [α120; .

The microorganism used in the present invention is a typical microorganism known as a so-called glutamic acid producing bacterium, namely Brevibacterium.
The genus or Corynebacterium
The microorganism belonging to the genus Rium is used as a parent strain and is collected by conventional mutation induction methods and screening methods.

Specifically, ultraviolet irradiation and N-methyl-N'-nitro-N
-Collected by methods such as nitromesoguanidine treatment.

An experimental example of the method for inducing the mutant strain is as follows.

Experimental Example 1 Corynebacterium glutamicum (ATCC-132
After UV irradiation treatment, L-α-amino-
It was spread on an agar medium supplemented with 0.2% ε-caprolactam (LAC) and cultured at 30°C for 4 to 10 days. The medium composition was as shown in Table 1 and J3.

Table 1 Glucose 10 0r.

Ammonium sulfate 1.5! llr.

Urea 1゜5 gr.

KH2PO41, Ogr.

K2HPO43,0 (lr.

tVIl)SO4・71120
0.1 gr.

Ca Cl 2 ・2H200, OO1+r.

Na CI 0.005 Or.

Fe 304-7H200,005or.

M n S 04 ・ 4 F12 0
0. 005 gr.

d-Biotin 30 μ9 Thiamine Hydrochloride Ju 100 μ9 Polypeptone
3 0.003Or.

Agar 20 (Jr.

Make 1 liter of the above composition with ion-exchanged water, and add NaOH
01-1 was adjusted to 6.8 and used as a culture medium. Among the colonies that appeared in the above culture, those with high L-lysine production ability were selected, and one of them was Corynebacterium glutamicum (Corynebacterium glutamicum).
glutamicum) genus (L A C1 mutant bacteria T
M-1107 (Feiko Kenboku ¥j7j47693) was obtained.

Brevibacterium lactofermentum (B revibacterium lactofermentum) was prepared in a similar manner.
rmentum) ATTCG-13869 (hereinafter TM-
Brevibacterium lactofermentum (B.
revivactrium+ lactfermen
tum) LΔC-resistant mutant strain TM-1108 (Feikoken Bacteria No. 7694) was obtained.

Experimental Example 2 A medium was prepared by adding LAG in the amount shown in Table 3 to the liquid medium shown in Table 2. This medium was sterilized by a conventional method and cultured in test tubes containing 10 ml each at 30° C. for 48 hours with aeration. To grow the bacteria, use 550I11μ after diluting the culture solution 2 times.
It was measured by the absorbance of

Table 2 Glucose 15 or.

Mushoku 　　　　　　　2. Ogr.

Urea 2. Ogr.

K1-12PO42, Ogr.

K2HPO41, Oor.

tvlg SO4・7f-1200,1 (Jr.

Ca　C1’　2H200,001! llr.

Na CI 0.005gr.

Fe　SO4・7H200,005or.

Mn SO4・ 4 F+2 0
0. C05gr.

d-Biotin 50 μ9 Thiamine Hydrochloride 100 μq Polypeptone S
O,OO3gr.

The above composition was made up to 1 liter with ion-exchanged water, and NaO
The pH was adjusted to 6.8 in step 1 and used as a culture medium.

Table 3 shows the growth rate of each strain obtained by these cultures.

Table 3 Concentration $1117 1107 1118 11080
0.413 0.451 0.392 0.38
81.0 0.081 0.215 0.097
0.181 To produce L-lysine using the jqed microorganisms as in the above experimental example, a normal nutrient medium containing a carbon source, a nitrogen source, inorganic salts, and nutritional substances required by the microorganisms is used. use. Carbon sources include sugars such as glucose, sucrose, molasses containing these, blackstrap molasses, and starch hydrolysates, and hydrolyzed sugar solutions obtained by hydrolyzing cellulose such as juices, fruit lees, wood, and paper. In addition to various sugars such as, organic acids such as acetic acid, alcohols such as ethanol, hydrocarbons, etc. can also be used.

As the nitrogen source, ammonia gas, various organic and inorganic ammonium salts, urea, and others can be used. Fermentation conditions are good for aerated culture, and fermentation temperature is 2-4 to 40℃.
, the time is 2-7 days. l) Adjustment of H is carried out using inorganic or organic acidic and alkaline substances, such as urea, calcium carbonate, and ammonia scum. The range is 5~. L-lysine is usually collected from the fermentation liquid by an ion exchange resin method or other known methods. Depending on the type of medium, direct crystallization can also be used.

Hereinafter, the present invention will be described in detail with reference to Examples 6 [Example 1 Example 1 One platinum loop of inoculum was scraped off from the top of the slant, inoculated into 150 ml of the seed culture medium shown below, and aerated for 24 hours. The seeds were cultured and used as a seed culture solution. The culture medium was as shown in Table 4. (This page is blank) Table 4 Meat extract 5 gr.

Peptone 15gr.

NaCI 5 (Jr.

KH2PO45 (lr.

The above composition was made up to 1 liter with ion-exchanged water.

On the other hand, the main fermentation medium listed in Table 5 was poured into a 1 liter M small glass Jarf 7 mentor, adjusted to a total volume of 1 liter with the above seed culture solution, and sterilized by a conventional method. The above seed culture solution was added to these to make 1 liter of liquid medium, and then culture with aeration and stirring was started at 30°C.

(Thursday page blank below) Table 5 Glucose 110 (lr.

Ammonium sulfate 500r.

KH2PO41,2or.

Mg304・7t-Iz　O0.5gr.

F (3304.7H200, O05gr.

Mn Cl 2・4H200,005! Jr.

Ca　C12・2H200,005Or.

Na CI 0.005gr.

d-Biotin 100 μg Thiamine Hydrochloride 200 μg Polypeptone 3
15 (lr.

Antifoaming agent 0.19gr.

The time required to complete culturing varies depending on the type of bacteria, but it takes between 70 and 90 days.
It was time. The culture results are shown in Table 6.

Table 6 Bacterial strains used L-lysine accumulation concentration* TM-111732,7 110746,1 110839,2 *Lysine hepta] Concentration converted to n-acid (IJ/l) Example 2 Seed culture was prepared in the same manner as above. Fermentation by aeration and stirring was carried out in the same manner as in Example 1, except that the main fermentation medium shown in Table 7 was changed.

Table 7 Blackstrap molasses (sulfuric acid treatment) 220gr, /1 (sugar conversion n amount) (110gr, /I) Ammonium sulfate
50 (Jr, /1 Polypeptone S 15gr, /1) The above sulfuric acid treatment involves adding a small amount of sulfuric acid to the blackstrap molasses and heating (120°C,
10 minutes). The time required for fermentation to complete differed depending on the bacteria, but it took 90 to 110 hours. By 81rI9'? fj fn L/taL-
The amount of lysine is shown in Table 8.

Table 8 Bacterial strain used: Accumulated concentration of lysine (*) TM-1
11738,3 110748,2 110842,1 ;1: L-lysine monohydrochloride Expressed in hydrochloride degree (gr, /l) [Effect of light] According to the present invention, L-lysine accumulation ability is high, and moreover, it has a high ability to accumulate blackstrap molasses. When used, higher lysine accumulation is obtained.

Claims (1)

[Claims] Brevibacterium
The genus or Corynebacterium
cultivating a mutant bacterium belonging to the genus Rium) that is resistant to L-α-amino-ε-caprolactam and has the ability to produce and accumulate L-lysine, and collecting L-lysine accumulated in the culture medium. A method for producing L-lysine, characterized by: