JP2721975B2 - Method for producing L-lysine - Google Patents

Description

The present invention relates to a novel method for producing L-lysine by an enzymatic reaction.

According to the present invention, L-lysine can be efficiently produced with high yield.

L-lysine plays an important role in human and animal nutrition as one of the essential amino acids, and the demand for pharmaceuticals, foods, feed additives and the like has been rapidly increasing in recent years.

(Conventional technology and problems) As an industrial method for producing L-lysine, stereoisomers exist as in the case of other amino acids, and it is difficult to produce only the L-isomer by a chemical synthesis method. Depending on.

However, in the production method of L-lysine by a known fermentation method, the yield to sugar is low and the accumulation of L-lysine is limited, and a method for producing L-lysine more effectively from a new viewpoint is provided. Is strongly required.

(Structure and Effect of the Invention) The present invention relates to a microbial cell belonging to a coryneform bacterium, which is biotin-requiring and resistant to S- (2-aminoethyl) -L-(-) cysteine (hereinafter referred to as AEC). An object of the present invention is to provide a method for producing L-lysine, wherein an enzymatic reaction is carried out in an aqueous solution containing glucose to produce L-lysine in the solution, and L-lysine is collected therefrom.

According to the present invention, in an aqueous solution containing glucose,
When an enzymatic reaction is performed using microbial cells belonging to coryneform bacterium which have biotin requirement and AEC resistance as an enzyme source, L-lysine is produced in the aqueous solution and a high yield can be obtained.

The method of the present invention is an enzymatic method. Compared to the conventional fermentation method, even when a completely synthetic medium containing glucose is used as an aqueous solution containing glucose, sterilization of the medium required in the fermentation method can be performed. Since complicated operations are not required, production management is greatly facilitated.
-Lysine can be produced.

(Detailed Description of the Invention) The method of the present invention uses an enzyme reaction system to produce L-lysine under conditions without any growth of microbial cells.
The present invention provides a method for producing lysine, and such a method is a novel method which has never been known before.

The microorganism used in the present invention is biotin-requiring and AE
It has C resistance and belongs to coryneform bacteria. Examples of the microorganism used in the present invention include Brevibacterium flavum MJ-233-
L-11 (Microbial Research Laboratories No. 10094; hereinafter, MJ-233-L-11)
), Brevibacte flavum
rium flavum) MJ-233-L-12
No. MJ-233-L-12), and these bacteria are suitably used in the present invention.

The above MJ-233-L-11 and MJ-233-L-12
Is Brevibacterium flavum
flavum) MJ- (Microtechnical Lab. No. 1497: Microtechnical Lab. No. 30)
No. 68) [hereinafter referred to as MJ-233] as a parent strain, and a microorganism positively imparted with AEC resistance.

The microorganism belonging to the coryneform bacterium which has biotin requirement and AEC resistance seen in the present invention can be used as it is, or an immobilized product obtained by immobilizing them by a known method can be used. You can do it. Examples of the immobilization method include using a polymerizable monomer such as acrylamide or insolubilizing cells in a suitable carrier such as alginate or carrageenan.

The resistant mutant used in the present invention is obtained by the following operation.

Mutagenesis was induced in the L-isoleucine-producing strain by ultraviolet irradiation or treatment with a chemical agent (for example, N-methyl-N'-nitro-N-nitrosoguanidine or the like). Plate medium containing threonine (urea 0.2%, ammonium sulfate 0.7%, KH ₂ PO ₄ 0.05%, K ₂ HP
_{O 4 0.05%, MgSO 4 ·} 7H 2 O0.05%, FeSO 4 · 7H 2 O6mg /, Mn
_{SO 4 · 4~6H 2 O6mg /,} ZnSO 4 · 7H 2 O6mg /, biotin 20
0 μg /, thiamine hydrochloride 100 μg /, AEC2 g /, L-threonine 1.2 g /, agar 2.0%, glucose 2%), sterilized, cultured at 30 ° C. for several days, and separated into large colonies. , To obtain a resistant mutant. Typical resistant mutants include Brevibacterium flavum
terium flavum) MJ-233-L-11, and this strain is supplied to the Institute of Microbial Industry, National Institute of Advanced Industrial Science and Technology
No. has been deposited. This resistant strain is Brevibacterium flavum MJ-233.
(Microtechnological Research Institute No. 1497) derived as an AEC resistant strain.

MJ-233-L-11 (AEC resistant strain), MJ-233-L
Table 1 shows the relative growth of -12 (AEC-resistant strain) and its parent strain, MJ-233, with respect to AEC.

Media composition: urea 0.2%, ammonium sulfate 0.7%, KH ₂ PO ₄ 0.05%, K ₂ HPO _{4
0.05%, MgSO 4 · 7H 2} O 0.05%, FeSO 4 · 7H 2 O 6mg /,
_{MnSO 4 · 4~6H 2 O 6mg /} , ZnSO 4 · 7H 2 O 6mg /, biotin 200 [mu] g /, (the AEC added in an amount shown in Table 1) Thiamine hydrochloride 100 [mu] g / culture method: 24Fai large the medium 10ml After dispensing into test tubes and sterilizing at 120 ° C for 10 minutes, MJ-233-L-11 strain was inoculated, glucose was added under aseptic conditions at 2%, and shaking culture was performed at 30 ° C for 20 hours.

In the present invention, the term “AEC-resistant strain” or “microorganism having resistance to AEC” means that AEC 0.2% is added to the above-mentioned (Note-2) medium, and the relative growth rate when cultured with shaking at 30 ° C. for 20 hours is 80%.
Defined above. However, And

The above biotin requirement used in the method of the present invention and
The medium used for preparing microbial cells having AEC resistance and belonging to the coryneform bacterium is not particularly limited, and may be one used for general microorganisms.

Ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, urea and the like can be used alone or in combination as a nitrogen source of a medium used for preparing the microbial cells used in the present invention.

As the inorganic salt, potassium monohydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate and the like are used. In addition, if necessary for the growth of bacteria and L-lysine production, peptone, meat extract, yeast extract, corn steep liquor,
Nutrients such as casamino acids and various vitamins are added to the medium and used.

The cultivation is performed under aerobic conditions such as aeration stirring and shaking, and the culturing temperature is 20 to 40 ° C, preferably 25 to 35 ° C. The pH during the culturing is adjusted to 5 to 10, preferably around 7 to 8. The pH during the culturing is adjusted by adding an acid or an alkali.

The glucose concentration at the start of the culture is preferably 1 to 5% by weight, more preferably 2 to 3% by weight. The culture period is 0.5 to 3 days, and the optimal period is 1 to 2 days.

The cells are collected from the culture thus obtained, washed with water or a suitable buffer, and used for the enzymatic reaction of the method of the present invention.

In the method of the present invention, an enzymatic reaction is performed in an aqueous solution containing at least glucose in the presence of the microbial cells prepared above (including the immobilized product thereof). Here, the concentration of glucose added to the aqueous solution is 0.
It is 5 to 20% by weight, preferably 1 to 10% by weight.

As the aqueous solution, glucose-containing water or a buffer such as phosphoric acid or tris hydrochloride can be used as described above, but preferably, a complete synthetic medium containing glucose is used. Here, the complete synthetic medium is an aqueous solution containing an inorganic nitrogen source and / or an inorganic salt whose chemical structure is known. Examples of the inorganic nitrogen source of the complete synthetic medium used in the present invention include ammonia, ammonium chloride, ammonium sulfate, ammonium sulfate, and ammonium phosphate.Examples of the inorganic salts include potassium monohydrogen phosphate, potassium dihydrogen phosphate, Examples thereof include magnesium sulfate, manganese sulfate, and iron sulfate. These inorganic nitrogen sources and inorganic salts can be used alone or in combination of two or more.

The concentration of the inorganic nitrogen source and / or the inorganic salt as an aqueous solution may be in the same range as a medium used for culturing ordinary microbial cells, and is not particularly limited.

An example of a complete synthetic medium is (NH ₄ ) ₂ SO ₄ 2 g /; KH ₂
PO ₄ 0.5 g /; K ₂ HPO ₄ 0.5 g /; MgSO ₄・ 7H ₂ O 0.5 g /; FeSO _{4
· 7H 2 O 20ppm; MnSO 4} · 4~6H 2 O 20ppm pH 7 containing.
There are 6 aqueous solutions.

As described above, the complete synthetic medium used in the present invention includes:
It does not contain biotin or natural products containing biotin. Amino acids, vitamins, saccharides, and the like having a known chemical structure that does not contain biotin can be added.

The amount of the microbial cells prepared as described above used in the method of the present invention is not particularly limited, but is generally used at a concentration of 1 to 50% (wt / vol). it can.

In the present invention, the enzymatic reaction is carried out at a temperature of about 20 to about 50C, preferably about 30 to about 40C, usually for about 10 to about 72 hours.

The enzymatic reaction is carried out in an air or oxygen atmosphere in the reaction system so that the concentration of dissolved oxygen in an aqueous solution containing glucose used for the reaction, preferably a complete synthetic medium containing glucose becomes 0.05 ppm or more and 8 ppm or less. Is preferably supplied continuously or intermittently.

Separation and purification of L-lysine produced in the reaction solution injection obtained by the above reaction method can be performed in the same manner as separation and purification of amino acids in the case of fermentation. For example, a known ion exchange method or It can be performed by a precipitation method or the like.

EXPERIMENTAL EXAMPLES In the following experimental examples, the qualitative properties of L-lysine were confirmed by the Rf value of paper chromatography, the mobility of electrophoresis, and the biological activity value by microbial quantification. The quantification was performed using a microbioassay and high-speed liquid chromatography (Shimadzu LC-5A) in combination. In the following examples, “%” means “% by weight”.

Example-1 Medium (0.4% urea, 1.4% ammonium sulfate, KH ₂ P
O ₄ 0.05%, K ₂ HPO ₄ 0.05%, MgSO ₄・ 7H ₂ O 0.05%, CaCl ₂
・ 2H ₂ O 2ppm, FeSO ₄・ 7H ₂ O 2ppm, MnSO ₄・ 4〜6H ₂ O
_{2ppm, ZnSO 4 · 7H 2 O} 2ppm, NaCl 2ppm, biotin 200
μg /, Thiamine / HCl 100 μg /, Casamino acid 0.1
%, Yeast extract 0.1%) in a 500 ml Erlenmeyer flask, sterilized (pH 7.0 after sterilization), and then Brevibacterium flavum MJ-233-
L-11 (Microtechnical Laboratories No. 10094) was inoculated, glucose was aseptically added to a concentration of 5 g /, and cultured with shaking at 30 ° C. for 2 days.

Then, the culture medium (5% glucose, ammonium sulfate _{2.3%, KH 2 PO 4 0.05} %, K 2 HPO 4 0.05%, MgSO 4 · 7H 2 O0.
_{05%, FeSO 4 · 7H 2} O 20ppm, MnSO 4 · 4~6H 2 O 20ppm,
1000ml of biotin 200μg /, thiamine / HCl 100μg /, casamino acid 0.3%, yeast extract 0.3%)
After charging into a vented agitating tank and sterilizing (120 ° C., 20 minutes), 20 ml of the above preculture was added, and the rotation speed was 1000 rpm and the aerated amount was 1
The cells were cultured at vvm, a temperature of 33 ° C. and pH 7.6 for 24 hours.

After cultivation, after collecting cells by centrifugation from 500 ml of culture,
The cells washed twice with demineralized distilled water were added to the reaction solution [(NH ₄ ) ₂ SO
₄ 2 g /; KH ₂ PO ₄ 0.5 g /; KH ₂ PO ₄ 0.5 g /; MgSO ₄・ 7H ₂ O 0.
_{5g /; FeSO 4 · 7H 2} O 20ppm; MnSO 4 · 4~6H 2 O 20ppm; was suspended in 1000ml of pH7.6],; thiamine hydrochloride 100 [mu] g /
The suspension was charged into a 2-volume aeration stirred tank, 9 g of glucose was added, the number of revolutions was 300 rpm, the amount of aeration was 0.1 vvm, the temperature was 33 ° C., and the pH was
The reaction was performed at 7.6 for 24 hours.

After the completion of the reaction, L-lysine was quantified in the supernatant obtained by centrifugation (4000 rpm, 15 minutes, 4 ° C.).

After completion of the reaction, 500 ml of the culture solution was passed through a column of a strongly acidic cation exchange resin (H ⁺ type) to adsorb L-lysine, washed with water, eluted with 0.5N aqueous ammonia, and then subjected to L-lysine extraction. The mixture was concentrated and L-lysine crystals were precipitated with cold ethanol. The results are shown in Table 1 below.

Example 2 Brevibacterium flavum MJ-233-L-12 under the same conditions as in Example 1
(Microbial Lab. No. 10095) and Example 1
After reacting under the same conditions as described above, L-lysine in the supernatant was quantified. Further, L-lysine crystals were precipitated in the same manner as in Example-1. The results are shown in Table 2.

Under the same conditions as in Example-1, Brevibacterium
Flavam (Brevibacterium flavum) MJ-233 was cultured, and reacted under the same conditions as in Example 1. After quantifying L-lysine in the supernatant, the yield was 0.06 g /.

 ──────────────────────────────────────────────────続 き Continuing from the front page (72) Inventor Masato Terasawa 8-3-1 Chuo, Ami-cho, Inashiki-gun, Ibaraki Pref. Inside the Central Research Laboratory of Mitsubishi Yuka Co., Ltd. (72) Inventor Hideaki Yukawa 8 Ami-cho, Inashiki-gun, Ibaraki Chome 3-1, Mitsubishi Yuka Corporation Central Research Laboratory

Claims (2)

(57) [Claims] An enzyme reaction in an aqueous solution containing glucose in the presence of a microbial cell belonging to coryneform bacterium, which is biotin-requiring and has S- (2-aminoethyl) -L-(-) cysteine resistance. And producing L-lysine in the solution, and collecting L-lysine from the solution. 2. The method according to claim 1, wherein the aqueous solution is a completely synthetic medium.