Process for preparation of R-(-)-3-halogeno-1,2-propanediol by treatment with microorganism

Abstract: A process for preparation of R-(-)-3-halogeno-1,2-propanediol which comprises cultivating in a medium containing racemate 3-halogeno-1,2-propanediol a bacterium, which, when cultivated in a medium containing racemate 3-halogeno-1,2-propanediol as a sole carbon source, can grow and proliferate, has an ability to assimilate S-(+)-3-halogeno-1,2-propanediol preferentially compared to R-(-)-3-halogeno-1,2-propanediol and belongs to the genus Pseudomonas , ot its culture cells; and recovering R-(-)-3-halogeno-1,2-propanediol from the resulting culture broth.

Description: This invention relates to a process for separatively obtaining optically active R-(-)-3-halogeno-1,2-propanediol from racemic 3-halogeno-1,2-propanediol using a microorganism. This invention further relates to a pure culture of a bacterium belonging to the genus Pseudomonas having the ability to assimilate S-(+)-3-halogeno-1,2-propanediol preferentially compared to R-(-)-3-halogeno-1,2-propanediol. R-(-)-3-halogeno-1,2-propanediol is a useful substance as a raw material for synthesis of optically active pharmaceuticals and physiologically active substances. R-(-)-3-halogeno-1,2-propanediol can, for example, be utilized for synthesis of optically active beta-adrenergic blockers, L-carnitine and the like. Further, R-(-)-3-halogeno-1,2-propanediol can be converted to R-(+)-glycidol, a useful intermediate for synthesis of optically active pharmaceuticals and pesticides as well as physiologically active substances and Ferroelectric Liquid Crystals. As processes for preparation of this useful R-(-)-3-halogeno-1,2-propanediol the following processes have hitherto been known. The process of Hayan F. Jones to obtain it from methyl-5-chloro-5-deoxy-alpha-L-arabinofuranoside (Chemistry and Industry, 15 , P533, 1978) and the process of H. Jackson, et al. to obtain it from 1,2,5,6-di-acetonyl-D-mannitol (Chem.-Biol. Interaction, 13 , P193, 1976) are chemical synthetic processes, and, in both cases, the raw materials are hard to obtain, high synthetic technique is necessitated and the steps are complicated, and thus these processes are industrially unsuitable. On the other hand, Takahashi et al. proposed a process to obtain optically active 3-chloro-1,2-propanediol using a microorganism (EP-A-0224246 and EP-A-0286059). In this process of using a microorganism, extremely many kinds of microorganisms are proposed and several microorganisms belonging to the genus Pseudomonas are included among them. These processes mainly utilize the reaction that S-(+)-3-halogeno-1,2-propanediol is oxidatively decomposed and metabolized by action of a microorganism. However, these processes have the following problems. Although Pseudomonas fragi IFO 3458, Pseudomonas crucivuiae IFO 12047 and Pseudomonas chlororaphis IFO 3904 disclosed in the above EP-A-0224246 can metabolize S-(+)-3-halogeno-1,2-propanediol, as is the case with other microorganisms, they cannot grow and proliferate in a completely synthetic medium containing racemic 3-halogeno-1,2-propanediol as a sole carbon source and an inorganic nitrogen such as ammonium sulfate or ammonium nitrate as a nitrogen source. Therefore, when reaction is carried out utilizing any of the above microorganisms, the process must be adopted which comprises proliferating the cells in advance in a nutrient medium to obtain lots of cells and making the cells after washing act on racemic 3-halogeno-1,2-propanediol, as disclosed in the examples of the above patent. If the above process is not adopted, it becomes necessary to add racemic 3-halogeno-1,2-propanediol in a medium which contains other nutrients and wherein the microorganism can grow, and this latter process is not a preferred process in view of reaction efficiency or purification of the product. EP-A-0207636 discloses the preparation of S-(-)-2,3-dichloro-1-propanol by cultivating an R-(+)-2,3-dichloro-1-propanol-assimilating strain belonging to the genus Pseudomonas in a culture medium containing racemic 2,3-dichloro-1-propanol. Thus, the object of the invention is to provide a process capable of preparing highly pure R-(-)-3-halogeno-1,2-propanediol economically, inexpensively and technically simply, compared to the aforesaid usual processes. As a result of vigorous studies to find a microorganism which can assimilate S-(+)-3-halogeno-1,2-propanediol preferentially compared to R-(-)-3-halogeno-1,2-propanediol and can grow and proliferate when cultivated in a medium containing racemic 3-halogeno-1,2-propanediol as a sole carbon source, the present inventors succeeded in isolation from the soil of a microorganism capable of attaining the above object of the invention and accomplished the invention. Thus, the invention provides a process for the preparation of R-(-)-3-halogeno-1,2-propanediol which comprises cultivating in a medium containing racemic 3-halogeno-1,2-propanediol a bacterium which, when cultivated in a medium containing racemic 3-halogeno-1,2-propanediol as sole carbon source, can grow and proliferate, has an ability to assimilate S-(+)-3-halogeno-1,2-propanediol preferentially compared to R-(-)-3-halogeno-1,2-propanediol and belongs to the genus Pseudomonas , or its culture cells; and recovering R-(-)-3-halogeno-1,2-propanediol from the resulting culture broth. According to the process of the invention, even when mass production ofR-(-)-3-halogeno-1,2-propanediol is undertaken, there is no need to cultivate a large number of cells separately and collect the resulting cells, and it is sufficient to cultivate the bacterium in an amount enough as a seed strain and inoculate it into the medium. In assimilation of S-(+)-3-halogeno-1,2-propanediol by any of the bacteria which can be used in the invention and belong to the genus Pseudomonas , de-hydrohalogenation reaction of S-(+)-3-halogeno-1,2-propanediol is carried out and as a result hydrohalogenic acid is formed. Suitable as 3-halogeno-1,2-propanediols used in the invention are 3-chloro-1,2-propanediol and 3-bromo-1,2-propanediol. The morphological and physiological properties of the three representative bacteria, which were isolated and recovered from the soil by the inventors and can be used in the invention, are set forth below in Table 1. By classification based on the results of Table 1 according to Bergey's Manual of Systematic Bacteriology 9th edition, it was revealed that all the above strains belong to the genus Pseudomonas because they are Gram-negative aerobic rods, have polar flagella, and are oxidase-positive and catalase-positive. Although there can be mentioned as their closely related strains Pseudomonas acidoborans , Pseudomonas testosteroni , Pseudomonas delafieldii , Pseudomonas facilis , Pseudomonas saccharophila , Pseudomonas flava , Pseudomonas pseudoflava and Pseudomonas palleronii , the above strains differs from Pseudomonas acidoborans and Pseudomonas testosteroni in that all the former strains can utilize D-glucose as a carbon source whereas the latter strains cannot utilize D-glucose as a carbon source, from Pseudomonas delafieldii , Pseudomonas facilis , Pseudomonas saccharophila and Pseudomonas flava in that all the former strains can utilize p-hydroxybenzoate as a carbon source whereas the latter strains cannot utilize p-hydroxybenzoate as a carbon source, and from Pseudomonas pseudoflava and Pseudomonas palleronii in that all the former strains form no carotenoid pigment whereas the latter strains form carotenoid pigments. Thus, all the above strains do not accord with the known strains in characteristics, and were believed to be novel strains and named Pseudomonas sp. DS-K-2D1, Pseudomonas sp. DS-K-9D1 and Pseudomonas sp. DS-K-14A4. Further, although these strains are mutually analogous strains each belonging to the genus Pseudomonas , Pseudomonas sp. DS-K-2D1 and Pseudomonas sp. DS-K-14A4 differ from Pseudomonas sp. DS-K-9D1 in that the former strains reduce litmus milk to white whereas the later strain does not reduce litmus milk to white, and Pseudomonas sp. DS-K-2D1 differs from Pseudomonas sp. DS-K-14A4 in that, in the growth condition in the MGPB agar, the raised condition of colonies is convex at the center in the former whereas the raised condition of colonies is convex in the latter. Thus, the above strains were found as three mutually different strains. The above three strains were deposited with Fermentation Research Institute, Agency of Industrial Science and Technology, Japan on November 15, 1989, and, thereafter, their control was transferred in the Fermentation Research Institute on September 12, 1990 in accordance with Budapest Treaty on the international recognition of the deposit of microorganism for the purpose of patent procedure. Deposit numbers of the respective strains are set forth below. Pseudomonas sp. DS-K-2D1 Pseudomonas sp. DS-K-9D1 Pseudomonas sp. DS-K-14A4 FRI (Nov. 15, 1989) FERM P-11109 FERM P-11108 FERM P-11110 FRI under Budapest Treaty (Sep. 12, 1990) FERM BP-3096 FERM BP-3095 FERM BP-3097 Strains which can be used in the invention and belong to the genus Pseudomonas are not limited to the above three strains. Namely, other strains can similarly be used so long as they have an ability to assimilate S-(+)-3-halogeno-1,2-propanediol preferentially compared to R-(-)-3-halogeno-1,2-propanediol and can grow and proliferate in a medium containing racemate 3-halogeno-1,2-propanediol as a sole carbon source. Specifically, the process of the invention can be carried out either by cultivating any of the above bacteria in a synthetic medium containing racemic 3-halogeno-1,2-propanediol as a substantially sole carbon source, inorganic nitrogens (various ammonium salts or nitrates) as nitrogen sources and further inorganic salts, and recovering the remaining R-(-)-3-halogeno-1,2-propanediol from the culture broth, or by cultivating any one of the above bacteria in a medium containing carbon sources, nitrogen sources, organic nutrient sources and inorganic sources and usually frequently used, for example, a bouillon medium or a peptone medium, inoculating the thus obtained culture broth or culture cells into a medium containing racemate 3-halogeno-1,2-propanediol as a sole carbon source, further carrying out cultivation or making the cells act, and then recovering the remaining R-(-)-3-halogeno-1,2-propanediol from the culture broth. As carbon sources, there can be used, besides racemate 3-halogeno-1,2-propanediol, alcohols such as glycerol, saccharides such as D-glucose, D-fructose and D-galactose, and organic acids such as citric acid, maleic acid, malic acid and fumaric acid and their salts. It is advantageous in the process of the present invention to carry out cultivation in a medium containing racemic 3-halogeno-1,2-propranediol as substantially the only carbon source. Further, as nitrogen sources there can be used those usually used in cultivation of cells. Examples thereof include inorganic nitrogens such as ammonium sulfate, ammonium nitrate and ammonium phosphate, and organic nitrogens such as urea, peptone, casein, yeast extract, meat extract and corn steep liquor. There can further be used generally known inorganic salts such as phosphates, magnesium salts, potassium salts, manganese salts, zinc salts and copper salts. Cultivation of the invention can be carried out by conventional conditions and means. Namely, the cultivation is typically carried out at a cultivation temperature of about 20 to 40°C, preferably 25 to 37°C, Typically the pH is about 5 to 10, preferably 5.5 to 9.5. cultivation is generally carried out under aerobic conditions, for example using a means of shaking cultivation or aeration stirring cultivation or the like. The substrate concentration in the reaction mixture is preferably of the order of 0.1 to 10% (v/v), and although the reaction time varies depending on the substrate concentration and other reaction conditions, it is preferably 48 to 80 hours. It is preferred that the reaction is discontinued at the time when the remaining substrate analysed by gas chromatography or the like has been reduced by up to 50%, namely at the time when S-(+)-3-halogeno-1,2-propanediol was substantially assimilated. Recovery and purification of the remaining R-(-)-3-halogeno-1,2-propanediol can be carried out as follows. Namely, the purification and recovery can be carried out by separating the culture broth after completion of the cultivation into the microbial cells and the supernatant by centrifugation, a flocculant agent or the like, adsorbing on active carbon R-(-)-3-halogeno-1,2-propanediol remaining in the supernatant, and then either eluting it with acetone and then subjecting the eluate to vacuum distillation, or extracting it with a solvent such as ethyl acetate and then subjecting the extract to vacuum distillation. Strains belonging to the genus Pseudomonas which can grow and proliferate in a medium containing racemic 3-halogeno-1,2-propanediol as sole carbon source and which have an ability to assimilate S-(+)-3-halogeno-1,2-propanediol preferentially compared to R-(-)-3-halogeno-1,2,-propanediol are novel. Pure cultures of such strains are also provided by the invention. As these strains, there can particularly preferably be used those named Pseudomonas sp. DS-K-9D1, Pseudomonas sp. DS-K-2D1 and Pseudomonas sp. DS-K-14A4. Example The invention is specifically described below by examples. % in the examples indicates % by weight unless otherwise noted. Example 1 100 ml of a medium having the composition of ammonium sulfate 0.5 % disodium hydrogenphosphate 0.1 % dipotassium hydrogenphosphate 0.1 % sodium dihydrogenphosphate 0.2 % magnesium sulfate 0.05 % iron sulfate, copper sulfate trace and manganese sulfate calcium carbonate 0.45 % pH 6.8 poured in a 500-ml Sakaguchi flask was sterilized at 121°C for 15 minutes, and then racemic 3-chloro-1,2-propanediol was added thereto to 1.0 % (v/v) to prepare a medium containing racemic 3-chloro-1,2-propanediol as a sole carbon source. One platinum loopful of a slant agar culture of Pseudomonas sp. DS-K-2D1, one of the microorganisms shown in Table 1, was inoculated into the above medium and cultivated with shaking for 4 days under the conditions of 30°C and 130 rpm. After completion of the cultivation, the culture broth was taken out and subjected to centrifugation to remove the cells and obtain the supernatant. This supernatant was concentrated up to about 20 ml and the concentrate was extracted with ethyl acetate. The extract was dehydrated with anhydrous magnesium sulfate and kept under reduced pressure to obtain 0.4 g of 3-chloro-1,2-propanediol as an oily substance. Identification of this substance was carried out by gas chromatography. When the substance was compared with commercially available racemic 3-chloro-1,2-propanediol (product of Tokyo Kasei Co.) using a column carrier PEG-20MP, 60-80 mesh, the retention times of both substances were utterly identical. The specific rotation of the present substance and the specific rotation of (R)-3-chloro-1,2-propanediol (literature value) are as follows. The present substance [α] 22 D = -7.75° (C=1, H₂O) Literature value [α] 22 D = -6.9° (C=2, H₂O) Further, after the present substance was tosylated according to a conventional method, HPLC analysis was carried out under the conditions of room temperature, a flow rate of 1.0 ml/min and a wavelength of 235 nm using an optical isomer-separating column [CHIRALCEL OC column (25 cm x 0.46 cm I.D.)] (produced by DAICEL CHEMICAL INDUSTRIES, LTD.) and hexane-isopropanol (95:5) as a solvent. Retention times by this analytic method are 79 minutes for the S form compound and 89.8 minutes for the R form compound, and the present substance exhibited a retention time corresponding to that of the R form compound and its enantiomer excess was 98 % e.e. or more. Examples 2 and 3 The same operations as in Example 1 were carried out except that the strain was replaced by Pseudomonas sp. DS-K-9D1 or Pseudomonas sp. DS-K-14A4. When analyzed according to the various analytical methods as shown in Example 1, each of the obtained substances was revealed to be R-(-)-3-chloro-1,2-propanediol having an enantiomer excess of 98 % e.e. or more. The results of Examples 2 and 3 are shown together below. Example No. Strain [α] 22 / D (C=1, H₂O) Yield (g) 2 DS-K-9D1 -7.70° 0.38 3 DS-K-14A4 -7.71° 0.45 Example 4 2.5 l of the medium having the composition of ammonium sulfate 0.5 % disodium hydrogenphosphate 0.02 % dipotassium hydrogenphosphate 0.02 % sodium dihydrogenphosphate 0.04 % magnesium sulfate 0.05 % iron sulfate, copper sulfate trace and manganese sulfate pH 6.8 poured in a 5-liter cultivation apparatus (jar fermentor) was sterilized at 121°C for 15 minutes, and then racemic 3-chloro-1,2-propanediol was added thereto to 1.0 % (v/v) to prepare a medium containing racemic 3-chloro-1,2-propanediol as a sole carbon source. Then, Pseudomonas sp. DS-K-2D1, one of the microorganisms indicated in Table 1, was previously cultivated with shaking at 30°C for 24 hours in a nutrient medium comprising 1.0 % of peptone, 1.0 % of yeast extract and 1.0 % of glycerol, pH7.2, and the culture broth was aseptically inoculated into the above medium containing racemic 3-chloro-1,2-propanediol as a sole carbon source so that its amount became 2 % (v/v). Aerobic stirring cultivation was carried out for 4 days under the following conditions: Temperature 30°C Aeration amount 0.5 l/min agitation 500 rpm Measurement and control of pH were carried out by a linked pH meter and the pH was controlled at 6.8 with 3N-NaOH. After completion of the cultivation, the culture broth was taken out and subjected to centrifugation to remove the cells and obtain the supernatant. The supernatant was treated in the same manner as in Example 1 to obtain 14.9 g of 3-chloro-1,2-propanediol. The obtained substance was subjected to the various analyses as shown in Example 1, and as a result it was revealed that the substance was R-(-)-3-chloro-1,2-propanediol having an enantiomer excess of 98 % e.e. or more. The specific rotation [α] 22 / D of this substance was -7.75° (C=1, H₂O). Examples 5 and 6 The same operations as in Example 4 were carried out except that the strain was replaced by Pseudomonas sp. DS-K-9D1 or Pseudomonas sp. DS-K-14A4 shown in Table 1. By the various analyses shown in Example 1, each of the obtained substances was revealed to be R-(-)-3-chloro-1,2-propanediol having an enantiomer excess of 98 % e.e. or more. The results of Examples 5 and 6 are shown below in a lump. Example No. Strain [α] 22 / D (C=1, H₂O) Yield (g) 5 DS-K-9D1 -7.74° 15.0 6 DS-K-14A4 -7.70° 13.5 Example 7 100 ml of a nutrient medium having the composition of 1.0 % of peptone, 1.0 % of yeast extract and 1.0 % of glycerol, pH7.2 in a 500-ml Sakaguchi flask was sterilized at 121°C for 15 minutes, and then one platinum loopful of a slant agar culture of Pseudomonas sp. DS-K-2D1 shown in Table 1 was inoculated thereinto. Cultivation was carried out at 30°C for 24 hours with shaking, the cells and the supernatant were separated by centrifugation and the supernatant was discarded. The resulting cells were washed a few times with a 50 mM phosphate buffer, pH7.0. The cells were then suspended in 100 ml of the medium containing racemic 3-chloro-1,2-propanediol as a sole carbon source shown in Example 1, and reaction was carried out at 30°C and 130 rpm for 3 days. After completion of the reaction, the cells were removed by centrifugation to obtain the supernatant. The supernatant was treated in the same manner as in Example 1 to obtain 0.4 g of 3-chloro-1,2-propanediol. The substance was subjected to the various analyses as shown in Example 1, and as a result it was revealed that the substance was R-(-)-3-chloro-1,2-propanediol having an enantiomer excess of 98 % e.e. or more. The specific rotation [α] 22 / D the substance was -7.75° (C=1, H₂O). Examples 8 and 9 The same operations as in Example 7 were carried out except that the strain was replaced by Pseudomonas sp. DS-K-9D1 or Pseudomonas sp. DS-K-14A4 shown in Table 1. By the various analyses as shown in Example 1, each of the obtained substances was revealed to be R-(-)-3-chloro-1,2-propanediol having an enantiomer excess of 98 % e.e. or more. The results of Examples 8 and 9 are shown below in a lump. Example No. Strain [α] 22 / D (C=1, H₂O) Yield (g) 8 DS-K-9D1 -7.74° 0.51 9 DS-K-14A4 -7.70° 0.45 Examples 10 to 12 The same experiments as in Examples 1 to 3 were carried out except that racemic 3-chloro-1,2-propanediol was replaced by racemic 3-bromo-1,2-propanediol. Experimental methods and other operations were carried out according to Example 1. The results of Examples 10 to 12 are shown below in a lump. Example No. Strain [α] 22 / D (C=1, H₂O) Yield (g) 10 DS-K-2D1 -3.80° 0.61 11 DS-K-9D1 -3.75° 0.65 12 DS-K-14A4 -3.77° 0.55 In this connection, the literature value of the specific rotation of (R)-3-bromo-1,2-propanediol is as follows: [α] 25 D = -3.94° (C=5.07, CHCl 3 ). Further, each of R-(-)-3-bromo-1,2-propanediols obtained by Examples 10 to 12 was tosylated by a conventional manner, and the tosylated compound was subjected to HPLC analysis under the conditions of room temperature, a flow rate of 1.0 ml/min and a wavelength of 235 nm using an optical isomer-separating column [CHIRALCEL, OC column (25 cm x 0.46 cm I.D.)] (produced by DAICEL CHEMICAL INDUSTRIES, LTD.) and hexane-isopropanol (95:5) as a solvent. Retention times in this analysis are 98.9 minutes for the S-form compound and 115.4 minutes for the R-form compounds, and each of the above substances exhibited a retention time corresponding to the R-form compound and had an enantiomer excess of 96 % e.e. or more. Examples 13 to 15 Experiments were carried out in accordance with Examples 4 to 6 except that racemic 3-chloro-1,2-propanediol was replaced by racemic 3-bromo-1,2-propanediol. Experimental methods and other operations were carried out according to Example 4. By the various analyses as shown in Example 10, each of the obtained substances was revealed to be R-(-)-3-bromo-1,2-propanediol having an enantiomer excess of 96 % e.e. or more. The results of Examples 13 to 15 are shown below in a lump. Example No. Strain [α] 22 / D (C=1, H₂O) Yield (g) 13 DS-K-2D1 3.77° 17.1 14 DS-K-9D1 -3.74° 15.5 15 DS-K-14A4 -3.78° 16.8 Examples 16 to 18 Experiments were carried out in accordance with Examples 7 to 9 except that racemic 3-chloro-1,2-propanediol was replaced by racemic 3-bromo-1,2-propanediol. Experimental methods and other operations were carried out according to Example 7. By the various analyses as shown in Example 10, each of the obtained substances was revealed to be R-(-)-3-bromo-1,2-propanediol having an enantiomer excess of 96 % e.e. or more. The results of Examples 16 to 18 are shown below in a lump. Example No. Strain [α] 22 / D (C=1, H₂O) Yield (g) 16 DS-K-2D1 -3.77° 0.62 17 DS-K-9D1 -3.78° 0.59 18 DS-K-14A4 -3.74° 0.66 In accordance with the invention, it is possible to prepare R-(-)-3-halogeno-1,2-propanediol by a method wherein inexpensive raw materials can be utilized and which is industrially convenient, namely by assimilating S-(+)-3-halogeno-1,2-propanediol preferentially from racemic 3-halogeno-1,2-propanediol utilizing a bacterium of the genus Pseudomonas .

Claims: A process for the preparation of R-(-)-3-halogeno-1,2-propanediol which comprises cultivating in a medium containing racemic 3-halogeno-1,2-propanediol a bacterium which, when cultivated in a medium containing racemic 3-halogeno-1,2-propanediol as sole carbon source, can grow and proliferate, has an ability to assimilate S(+)-3-halogeno-1,2-propanediol preferentially compared to R-(-)-3-halogeno-1,2-propanediol and belongs to the genus Pseudomonas , or its culture cells; and recovering R-(-)-3-halogeno-1,2-propanediol from the resulting culture broth. A process according to claim 1 wherein the bacterium is Pseudomonas sp. DS-K-9D1, Pseudomonas sp. DS-K-2D1 or Pseudomonas sp. DS-K-14A4, deposited with Fermentation Research Institute, Agency of Industrial Science and Technology, Japan as Deposit Nos. FERM BP-3095, FERM BP-3096 and FERM BP-3097, respectively. A process according to claim 1 or 2 wherein the 3-halogeno-1,2-propanediol is 3-chloro-1,2-propanediol or 3-bromo-1,2-propanediol. A process according to claim 1, 2 or 3 wherein the cultivation is carried out under aerobic conditions. A process according to any one of the preceding claims wherein the cultivation is carried out at a temperature of 20°C to 40°C. A process according to any one of the preceding claims wherein the cultivation is carried out at a pH of 5 to 10. A process according to any one of the preceding claims wherein the cultivation is carried out in a medium containing racemic 3-halogeno-1,2-propanediol as substantially the only carbon source. A pure culture of a strain belonging to the genus Pseudomonas which can grow and proliferate in a medium containing racemic 3-halogeno-1,2-propanediol as sole carbon source and which has an ability to assimilate S-(+)-3-halogeno-1,2-propanediol preferentially compared to R-(-)-3-halogeno-1,2-propanediol. A pure culture according to claim 8 wherein the strain is Pseudomonas sp. DS-K-9D1 (FERM BP-3095), Pseudomonas sp. DS-K-2D1 (FERM BP-3096) or Pseudomonas sp. DS-K-14A4 (FERM BP-3097).

Inventors: KASAI NAOYA, SUZUKI TOSHIO, KASAI, NAOYA, SUZUKI, TOSHIO
Applicants: DAISO CO LTD, DAISO CO., LTD.
Application year: 1990
Main classification: C12P41
IPC codes: C12P41, C12R1, C12P7