JPH0451159B2 - - Google Patents

Description

[Detailed description of the invention]

TECHNICAL FIELD The present invention relates to a biochemical production method of (+)-trans primary chrysanthemum acid. More specifically, it belongs to the genus Arthrobacter, Flavobacterium, Rhodotorula, Rhodosporidium, Nocardia or Thermomyces, and has the general formula () (In the formula, R represents a lower alkyl group.) For the (±)-primary chrysanthemum acid ester,
A feature of this method is that an esterase produced by a microorganism having an asymmetric hydrolyzing ability is applied to the ester to asymmetrically hydrolyze it and split it into (+)-trans primary chrysanthemum acid and its enantiomer ester. The present invention relates to a biochemical production method of (+)-trans primary chrysanthemum acid. Chrysanthemum acid is a carboxylic acid represented by the following formula (), and is a compound constituting the acid component of low-toxicity, fast-acting insecticidal esters collectively called pyrethroids, such as allethrin, tetramethrin, resmethrin, flamethrin, and phenothrin. Daiichichrysanthemum acid has asymmetric carbon atoms at the C ₁ and C ₃ positions, and those with an absolute configuration of R at the C ₁ position have (+) optical rotation (in a specific solvent). Therefore,
It is called (+)-primary chrysanthemum acid, and those whose absolute configuration at the C ₁ position is S are called (-)-primary chrysanthemum acid. Regarding the insecticidal efficacy of these pyrethroid esters, only (+)-phenolic acid is effective, and (-)-phenolic acid is almost ineffective. Furthermore, although it is difficult to generalize the relationship between the efficacy of cis and trans isomers depending on the target pest and the nature of the efficacy, (+)-trans pyrethroids are more effective than pyrethroid cis-(+)-cis pyrethroids. They often have superior knock-down efficacy or lethal efficacy (Kosuke Yoshioka, Organic Synthetic Chemistry, Vol. 38, No. 12, 1980). Therefore, it is very important to industrially advantageously produce (+)-phenolic acid. By the way, the currently known method for producing (+)-daisylchrysanthemum acid is mainly an organic synthetic chemical resolution method, but it requires relatively expensive optically active reagents or involves complicated steps. At present, there is a desire to develop a more economically advantageous optical resolution method in view of the requirements. The present inventors have conducted repeated research to develop a method for producing (+)-phenolic acid, which can be an economically advantageous production method, and have found that Arthrobacter, Flavobacterium, Rhodotorula, Rhodosporidium spp. An esterase produced by a microorganism belonging to the genus Nocardia or Thermomyces can act on the (±)-primary chrysanthemum acid ester represented by the general formula () to optically specifically asymmetrically hydrolyze it. This discovery led to the completion of the present invention. That is, the present invention relates to (±)-primary chrysanthemum acid esters belonging to the genus Arthrobacter, Flavobacterium, Rhodotorula, Rhodosporidium, Nocardia, or Thermomyces and represented by the general formula (). Then, an esterase produced by a microorganism capable of asymmetric hydrolysis is allowed to act on the ester, and the ester is optically specifically asymmetrically hydrolyzed into (+)-trans primary chrysanthemum acid and its enantiomer ester. The present invention provides a novel biochemical method for producing (+)-trans primary chrysanthemum acid by decomposition, which is advantageous in terms of purity. Next, the method of the present invention will be explained. The (±)-primary chrysanthemum acid ester represented by the general formula () used as a raw material of the present invention is 2,5-
Obtained by reacting dimethyl-hexa-2,4-diene with various diazoacetic esters,
Ethyl ester is the most common because of its availability. Esterases that can be used in the present invention belong to the genus Arthrobacter, Flavobacterium, Rhodotorula, Rhodosporidium, Nocardia, or Thermomyces, and are , is an esterase produced by microorganisms with asymmetric hydrolysis ability. Particularly useful microbial strains in the present invention are illustrated below. (1) Arthrobacter globifuormis
Arthrobacter globiformis IF−12958 (2) Flavobacterium esteroaromaticum IF
−3751 (3) Rhodotorula rubra
IF−0714 (4) Rhodotorula minuta var.texensis IF
−1102 (5) Rhodosporidium toruloides
Rhodosporidium toruloides IF−0871 (6) Nocardia asteroides
asteroides IF−3424 (7) Nocardia erythropolis
erythropolis IF−12682 (8) Thermomyces lanuginosus
Thermomyces lanuginosus IF−9738 All of these strains are American Type.
It is preserved at the Culture Collection (ATCC) or the Fermentation Research Institute ( IFO ) in Osaka City, and can be obtained through these institutions. The above-mentioned microorganisms are cultured in a liquid culture according to a conventional method, for example, by inoculating the microorganisms into a sterilized liquid medium.
Reciprocating shaking culture can be performed at -40°C for 1 to 8 days, and solid culture can also be performed if necessary. The composition of the medium is not particularly limited as long as it is used for the cultivation of ordinary microorganisms and can be used by the above microorganisms.For example, carbon sources and nitrogen sources include glycose, starch, dextrin, molasses, fats and oils. Soybean flour, defatted soybean flour, fatty soybean meal, cornstarch liquor, etc. can be used. Further, as inorganic salts, ammonium sulfate, dipotassium phosphate, magnesium sulfate, urea, etc. can be used. In some cases, it is also possible to add primary chrysanthemum acid ester or fatty acid ester to the medium. When carrying out the method of the present invention, the asymmetric hydrolysis reaction of the (±)-primary chrysanthemum acid ester is carried out using a culture solution in which the microorganism is cultured, a culture solution, a bacterial cell suspension, an esterase oil extract, or a concentrated solution. This is carried out by mixing the (±)-primary chrysanthemum acid ester with an aqueous solution containing an esterase-containing product or a processed product thereof, such as crude esterase or purified esterase, and stirring or shaking the mixture. If necessary, a non-ester surfactant may be added, and the enzyme may be immobilized for use. Further, the appropriate reaction temperature at this time is 10 to 65°C, and 20 to 50°C is preferable since the stability of esterase tends to decrease at high temperatures and the reaction rate is slow at too low temperatures. In addition, the PH during the reaction is PH4-9, preferably PH7
Preferably nearby. Next, after carrying out the asymmetric hydrolysis reaction in this manner, the liberated Daishu chrysanthemum acid and unreacted ester are separated and recovered. For this separation and recovery, operations such as solvent extraction, column chromatography, and fractional distillation can be appropriately employed. For example, the reaction solution is extracted with an organic solvent such as chloroform, ether, benzene, or toluene, and the extract is fractionally distilled under reduced pressure to separate and obtain free chrysanthemum acid and unreacted ester. Next, the present invention will be explained in more detail with reference to Examples, but the present invention is not limited thereto. Examples 1 to 9 Liquid medium in a 500 ml flask with a shoulder [For bacteria (Examples 1 to 5), sweetened bouillon medium (10.0 g of glucose, 5.0 g of peptone, 5.0 g of meat extract in 1 part of water,
Dissolve 3.0g of salt to adjust the pH to 7.2. ), for molds and yeasts (Examples 6 to 9), malt extract, yeast extract medium (1 part water, 5.0 g peptone, glucose
Dissolve 10.0g, malt extract 3.0g, and yeast extract 3.0g, and adjust the pH to 6.5. )] After sterilization, two loopfuls of each microorganism listed in Table 1 were inoculated from the slant culture, and cultured with reciprocating shaking at 30°C for 80 hours. Next, 1.0 g of (±)-ethyl monochrylate (cis/trans ratio; 35/65) was added, and the mixture was shaken at 30°C.
The reaction was allowed to proceed for 40 hours. After the reaction, the reaction product was extracted with ethyl ether. The extract was analyzed by gas chromatography (column: 10% FFAP, 2.1 m, 170°C), and the hydrolysis rate was calculated from the peak area ratio of primary chrysanthemum acid and primary ethyl. Concentrate the extract and perform fractional distillation under reduced pressure.
Free chrysanthemum acid and unreacted ethyl chrysanthemum acid were separated and obtained. 5 mg of the obtained free chrysanthemum acid was dissolved in 1 ml of toluene, and equimolar amounts of thionyl chloride, pyridine and (+)-2-octanol were added and reacted. Isomer analysis was performed using gas chromatography (column: 10% DCQF-1, 5.1 m, 140°C) as a diastereomer of octanol. The results are shown in Table 1 below.

【table】

[Table] Example 10 Bacteria were collected by centrifugation from 200 ml of Arthrobacter globisformis (IFO-12958) culture solution prepared in the same manner as in Example 1, and washed twice with distilled water. After, 0.1M concentration NaH ₂ PO ₄ −
It was suspended in 30 _ml of _Na2HPO4 buffer (PH7.0). This bacterial cell suspension is treated with an ultrasonic cell disruption device,
The bacterial cells were crushed and the bacterial cell fragments were separated and removed by centrifugation to obtain a crude enzyme solution. To 20 ml of this crude enzyme solution was added 0.5 g of (±)-ethyl monochrylate, and the mixture was reacted at 40° C. for 24 hours with stirring. Thereafter, separation analysis was performed in the same manner as in Example 1, and the hydrolysis rate and the isomer ratio of free chrysanthemum acid were determined. The results are shown in Table 2.

【table】

Claims (1)

[Scope of Claims] 1 Belongs to the genus Arthrobacter, Flavobacterium, Rhodotorula, Rhodosporidium, Nocardia or Thermomyces, and has the general formula (In the formula, R represents a lower alkyl group.) For the (±)-primary chrysanthemum acid ester,
The method is characterized in that an esterase produced by a microorganism having an asymmetric hydrolyzing ability is applied to the ester to asymmetrically hydrolyze it and split it into (+)-trans primary chrysanthemum acid and its enantiomer ester. (+)
-Biochemical production method of trans-daisy chrysanthemum acid.