JPH0297394A - Treatment of enzymatic catalyst - Google Patents

Abstract

PURPOSE:To obtain L-valine useful as a drug, etc., in high yield and efficiency by subjecting a microbial cell belonging to a biotin-requiring coryne-type bacterial strain to a specific treatment before the enzymatic reaction with said microbial cell in a glucose-containing aqueous solution. CONSTITUTION:Cells of a microorganism belonging to a biotin-requiring coryne- type bacterial strain [preferably Brevibacterium flavum MJ-233 (FERM BP-1497), etc.] are immersed in an aqueous solution containing 1-20vol.% of ethanol. L-valine can be produced by the enzymic reaction in a glucose-containing aqueous solution using said microbial cell as an enzyme source. The treatment with the ethanol-containing aqueous solution is carried out preferably at 30-40 deg.C and pH7-8 for 3-25hr.

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, L-valine can be efficiently produced with high yield.

(2) -Valine plays an important role in the nutrition of humans and animals as one of the essential amino acids, and the 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, L-valine is produced by chemical synthesis method.
It is difficult to manufacture only one piece, and fermentation is mainly used.

However, in the production of L-valine using the known fermentation method, there is a limit to the accumulation of L-valine, and from a new perspective,
- There is a need to provide a method for accumulating and producing 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 microbial cells belonging to coryneform bacteria that require biotin as an enzyme source (Japanese Patent Application No. 101677/1988)
(No. Publication).

(Structure and Effects of the Invention) The present inventors performed an enzymatic reaction on microbial cells belonging to biotin-requiring coryneform bacteria in an aqueous solution containing glucose to produce L-valine in the solution.
It was discovered that L-valine can be produced in high yield by immersing the microbial cells in advance in an aqueous solution containing 1 to 20% by volume of ethanol, leading to the present invention. According to the present invention, when producing L-valine through an enzymatic reaction using microorganisms belonging to biotin-requiring coryneform bacteria as an enzyme source in water/8 solution containing glucose, the microorganisms are prepared in advance. By immersing L-valine in an aqueous solution containing 1 to 20% ethanol by volume, the amount of glucose consumed can be significantly reduced, and as a result, the yield of L-valine to glucose can be greatly improved. Ta. Therefore, according to the method of the present invention, -valine can be produced industrially and efficiently.

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

The microorganism used in the present invention belongs to biotin-requiring coryneform bacteria. This includes L-isoleucine producing bacteria. Examples of the microbial cells used in the present invention include Brevibacterium flavum (Br
evibacterium flavum) MJ-2
33 (Feikoken Joyori No. 1497), Brevibacterium flavum
um) M, J-233-AB-41 (Feikoken Joyori No. 1498), Brevibacterium flavum (Bre
vibacterium flavum) MJ
233ABT-11 (Feikoken Joyori No. 1500) and Brevibacterium flavum (Brevibacterium flavum).
riumflavum) MJ-233-ABD-21
, (Feikoken Joyori No. 1499), etc., and these bacteria are preferably used in the present invention.

In addition, the above (Feikoken Joyori No. 1498 (Feikoken Byoyori No. 3812)) is a DL version using (Feikoken Joyori No. 1497 (Feikoken Byoyori No. 3068)) as the parent strain. - It is an ethanol-assimilating microorganism that has been actively given resistance to α-aminobutyric acid (see columns 3 and 4 of Japanese Patent Publication No. 59-28398). Microtechnology Research Institute No. 1500 (Feikoken Research Institute No. 84)
No. 23) is a mutant strain with high I, -α-aminobutyric acid transaminase activity using the parent strain (Feikoken Joyori No. 1497) (see JP-A-62-519989). In addition, FEIKEN JYOYO NO.
No. 5) is a D using (Feikoken Joyori No. 1497) as the parent stock.
-α-Aminobutyric acid deaminase is a highly active mutant strain (see Japanese Patent Application Laid-Open No. 177993/1983).

In addition to these microbial cells, Brevibacterium ammonium
niagenes>ATCC6871, ATCC13
745, ATCC 13746, Brevibacterium
Brevibacterium div
aricatum) ATCC14020, etc. can also be used.

The microbial cells belonging to the biotin-requiring coryneform bacteria found in the present invention can be used as they are, or they can be immobilized by known methods. As a method for this immobilization, there are methods such as 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.

Glucose or the like is used as a carbon source for the culture medium used to prepare the microbial cells used in the present invention. As the nitrogen source, ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, urea, etc. can be used alone or in combination.As the inorganic salt, potassium hydrogen phosphate, potassium dihydrogen phosphate, magnesium sulfate, etc. can be used. In addition, nutrients such as peptone, meat extract, yeast extract, corn stable liquor, casamino acid, and various vitamins are added to the medium if necessary for the growth of bacteria and L-isoleucine.

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

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

Cells are collected from the culture thus obtained, washed with water or an appropriate buffer, and the washed cells are used in the method of the present invention.

In the method of the present invention, the microbial cells prepared above are immersed in a solution containing ethanol, and then subjected to an enzymatic reaction in an aqueous solution containing at least glucose in the presence of the treated microbial cells, Produces L-valine.

The concentration of ethanol used to treat microbial cells is 1 to 1.
20% by volume, preferably 5-15% by volume is suitably used.

As the solution used for treating the bacterial cells, water or a suitable buffer can be used, and a nitrogen source, inorganic salts, vitamins, etc. can be added as necessary.

The concentration of microbial cells during treatment is 0.5 to 50% by weight, preferably 1 to 30% by weight.

The temperature during treatment is 10 to 60°C, preferably 30 to 4°C.
It is 0°C. The pH of the treatment liquid is 5 to 10, preferably 7.
to 8 are preferably used. Processing time is 1 to 30 hours.
Preferably it is 3 to 25 hours.

The treated microbial cells prepared as described above are used in the enzyme reaction, but an immobilized product of the microbial cells can also be subjected to the reaction.

The immobilized bacterial cells of the present invention can be prepared by appropriately selecting from known immobilization methods, such as entrapment methods using acrylamide, alginate, carrageenan, etc., and ionic bonding methods using DEAE-Sephadex, DEAE-cellulose, etc.

The reaction solution is usually a completely synthetic medium, but
Here, the completely synthetic medium is an aqueous solution containing an inorganic nitrogen source and/or an inorganic salt with a known chemical structure. Examples of inorganic nitrogen sources for the fully synthetic medium used in the present invention include ammonia, ammonium chloride, ammonium sulfate, ammonium nitrate, ammonium phosphate, etc., and examples of inorganic salts include potassium hydrogen phosphate, potassium dihydrogen phosphate, Examples 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.

An example of a completely synthetic medium is (NH,), 30.23
g/j2, KH2PO40, 5g/l, KHPO40,
5g/j! ,,Mg5O,・7H200,5g/*,,
FeSO4' 7H20201)pmXMnSO4・4
There is an aqueous solution with pH 7.6 containing ~6H2020pI)m.

As mentioned above, the fully synthetic medium used in the present invention does not contain biotin or natural products containing biotin. Amino acids, vitamins, sugars, etc. with known chemical structures that clearly do not contain biotin can be added.

The amount of 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). be able to.

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.

1. The enzyme reaction is carried out in an aqueous solution containing glucose used in the reaction, preferably in a fully synthetic medium containing glucose as described above, where the dissolved oxygen concentration is 0°O5 ppm or more, 8
It is preferable to continuously or intermittently supply air or oxygen into the reaction system so that the concentration is ppm or less.

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

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 (Leuc
onostoc mesenteroides) A
The microbioassay method using TCC8042 and high performance liquid chromatography (Shimabara LC-5A) were used in combination. The residual amount of glucose was determined using a glucose determination kit (Glucose C-Test Wako, manufactured by Wako Tsumugi Kogyo Co., Ltd.). In addition, in the following experimental examples, % means weight %.Example 1 Medium (urea 0.4%, ammonium sulfate 1.4%, KH
2Po, 0.05%, K2 HP 040.

05%, MgSO4・TI (200,05%, CaCj
l! 2 H2H202ppm,,FeSO4・7H2
02ppm=MnSO4・4~6H2O2ppm, Z
nSO4・7H 202ppm NaCIl 2ppm, biotin 200μg/β, thiamine・H (1!100μg
/ j2, casamino acid 0.1%, yeast extract 0.1%
)100rr+j! 500m1! After dispensing into Erlenmeyer flasks and sterilizing (pH 7.0 after sterilization) Brevibacterium fl.
avum) M J-233 (Feikoken Joyori No. 149
No. 7) was inoculated, glucose was added aseptically to a concentration of 5 g/β, and shaking culture was performed at 30° C. for 2 hours.

Next, the main culture medium (glucose 5%, ammonium sulfate 2.3%, KH2PO40.05%, KHP○40.0
5%, MgSO4・7H200,05%, FeSO4・
7H2020ppm, MnSO4・4~6H2020p
pm, biotin 200. jig/fi, thiamine--H
C11l00p/R, casamino acids 0.3%, yeast extract 0.3%) was charged into a 21 volume aerated stirring tank, and after sterilization (120°C, 20 minutes), 2 of the preculture was added.
0mj2 was added and the rotation speed was 11,000 rpm.

Culture was carried out for 24 hours at aeration rate of 1 vvm, temperature of 33° C., p) (7,6).

After the cultivation is completed, the culture is 100rnj! Bacteria were collected by centrifugation. This was washed twice with demineralized distilled water, and the resulting bacterial cells were added to 100 mL of each ethanol-containing aqueous solution shown in Table 1.
((NH,)2So, 0.2g.

KH2PO40,05g-MgSO4・7H200,
05g] and immersed at 33°C for 3 hours. The immersion-treated bacterial cells were collected by centrifugation, washed with demineralized distilled water, and the treated bacterial cells were added to the reaction solution [glucose 100 g
/l-KH2PO40゜5g, Ni2HPO40.5g
/β, MgSO47H200,5g/n= Fe3O4
H7H202oppm, thiamine-HCl salt 100μg
/It (pH8,0))50m7! suspended in. Further, to adjust the pH, dry heat sterilized (heated at 150° C., 5 hours) calcium carbonate was added at a concentration of 50 g/β. The reaction was carried out using a 500ml flask at 33°C and at 2 rotations.
A shaking reaction was performed at 2 Orpm for 40 hours. After the completion of the reaction, the supernatant was sterilized by centrifugation (4000 rpm, 15 minutes, 4°C), and the amount of valine and remaining glucose in the supernatant was quantified.

The results are shown in Table 1.

The remaining amount of valine in the table 6 - Valine production x 100 100g/j2 Glucose residual concentration f la
vum) MJ-233-AB 41 (aσKajyoyo
No. 1498) was cultured, and after reacting with Ibokawaretsu-1 in a giant bullfight, L-valine and glucose in the supernatant were 100 g/j! Glucose residual concentration x 100 Brevibacterium flavum (Brevibacterium rI)
avum) MJ-233-ABT-11 (IkH5f'
Brevibacterium f.
lavum) MJ-233-ABD 21 Watakyumukyori No. 1499) was cultured and reacted with the same type of cow as J-1. Is it a good fortune with a ridged fin? Mr. H is shown in Table 4.

Table 4 Balin d child δ adjustment Barin beef plan ×100 100g/7! Glucose residual concentration

Claims (2)

[Claims] (1) When carrying out an enzymatic reaction in an aqueous solution containing glucose in the presence of microbial cells belonging to biotin-requiring coryneform bacteria to produce L-valine in the solution, A method for treating an enzyme catalyst, comprising immersing it in an aqueous solution containing ~20% by volume of ethanol. (2) causing an enzymatic reaction in an aqueous solution containing glucose in the presence of an enzyme catalyst to produce L-valine in the aqueous solution;
In the L-valine production method for collecting L-valine from now on, it is necessary to immerse microbial cells belonging to biotin-requiring coryneform bacteria to be used in the enzyme catalyst in advance in an aqueous solution containing 1 to 20% by volume of ethanol. Characterized by L-
Method of manufacturing valine.