JPS6120274B2 - - Google Patents

Description

[Detailed description of the invention]

The present invention relates to a method for producing α-keto acids using the action of microbial enzymes. α-keto acids corresponding to essential amino acids are useful medicines for renal dysfunction such as uremia. As a result of various studies on the production method of α-keto acids, the present inventors found that the general formula [In the formula, R is -CH ₂ R ₁ or

[Formula] (where R ₁ is -H, -OH, -COOH, -CH ₂ COOH,

【formula】

[Formula] −CH ₂ SCH ₃ , −CH ₂ CH ₂ CH ₂ NH ₂ ,

【formula】

【formula】

[Formula] and R ₂ is -CH ₃ or C ₂ H ₅ ). ] By treating the D-isomer of the α-amino acid represented by or its salt with a culture, bacterial cells, or a processed product of a microorganism belonging to the genus Dorigonopsis in the presence of catalase as necessary, the above-mentioned amino acid is obtained. In addition, the racemic form of the above-mentioned amino acids may be added to cultures of microorganisms belonging to the genus Trigonopsis, bacterial cells or processed products thereof, and α-keto acids.
- If necessary, a culture of a microorganism, a bacterial cell, or a processed product thereof having the ability to convert the L-form of an amino acid or a salt thereof into an α-keto acid is allowed to act in the presence of catalase to convert the corresponding α −
They discovered that it becomes a keto acid and completed the present invention. As the microorganism used in the present invention, any microorganism belonging to the genus Trigonopsis can be used as long as it has the ability to convert the above-mentioned amino acids into the corresponding α-keto acids, but a preferred microorganism is Trigonopusis variabilis (IFO).・755
There is. The medium for culturing the microorganisms used in the present invention may be any natural medium, medium, or synthetic medium as long as it contains moderate amounts of carbon sources, nitrogen sources, inorganic substances, and the like. Carbon sources include carbohydrates such as glucose, fructose, sucrose, maltose, galactose, ribose, molasses, and starch hydrolysates, hydrocarbons such as normal paraffin and kerosene, alcohols such as methanol, ethanol, glycerin, and sorbitol, pyrupic acid, Examples include organic acids such as lactic acid and acetic acid. Nitrogen sources include inorganic nitrogen compounds such as ammonia, ammonium chloride, ammonium phosphate, ammonium sulfate, ammonium carbonate, ammonium acetate, ammonium nitrate, and sodium nitrate, various amino acids, urea, peptone, meat extract, yeast extract, corn steep liquor, casein, Examples include nitrogen-containing substances such as protein hydrolysates and fruit meal. Inorganic substances include phosphates such as potassium phosphate, dipotassium phosphate, monosodium phosphate, and disodium phosphate;
Examples include sulfates and hydrochlorides of magnesium and manganese. The microorganisms used in the present invention may be cultured for 1 to 5 days under aerobic conditions at a temperature of 20 to 40° C. and near neutral pH. The culture fluid thus obtained, the bacterial cells collected from the culture fluid, or those activated therefrom are used in the α-keto acid production reaction. The activation treatment here refers to a cell-free extract obtained by physically or chemically treating bacterial cells, an enzyme purified from the cell-free extract using a known method, or a cell-free extract obtained by activating bacterial cells as they are. Refers to activated bacterial cells. Examples of activation methods include ultrasonic treatment, French press treatment, -
Method of freezing and then thawing at 10℃ or below, treatment with organic solvents such as ethanol, n-butanol, benzene, toluene, acetone, etc., cetyltrimethylammonium bromide, sodium desoxycholate, sodium decyl sulfate, sorbitan monolaurate, digitonin, triton ×
There are surfactant treatments such as 100, high osmotic solution treatments, etc. Among these, the freeze-thaw method and the toluene treatment method are easy to operate and can sufficiently increase enzyme activity. When carrying out the present invention, catalase is also produced during the cultivation of the bacteria used, so in principle there is no need to add catalase. requires the addition of catalase. In addition, it is the generated α that requires catalase.
- To prevent oxidative decarboxylation of keto acids. The reaction of the present invention is usually completed in 3 to 6 hours at a pH of 7 to 9 and a temperature of 28 to 35°C. Moreover, this reaction is preferably carried out under aerobic conditions. The target product accumulated in the culture solution or reaction solution can be easily extracted, purified and isolated by a combination of a method using a strongly acidic ion exchange resin and a method of producing and precipitating an insoluble salt. In addition, in the present invention, when the racemic form of the amino acid described above is used as a raw material, known L-α-amino acids are mixed with α- The corresponding α-keto acid can be obtained by reacting, purifying, and isolating in the same manner as the above-mentioned method in the simultaneous presence of a culture, bacterial cells, or processed products of microorganisms that have the ability to produce a keto acid. can. This method has very high industrial value since it is not necessary to optically resolve the starting material α-amino acid. Examples of microorganisms that have the ability to convert L-α-amino acids into α-keto acids include Proteus vulgaris Hauser.
IFO・3851, Sarcina lutea (Sarcina,
lutea) IFO・3030, Acetobacter suboxydans (Acetobacter suboxydans) IFO・
3172, Neurospora cratusa
crassa), etc. Next, the present invention will be specifically explained with reference to Examples.
In addition, alpha-keto acid was quantified by the following method. 1ml for 5ml of test solution containing 0.2-1.5μmol of keto acid
dinitrophenylhydrazine reagent (500 μmol
dinitrophenylhydrazine dissolved in 100 ml of 2N hydrochloric acid) and left at 30°C for 10 minutes. Next, 4 ml of water and 10 ml of 2.2N caustic soda were added, stirred, and the absorbance was measured at 420 nm. On the other hand, the same operation as above is performed using 5 ml of water instead of the test solution, and the absorbance derived from dinitrophenylhydrazine is measured. Now, the absorbance of the detection is A, the absorbance due to 1 μmol of keto acid is k, and μmol of α-keto acid in the test solution.
If the number is x and the absorbance of 1 μmol of dinitrophenylhydrazine is d, then A=kx+d(5.0−
x) Therefore, the number x of μmol of α-keto acid is expressed as x=(A-5d)/(k-d). Example 1 Glucose 2%, potassium nitrate 0.5%, inoculated with Trigonopsis variabilis IFO 755 strain,
Potassium phosphate 0.4%, magnesium sulfate 0.4
%, calcium chloride 0.05%, biotin 0.000002
%, Thiamine Hydrochloride 0.00001%, DL-Alanine
100 ml of a medium containing 0.3% (PH 6.0 before sterilization) was placed in a 500 ml triangular Kolben, and cultured with shaking at 27°C for 48 hours. On the other hand, glucose 2%, sucrose 2%
%, cornstarch liquor 1%, potassium nitrate
0.5%, potassium phosphate 0.4%, magnesium sulfate 0.4%, calcium chloride 0.05%, bithion
Main culture medium consisting of 0.00002%, thiamine hydrochloride 0.00001%, DL-methion 0.4% (PH6.0 before sterilization)
Pour 100ml into a 500ml Erlenmeyer flask and sterilize it.
This was inoculated with 1 ml of the above preculture solution and cultured with shaking at 27°C for 72 hours. The culture solution thus obtained was centrifuged to collect the bacteria, 1.5 ml of toluene was added to the bacteria, and the mixture was shaken at 30°C for 30 minutes. Then, 2 ml of water was added and the mixture was further shaken for 1 hour. The treated bacterial cells were collected by centrifugation and suspended in 20 ml of 0.1 mol pyrophosphate buffer (PH8.1) to prepare an enzyme solution. D-leucine was added to this enzyme solution at a concentration of 2% (400 mg) and reacted aerobically with shaking at 30°C for 3 hours, resulting in a conversion rate of 90% (mol of α-keto acid produced). number/number of moles of α-amino acid in the raw material)
The formation of α-keto-γ-methylvaleric acid was observed. Next, the bacterial cells are removed to stop the reaction, and the reaction solution is treated with a strongly acidic ion exchange resin (Diaion PK208).
(manufactured by Mitsubishi Chemical Industries, Ltd.) and unreacted D-
After adsorbing and removing leucine, decolorizing with activated carbon (purified Shirasagi, Takeda Pharmaceutical Co., Ltd.), and then decolorizing with calcium hydroxide.
The pH was adjusted to 4.5, the mixture was concentrated under reduced pressure, and the precipitated white crystals were collected by filtration. The dry weight of the crystals obtained was 260 mg.
(purity 100%), and the production yield was 81% (number of moles of α-keto acid obtained/number of moles of α-keto acid produced). Example 2 In place of D-leucine in Example 1, D-
Arginine, D-lysine, D-histidine, D-
Glutamic acid, D-aspartic acid, D-alanine, D-valine, D-isoleucine, D-serine, D-methionine, D-phenylalanine, D
The same reaction as in Example 1 was carried out using -tryptophan, and the corresponding α-keto acids were obtained as shown in the table below.

[Table] Obyric acid

[Table] Percent phenylara phenylpyruvic acid 88%
Nintryptophore indolepyruvate 90%
Example 3 A medium containing 1% peptone, 1% meat extract, and 0.5% salt (PH7 before sterilization) was placed in a 500ml triangular container.
After preparing 100ml and sterilizing it at 120℃ for 30 minutes,
Proteus vulgaris Hauser strain (IF
O.3851) was inoculated and cultured with shaking at 30°C for 24 hours. Trigonopsis variabilis IFO 755 obtained in the same manner as in Example 1 was added to the thus obtained culture solution.
Add 20 ml of activated cell suspension of the strain (equivalent to 100 ml of culture solution) and DL-leucine to a final concentration of 2% (2.4 g), and shake aerobically at 30°C for 3 hours. When the reaction is carried out, α-keto-
Formation of γ-methylvaleric acid was observed. The bacterial cells were removed to stop the reaction, and the target product was isolated from the reaction solution in the same manner as in Example 1. The dry weight of the obtained white crystals was 1.5 g (100% purity).
The production yield was 80%. Example 4 Trigonopsis obtained in the same manner as in Example 1
Activated bacterial cell suspension of IFO 755 strain 20
Add D-leucine to a concentration of 2% (400 mg), add 9000 ^u of catalase, and incubate at 30°C.
When reacted for 3 hours with aerobic shaking, α-keto-γ-methylvaleric acid was produced at a conversion rate of 95%.

Claims (1)

[Claims] 1. General formula [In the formula, R is -CH ₂ R ₁ or [Formula] (where R ₁ is -H, -OH, -COOH, -CH ₂ COOH,
[Formula] [Formula] -CH ₂ SCH ₃ , - CH ₂ CH ₂ CH ₂ NH ₂ , [Formula] [Formula] [Formula] and R ₂ is -CH ₃ , C ₂ H ₅ ) . ] The general formula R is characterized in that the D-form of the α-amino acid represented by the formula R or a salt thereof is treated with a culture of a microorganism belonging to the genus Trigonopsis, cells, or a treated product thereof in the presence of catalase if necessary. -COCOOH (In the formula, R has the same meaning as above.) A method for producing an α-keto acid represented by the formula: -COCOOH. 2 General formula [In the formula, R is -CH ₂ R ₁ or [Formula] (where R ₁ is -H, -OH, -COOH, -CH ₂ COOH,
[Formula] [Formula] -CH ₂ SCH ₃ , -CH ₂ CH ₂ CH ₂ NH ₂ , [Formula] [Formula] [Formula], and R ₂ is -CH ₃ and C ₂ H ₅ . ) is shown. ]
A culture of a microorganism belonging to the genus Trigonopsis,
Bacterial cells or their processed products and cultures of microorganisms capable of converting L-forms of α-amino acids or salts thereof into α-keto acids, microbial cells or their processed products, as necessary, in the presence of catalase. 1. A method for producing an α-keto acid represented by the general formula R-COCOOH (wherein R has the same meaning as above).