JPH04234991A - New microorganism - Google Patents

Abstract

PURPOSE:To provide a method for biologically producing (+)-chrysanthemumic acid ester or (+)-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid by the use of a microorganism. CONSTITUTION:Arthrobacter SC-6-98-28 strain (FERM P-11851) having a high esterase activity and obtained by treating Arthrobacter.globiformis IFO-12958 strain with NTG. The microorganism hydrolyzes racemic chrysanthemumic acid or racemic 3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid ester into the (+)-chrysanthemumic acid or the (+)-3-(2,2-dichlorovinyl)-2,2- dimethylcyclopropanecarboxylic acid.

Description

Detailed Description of the Invention [0001] [Industrial Application Field] The present invention relates to Arthrobacter (Arthrobacter
Arthrobacter strain SC-6-98-28, which is a microorganism belonging to the genus
+)-Chrysanthemum acid or (+)-3-(2,2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid (
The present invention relates to a biochemical production method of DV acid (hereinafter abbreviated as DV acid). More specifically, Arthrobacter globiformis (Arthrobacter globiformis)
ormis) IFO-12958 strain to N-methyl-N'
- Microorganism Arthrobacter strain SC-6-98-28 obtained by mutagenesis treatment with nitro-N-nitrosoguanidine (hereinafter abbreviated as NTG) and (+)-chrysanthemum acid obtained using the microorganism. Or it relates to a biochemical production method of (+)-DV acid. [Prior Art] Chrysanthemum acid represented by the general formula 3 [Chemical 3] (In the formula, X represents a methyl group or a chlorine atom. This formula does not represent a steric relationship.) Or (+)-DV acid is a compound constituting the acid component of low toxicity, fast-acting insecticidal ester compounds collectively called pyrethroids, such as allethrin, tetramethrin, resmethrin, flamethrin, phenothrin, and permethrin. Chrysanthemum acid and DV acid exist in two types of isomers called (+) and (-) isomers based on the asymmetric carbon at the C1 position. Regarding insecticidal efficacy as a pyrethroid, it is known that the (+) form is effective and the (-) form is almost ineffective (Kosuke Yoshioka, Synthetic Organic Chemistry, 3rd Edition).
Volume 8, No. 12, 1980). Therefore, it is very important to industrially advantageously produce (+)-chrysanthemum acid or (+)-DV acid. [0005] The method of producing (+)-chrysanthemum acid or (+)-DV acid using an organic synthetic chemical resolution method requires expensive optically active reagents or complicated steps. It has its drawbacks. On the other hand, the esterase produced by microorganisms is expressed by the general formula 4: ), which is a compound represented by racemic chrysanthemum acid ester or racemic DV acid ester, is stereoselectively asymmetrically hydrolyzed to produce (+)-chrysanthemum acid (or +)-DV acid)
and its enantiomer ester (+
)-Chrysanthemum acid or (+)-DV acid biochemical production methods are known. This biochemical production method is superior to synthetic organic chemical production methods in that it does not require expensive reagents or complicated steps. [0007] The present inventors have already identified Arthrobacter globiformis IFO-12958.
The stock is represented by the general formula 5 [Chemical formula 5] (wherein, R represents a C1 to C4 alkyl group, and X represents a methyl group or a chlorine atom. This formula does not represent a steric relationship.) By stereoselectively asymmetrically hydrolyzing racemic chrysanthemum acid ester or racemic DV acid ester shown by
+)-Chrysanthemum acid or (+)-DV acid is produced,
It has been found that this microorganism is extremely useful in the biochemical production method of (+)-chrysanthemum acid or (+)-DV acid. (JP-A-59-210892, JP-A-63-2510
(No. 99) [0008] However, the hydrolytic activity per amount of bacterial cells is
It is not necessarily high enough, and there are problems such as a large amount of bacterial cells being required for industrial use of the asymmetric hydrolysis reaction. Means for Solving the Problems The present inventors have discovered that Arthrobacter globiformis (Arthrobacter globiformis)
We have been conducting intensive studies to create a microorganism with high racemic chrysanthemum acid ester hydrolysis activity per cell mass by mutagenesis of the IFO-12958 strain (E. r globif-ormis). As a result, Arthrobacter globiformis (
Arthrobacter glob-iformis
) After mutagenesis of IFO-12958 strain by NTG treatment, the hydrolysis activity of racemic chrysanthemum acid ethyl ester per bacterial mass was determined for the mutant strain grown on agar medium containing racemic chrysanthemum acid ethyl ester. We made sincere efforts to obtain microorganisms with high quality, and obtained the microorganism Arthrobacter SC-6-98-28 strain of the present invention. Microorganism Arthrobacter SC-6 of the present invention
-98-28 strain was submitted to the Institute of Microbial Technology, Agency of Industrial Science and Technology, as part of the Microbiological Research Institute No. 11851 (FERM P-11851).
) has been deposited as. The present invention will be explained in more detail below. In the present invention, Arthrobacter globiformis I
Mutagenesis treatment of the FO-12958 strain by NTG treatment was performed according to a conventional method. That is, Arthrobacter globiformis IFO-12958 strain is cultured using a known liquid medium, and when it reaches the logarithmic growth phase, bacterial cells are separated and obtained by centrifugation. After washing the cells with an appropriate buffer, they are treated with an aqueous NTG solution. Thereafter, the bacterial cells are separated by centrifugation, washed with an appropriate buffer, and inoculated onto an agar medium in which racemic chrysanthemum acid ethyl ester is suspended. The microbial cells were cultured, and the microbial strains that grew and formed colonies were harvested and again applied and inoculated onto an agar medium in which racemic chrysanthemum acid ethyl ester was suspended. The methyl butyrate hydrolysis activity of the microorganisms that grew and formed colonies here was measured, and the activity was determined to be similar to that of Arthrobacter globiformis (Arthrobacter globiformis).
rmis) that is higher than the IFO-12958 strain. The racemic ethyl chrysanthemum acid hydrolysis activity of the selected microorganisms was measured, and the microorganism with the highest activity was selected to obtain Arthrobacter strain SC-6-98-28. The bacteriological properties of Arthrobacter SC-6-98-28 strain are Arthrobacter globiformis IF.
Although it is exactly the same as the O-12958 strain, the chrysanthemum acid ethyl ester hydrolysis activity per bacterial cell amount is more than three times higher than that of the Arthrobacter globiformis IFO-12958 strain. In addition, Arthrobacter SC-6
-98-28 strain acts on racemic chrysanthemum acid ester (or racemic DV acid ester) and selectively hydrolyzes the (+) form of the ester, resulting in highly optically pure (+)-chrysanthemum acid or (+) )-DV acid is produced. Arthrobacter globiformis IF
O-12958 strain and Arthrobacter SC-6-98
-28 strain is cultured in liquid culture according to a conventional method, for example, by inoculating microorganisms into a sterilized liquid medium, and usually at 20 to 40°C for 1 to 30 minutes.
Shaking culture can be performed for 8 days. Moreover, solid culture can also be performed if necessary. The composition of the medium is not particularly limited as it is used for normal microbial culture; for example, glucose,
Starch, dextrin, molasses, fats and oils, soy flour, corn steep liquor
) etc. can be used. Moreover, ammonium sulfate, dipotassium phosphate, magnesium sulfate, urea, etc. can be used as an airless salt. In some cases, chrysanthemum acid ester, DV acid ester, or fatty acid ester can also be added to the medium. Using the microorganism of the invention, the general formula 6: (wherein, R represents a C1 to C4 alkyl group and X represents a methyl group or a chlorine atom. (This does not represent a relationship.) When carrying out the asymmetric hydrolysis reaction of racemic chrysanthemum acid ester (or racemic DV acid ester), which is a compound represented by
Esterase-containing materials such as culture broth of microorganisms, bacterial cell suspensions, esterase extracts or concentrates thereof, or processed products thereof, such as crude esterases, purified esterases, and aqueous solutions containing the general formula 7 7] (In the formula, R represents a C1 to C4 alkyl group, and X represents a methyl group or a chlorine atom. This formula does not represent a steric relationship.) This is carried out by mixing racemic chrysanthemum acid esters or racemic DV acid esters shown in and stirring or shaking the mixture. [0014] If necessary, it is also possible to use immobilized microorganisms or esterases. A suitable reaction temperature is 20 to 70°C, preferably 30 to 60°C.
It is. The pH during the reaction is pH 4 to 11, preferably p
H7-10. Next, after performing the asymmetric hydrolysis reaction in this manner, the produced carboxylic acid and unreacted ester are separated and recovered. During this separation and recovery, solvent extraction,
Operations such as column chromatography and fractional distillation can be employed as appropriate. For example, the reaction solution is extracted with an organic solvent such as methyl isobutyl ketone, ether, or toluene, and the extract is fractionally distilled under reduced pressure to separate the produced chrysanthemum acid or DV acid and the unreacted ester. In addition,
The unreacted ester is processed by a method such as racemization to convert it into a raw material of general formula 8 [Formula 8] (wherein, R represents a C1 to C4 alkyl group, and X represents a methyl group or a chlorine atom. This formula does not represent a steric relationship.) This leads to racemic chrysanthemum acid ester (or racemic DV acid ester) shown by [Examples] Next, the present invention will be explained in detail with reference to Examples and Reference Examples, but the present invention is not limited to these Examples. Example 1 Arthrobacter globiformis (Arthrob
Acter globiformis) IFO-129
58 strains were treated with soluble starch 1. 5%, polypeptone 0. 7
%, yeast extract 0. 5% and dipotassium phosphate 0. Culture was performed at 30°C using a liquid medium containing 5%. When the logarithmic growth phase was reached, 4 ml of the culture solution was collected, and the bacterial cells were separated and obtained by centrifugation. Add 5 ml of this bacterial body to 0.0 mL. 1M
After washing with succinic acid-NaOH buffer (pH 5.5), 0. The sample was treated with 0.04% NTG aqueous solution for 2 hours. The bacterial cells isolated by centrifugation were washed with 5 ml of phosphate buffer (pH 7.0), 2% agar was added to the medium with the above composition, and racemic chrysanthemum acid ethyl ester at a concentration of 10 mM was suspended. The cells were inoculated onto an agar medium. The microorganism strain that had grown and formed a colony after being cultured at 30°C was harvested, and again applied and inoculated onto an agar medium in which racemic chrysanthemum acid ethyl ester was suspended. The methyl butyrate hydrolysis activity of the microorganisms that grew and formed colonies here was measured, and the activity was determined to be similar to that of Arthrobacter globiformis (Arthrobacter globiformis).
globiformis) IFO-12958 strain was selected. The racemic ethyl chrysanthemum acid hydrolysis activity of the microorganisms selected here was measured, and the microorganism with the highest activity was selected.
6-98-28 strain was obtained. Example 2 In a 500ml Erlenmeyer flask, 100ml of liquid medium (30g of soluble starch, 7g of polypeptone, 5g of yeast extract, and 5g of dipotassium phosphate are dissolved in 1L of water, and adjusted to pH 5.0).
After sterilization, one platinum loopful was inoculated from a slope culture of SC-6-98-28 strain, and cultured with rotational shaking at 30° C. for 24 hours. 2. A sterilized liquid medium (60 g of soluble starch, 10 g of polypeptone, 2 g of yeast extract in 1 L of water,
g and 5 g of dipotassium phosphate to adjust the pH to 5.0. ), and 10 ml of the culture solution cultured in the Erlenmeyer flask described above was inoculated thereto. When cultured for 43 hours with aeration at 3 L/min and stirring at 750 rpm, the turbidity (OD660) of the culture solution was 19. It was 3. Bacteria were isolated from 100 ml of the culture solution obtained here by centrifugation. 25 m of the obtained bacterial cells
l's 0. Suspended in 5M carbonate buffer (pH 10), racemic primary chrysanthemum acid ethyl ester (trans form/cis form = 90
/10)0. Add 5g and stir at 40°C for 8 hours.
Made it react. After the reaction, the reaction solution was adjusted to pH 2.0 or less with concentrated hydrochloric acid, and then extracted with methyl isobutyl ketone. An internal standard substance (dimethyl phthalate) was added to the extract, and the mixture was subjected to gas chromatography (column: 10% Shinchr).
om F51 + 1% phosphoric acid, 2.1 m, 180 °C)
The molar amount of chrysanthemum acid produced was measured, and the hydrolysis activity of chrysanthemum acid ethyl ester per amount of bacterial cells was determined using the following formula.9. It was 5 μmol/hr/ml/OD. Hydrolysis activity was calculated according to the following formula. Hydrolytic activity (μmol/hr/ml
/OD) = Amount of chrysanthemum produced (μmol) ÷ Reaction time (hr) ÷ Amount of culture solution used for reaction (ml) ÷ Turbidity of culture solution (OD660) Example 3 Arthrobacter SC- 6-98-28 strain was cultured for 29 hours using a small fermenter in the same manner as in Example 2 (turbidity (OD660) of culture solution = 18.1).
By centrifuging 300 ml of the culture solution, the hydrolysis activity of racemic chrysanthemum acid ethyl ester per amount of bacterial cells was 10.5.
Microbial cells of μmol/hr/ml/OD were obtained. The obtained bacterial cells were added to 25 ml of 0.5 M carbonate buffer (pH 10).
), racemic chrysanthemum acid ethyl ester (trans form/
0.45 g (2.3 mmol) of cis isomer=95/5 was added thereto, and the mixture was stirred at 40° C. for 46 hours to react. Thereafter, the same operation as in Example 1 was performed, and the amount of chrysanthemum acid produced was 0.95 mmol. The chrysanthemum acid obtained here was induced into an amide compound with 3,5-dichloroaniline, and then subjected to liquid chromatography using an optically active column [Column: SUMICHIRAL OA-2100]
(Sumika Chemical Analysis Center), solvent: hexane/1,2-dichloroethane = 17/3], the stereoisomer type ratio was analyzed, and the ratio of (+) form/(-) form was 99.8/0.
It was 2. In addition, all of them were trans-isomers, and no cis-isomers were included. Example 4 The bacterial cells isolated from 400 ml of the culture solution obtained in Example 3 were suspended in 25 ml of 1M carbonate buffer (pH 10), and racemic DV acid ethyl ester (trans form/cis form = 55/45) was added, and the mixture was stirred at 50° C. for 50 hours to react. Thereafter, extraction was carried out in the same manner as in Example 1, and the stereoisomer contrast of the produced DV acid was measured. As a result, the ratio of (+) form/(-) form of the produced DV acid was 99.6/0.4, and the ratio of trans form/cis form was 95.8/4.2. Reference Example 1 Arthrobacter globiformis IFO-1295
Eight strains were cultured in the same manner as in Example 2, and the turbidity (OD66
0) A culture solution of 16.0 was obtained. Thereafter, in the same manner as in Example 2, the hydrolysis activity of primary chrysanthemum acid ethyl ester per amount of bacterial cells was determined to be 3.2 μmol/hr/ml.
/OD.

Claims (2)

[Claims] Claim 1: Arthrobacter SC-, a microorganism belonging to the genus Arthrobacter;
6-98-28 strain (FERM P-11851), its culture, microbial cells, or esterase extracted from microbial cells, was prepared using the general formula 1 [Formula 1] (wherein, R is a C1 to C4 alkyl group) and X represents a methyl group or a chlorine atom. This formula does not represent a steric relationship.) Racemic chrysanthemum acid ester or racemic 3-(2,
2-dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid ester) to asymmetrically hydrolyze it. Alternatively, it represents a chlorine atom. This formula does not represent a steric relationship.) (+)-Chrysanthemum acid or (+)-3-(2,2-
Biochemical production method of dichlorovinyl)-2,2-dimethylcyclopropanecarboxylic acid Claim 2: Arthrobacter SC-, which is a microorganism belonging to the genus Arthrobacter.
6-98-28 strain (FERM P-11851)