JPH0272888A - Production of l-valine - Google Patents

Abstract

PURPOSE:To obtain L-valine on an industrial scale in high efficiency without causing the proliferation of microorganisms by subjecting cells of a microorganism belonging to biotin-requiring coryne-form bacterium to enzymatic reaction under a specific condition in an aqueous solution containing glucose and a nitrogen source. CONSTITUTION:Cells of a microorganism belonging to biotin-requiring coryne- form bacterium is subjected to enzymatic reaction in an aqueous solution containing glucose and a nitrogen source. The reaction is carried out under a condition to keep the weight ratio of the glucose to the nitrogen of the nitrogen source in the reaction liquid to 5-40. The above microorganism is preferably Brevibacterium flavum MJ-233 (FERM BP-1497), etc., and the reaction is carried out preferably at 30-40 deg.C.

Description

DETAILED DESCRIPTION OF THE INVENTION (Industrial Field of Application) The present invention relates to a method for producing valine by enzymatic reaction.

According to the present invention, it is possible to efficiently produce ichinorin with high yield.

L-valine plays an important role in the nutrition of humans and animals as one of the essential amino acids, and demand for pharmaceuticals, foods, feed additives, etc. has increased rapidly in recent years.

(Prior art and issues) As for the industrial production method of L-valine, since stereoisomers exist as in the case of other amino acids, it is difficult to produce only L-valine using chemical synthesis methods, and the main method is fermentation. It's according to the law.

However, in the production of L-valine using the known fermentation method, there is a limit to the accumulation of L-valine.
There is a need to provide a method for producing significant amounts of valine.

The present inventors previously demonstrated that in an aqueous solution containing glucose,
A method for producing L-valine in a high yield in an aqueous solution by carrying out an enzymatic reaction using microorganisms belonging to coryneform bacteria that require biotin as an enzyme source (Japanese Patent Application No. 101677/1982)
is proposed.

(Structure and Effects of the Invention) In order to produce L-valine more efficiently, the present inventors have conducted extensive studies on the composition of the reaction solution, etc. As a result, the present inventors have found that a biotin-requiring Corine type product has been obtained. Microbial cells that succumb to Il bacteria,
When performing an enzymatic reaction in an aqueous solution containing glucose and a nitrogen source to produce I,-valine in the solution, the ratio of the weight of glucose in the solution to the weight of nitrogen in the nitrogen source (hereinafter referred to as C
We discovered that L-valine can be produced in high yield by carrying out an enzymatic reaction at a ratio of 5 to 40.
We have arrived at the present invention.

According to the present invention, when performing an enzyme reaction in an aqueous solution containing glucose and a nitrogen source using a biotin-requiring microorganism belonging to coryneform bacteria as an enzyme source, the C/N ratio in the aqueous solution is set to 5 to 5. By carrying out the reaction in the range of 40, it was possible to significantly reduce the amount of glucose consumed, and as a result, it became possible to significantly improve the yield of L-valine relative to glucose. Therefore, according to the method of the present invention, L-valine can be produced industrially and efficiently.

(Detailed Description of the Invention) The method of the present invention provides a method for producing valine using only an enzymatic reaction for producing valine under conditions that do not involve the growth of microbial cells.

The microorganisms used in the present invention belong to biotin-requiring coryneform bacteria, and are preferably ethanol-assimilating. This includes L-isoleucine producing bacteria. Examples of the microbial cells used in the present invention include Brevibacterium flavum (Breviba
cterium flavum) MJ-233 (Feikoken Joyori No. 1497), Brevibacterium flavum (Brevibacterium flavu+
w) MJ-233-AB-41 (lak Koken Joyori 1st
498), Brevibacterium flavum (Brev
ibacLerium flavu＋＊) M J
-233-A BT-11 (Feikoken Joyori No. 1500) and Brevibacterium flavum (Breviba
cterium flavu+w) MJ-233-
ABD-21 (T3 Koken Jyoyori No. 1499) and the like, and these flashes are preferably used in the present invention.

The above (Feikoken Joyori No. 1498) is an ethanol-assimilating microorganism that has been actively given DL-α-aminobutyric acid resistance using (Feikoken Joyori No. 1497) as its parent strain ( (See columns 3 and 4 of Japanese Patent Publication No. 59-28398).

(Feikoken Joyori No. 1500) is (@Koken Joyori No. 1)
497) as the parent strain, and is a mutant strain with high monoα-aminobutyric acid transaminase activity (Japanese Patent Application Laid-Open No. 62-51998).
In addition, (Feikoken Joyori No. 1499) is a D-α-aminobutyric acid deaminase high activity mutant strain derived from the parent strain (Feikoken Joyori No. 1497) (Japanese Patent Laid-Open Publication No. 61
(Refer to Publication No. 177993).

In addition to these microbial cells, Brevibacterium ammonium
niagenes) ATCC6871, ATCC13
745, ATCC 13746, Brevibacterium
Brevibacterium sodiva
ricatum) ATCC 14020, etc. The microorganisms belonging to biotin-requiring coryneform bacteria used in the present invention can be used as they are, or they can be immobilized by known methods. Immobilized products can also be used. This immobilization method includes using a polymerizable monomer such as acrylamide, or making the bacterial cells insoluble in a suitable carrier such as alginate or carrageenan.

The medium used for preparing the microbial cells belonging to the above-mentioned biotin-requiring coryneform bacteria used in the method of the present invention is as follows:
There are no particular limitations, and any material used for general microorganisms may be used.

As the nitrogen source for the culture medium used for the preparation of the microorganism cells used in the present invention, ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, urea, etc. can be used alone or in combination.As the inorganic salt, potassium phosphate-hydrogen can be used. , potassium dihydrogen phosphate, magnesium sulfate, etc. are used. In addition, if necessary for the growth of bacteria, etc.
Nutrients such as peptone, meat extract, yeast extract, cornstarch liquor, casamino acids, and various vitamins are added to the medium. Cultivation is performed under aerobic conditions such as aeration and shaking, and the culture temperature is 20-40°C, preferably 25-30°C.
Perform at 5°C. pH during culturing is 5 to 1O1, preferably 7
The pH is adjusted to around 8 to 8, and acid or alkali is added to adjust the pH during culturing.

Glucose concentration at the start of culture is preferably 1-5T%
, more preferably 2 to 3% by weight, and the culture period is 0.
．． 5-3 days, optimal period is 1-2 days.

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

In the method of the present invention, C/N ratio from 2 to 45, preferably from 5 to
The glucose concentration usually used is 05-20% by weight, preferably 1-10% by weight. The nitrogen concentration of the nitrogen source used in the reaction is usually 0°0.
The amount is 1 to 4% by weight, preferably 0.02 to 2% by weight.

As the aqueous solution, a completely synthetic medium is usually suitably used.
1 Here, the completely synthetic medium is an aqueous solution containing an inorganic nitrogen source and an inorganic salt with a known chemical structure. Inorganic nitrogen sources for the fully synthetic medium used in the present invention include ammonia,
Examples include ammonium chloride, ammonium nitrate, ammonium nitrate, and ammonium phosphate. Examples of inorganic salts include potassium hydrogen phosphate, potassium dihydrogen phosphate, 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.

An example of a completely synthetic medium is (NH4) 2 S04
23 g/j! ;KH2P 04 0. 5g/
β; K2HP O40, 5g/1; Mg
SO4・7H200,5g/12; FeSO4
H7H2020ppm; MnSO4・4~6H2020
There is an aqueous solution with a pH of 7.6 containing ppm.

As mentioned above, the completely synthetic medium used in the present invention does not contain biotin or natural products containing biotin, or contains amino acids, vitamins, m-types, etc. with chemical structures that clearly do not contain biotin. You can use it.

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% (w L / vol). Can be done.

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

- In the enzyme reaction described in F, the dissolved oxygen concentration in the aqueous solution containing glucose and nitrogen sources used in the reaction is o, O5pp.
Air or r! is added to the reaction system so that the concentration is at least m and at least sppm. ! It is preferable to supply the element continuously or intermittently.

The separation and purification of valine produced in the reaction solution obtained by the above reaction method can be performed in the same manner as the separation and purification of amino acids by fermentation, for example, by the known ion exchange resin treatment method, precipitation method, etc. This can be done by

'XJL Section' In the following experimental examples, the quality of L-valine was confirmed by the Rf value of paper chromatography, the mobility of electrophoresis, and the biological activity value of microorganism quantification. Quantification was performed using Leuconostoc mesenteroides (Leuconosto mesenteroides).
c mesenteroides) AT CC80
The microbioassay method using 42 was used in combination with high performance liquid chromatography (Takashi LC-5A). The residual amount of glucose was determined using a glucose determination kit (Nigercose C Test Wako, manufactured by Wako Pure Chemical Industries, Ltd.). In addition, in the following experimental examples, the expression % means weight %.

Example-1 Medium (urea 0.4%, ammonium sulfate 1.4%, K
Hz PO40.05%, K2 HP 040.

05%, MgSO4・7H200,05%, CaC1z
・2Hz 0 2ppm, Fe50°7H202p
pm, MnSO4-4~6H202ppm+ZnSO
4.7H202ppm.

NaCe 2pI)m, biotin 200μg/l,
Thiamin H (1!100μ (H7g, Casamino acid 0
．． 1%, yeast extract 0.1%) 100mj+ for 500m
Dispense into f volume Erlenmeyer flask and sterilize (sterilize 1 & pH 7,0)
After that, Brevibacterium flavum (Breviba
cLeriu+m flavum) M J -23
3 (Kaikoken Joyori No. 1497) was inoculated, and glucose was added aseptically to a concentration of 5 g// to culture with shaking at 30° C. for 2 days.

Next, main culture medium (glucose 5%, ammonium sulfate 2.3%, KH2PO40.05%, K, Hl) 0.0
．． 05%, Mg5O, 7H200, 05%, Fe50
．．・7H2020ppm, Mn5O4・4-6H2
020ppm, biotin 200μg/l, thiamin.
HCI 1100tt/R, casamino acid 0.3%,
Yeast extract 0.3%) was placed in a 21-volume aeration stirring tank, and after sterilization (120°C, 20 minutes), 'lQml' of the preculture was added, and the rotation speed was 1100.
Qrp, ventilation q l v V m, ? m degree 33℃,
Culture was carried out at pH 6 for 24 hours.

After the completion of the culture, each 100 ml of the culture was centrifuged to collect the bacteria, and the bacterial cells were washed twice with salt distilled water and added to the reaction solution [glucose 100 g/l, K H2P O40.5
g/l, K2 HPO40,5g/It, Mg5o,-
78200,5g/1. , FeSO47112020p
pm, thiamine-hydrochloride 100 μgel, (pH 8,
After suspending at 50 mf, a nitrogen source was added so that the C/N ratio was as shown in Table 1. Note that ammonium sulfate was used as a nitrogen source. In addition, to adjust the pH, add 50% calcium carbonate that has been dry heat sterilized (heated at 150°C for 5 hours).
It was added at a concentration of g/12. The reaction was carried out using a 500 ml square flask and shaking at 33° C. and a rotational speed of 22 rpm for 40 hours.

After the reaction, distal bone 1 itu (4000 rpm, 1
The amount of valine and the remaining amount of glucose in the supernatant liquid was sterilized at 4° C. for 5 minutes.

The results are shown in Table 1.

Table 1 The results are shown in Table 2.

2nd table - Valin d mixture δ machining (g/1) L-barrier 4 bullet d mouth * (g/l) Example-2 Brevibacterium flavum (Brevihactσ1111 fla 4) in Example-1 and Ii'JMk -M
J-233-AB-41Gf) tσkenjoyori No. 1498
Issue) will be carried out again as long as it is cultivated! II-I
The remaining amount of L-valine and glucose was determined.

Shinto 1-3 Real cap row-1 and *”M)'ld'L4 Brevibacterium flavum Φrevibacterius+ f
lavm MJ-233-ABT-11@σ Ogijoyori 1st
No. 500) was cultured and the fruit 8el-I
4 b How many rows I tri'? I was embarrassed and reacted in '44 cow.
Table 3 shows the determination results of L valine and glucose residual V, and Table 4 shows the results of heart measurement.

Table 1 Barin style Japanese d Fu length (g//) Shibarin 1 blade gap (g/l) J] What row - 4 Minoru 1-1 and eyes "14)'ld! Brevi with W Bacterium flavum rev i bacter i m
"1 avu+++)

Claims (1)

[Claims] (1) When microbial cells belonging to biotin-requiring coryneform bacteria are subjected to an enzymatic reaction in an aqueous solution containing glucose and a nitrogen source to produce L-valine in the solution,
The ratio of the weight of glucose in the reaction solution to the weight of nitrogen in the nitrogen source (
A method for producing L-valine, characterized in that an enzymatic reaction is carried out at a ratio of (glucose amount/nitrogen amount) in the range of 5 to 40.