Process for Producing Malonic Acid Derivatives

Home , Cyanoacetic acid, Ethyl cyanoacetate

Europäisches Patentamt

(19) European Patent Ofﬁce Ofﬁce européen des brevets (11) EP 1 008 655 A1

(12) EUROPEAN PATENT APPLICATION published in accordance with Art. 158(3) EPC

(43) Date of publication: (51) Int. Cl.7: C12P 7/62 14.06.2000 Bulletin 2000/24 (86) International application number: (21) Application number: 98904405.2 PCT/JP98/00711

(22) Date of ﬁling: 20.02.1998 (87) International publication number: WO 98/37219 (27.08.1998 Gazette 1998/34)

(84) Designated Contracting States: • ENOMOTO, Kanehiko CH DE ES FR GB IT LI Otake-shi, Hiroshima 739-060606 (JP) • ENDO, Takakazu, (30) Priority: 20.02.1997 JP 3650897 Mitsubishi Rayon Co Ltd 20.02.1997 JP 3651097 Yokohama-shi, Kanagawa 230-0053 (JP)

(71) Applicant: (74) Representative: HOFFMANN - EITLE Mitsubishi Rayon Co., Ltd. Patent- und Rechtsanwälte Tokyo 108-8506 (JP) Arabellastrasse 4 81925 München (DE) (72) Inventors: • OZAKI, Eiji , Otake-shi, Hiroshima 739-0606 (JP)

(54) PROCESS FOR PRODUCING MALONIC ACID DERIVATIVES

(57) A process for producing malonic monoesters represented by the following general formula (II): HOOCCH2COOR, wherein R represents alkenyl, aryl, aralkyl or C1-20 alkyl, characterized by hydrolyzing a cyanoacetate represented by the following general formula (I): NCCH2COOR, wherein R is as deﬁned above, by treating with a culture of a microorganism belonging to the genus Corynebacterium, Gordona or Rhodococcus and having a nitrilase activity or option- ally treated cells of the same. EP 1 008 655 A1 Printed by Xerox (UK) Business Services 2.16.7 (HRS)/3.6 12EP 1 008 655 A1

Description HOOCCH2COOR (II)

TECHNICAL FIELD wherein R is alkenyl, aryl, aralkyl or C1-20 alkyl, comprising treating a cyanoacetic acid ester [0001] The present invention relates to a method for 5represented by Formula (I): preparing malonic acid monoesters which are useful as intermediates in the synthesis of various chemical prod- NCCH2COOR (I) ucts, medicines, agricultural chemicals and so on. wherein R is as defined in Formula (II), with BACKGROUND ART 10 a culture, cells or a product from treated cells of a microorganism belonging to the genus [0002] As a method for preparing malonic acid Corynebacterium, Gordona or Rhodococcus and monoesters, chemical hydrolysis of malonic acid having nitrilase activity to thereby hydrolyze the diesters is commonly used. According to this method, cyanoacetic acid ester. however, it is difficult to separate the generated malonic 15 (2) The method of (1) above, wherein the acid monoester from the unreacted malonic acid diester cyanoacetic acid ester is continuously added to the and the malonic acid which is a by-product. Thus, it is reaction solution while maintaining the concentra- impossible to obtain highly pure malonic acid tion of the cyanoacetic acid ester in the solution in monoesters. the range from 0.01 to 10% by weight during the [0003] As a method for obtaining highly pure 20 hydrolysis. malonic acid monoesters, a method using Meldrum's (3) The method of (1) above, wherein the C1-20 alkyl acid as a raw material is known [see, for example, represented by R is C3-20 alkyl and the microorgan- Matoba Katsuhide et al., Chem. Pharm. Bull., 31 (8), ism having nitrilase activity is a microorganism 2955 (1983); or Rigo B. et al., Tetrahedron Lett., 30(23), belonging to the genus Rhodococcus. 3073 (1989)]. However, since this method uses expen- 25 (4) The method of (3) above, wherein the sive Meldrum' s acid, it cannot be said a practical cyanoacetic acid ester is continuously added to the method and is not suitable for industrial production. reaction solution while maintaining the concentra- [0004] As another method for obtaining highly pure tion of the cyanoacetic acid ester in the solution in malonic acid monoesters, a method is known in which the range from 0.01 to 10% by weight during the malonic acid diesters are treated with an enzyme or 30 hydrolysis. microorganism having an ability to hydrolyze ester (5) The method of (1) above, wherein the microor- bonds (Japanese Unexamined Patent Publication No. ganism having nitrilase activity is Corynebacterium 8-173174). However, the use of malonic acid diesters as nitrilophilus ATCC 21419. a raw material is disadvantageous in terms of cost. (6) The method of (1) above, wherein the microor- [0005] Therefore, development of a highly produc- 35 ganism having nitrilase activity is Gordona terrae tive method for preparing highly pure malonic acid MA-1 (FERM BP-4535). monoesters has been desired. (7) The method of (1) above, wherein the microorganism having nitrilase activity is Rhodococcus DISCLOSURE OF THE INVENTION rhodochrous ATCC 33025. 40 (8) A method for preparing a malonic acid [0006] It is the object of the present invention to pro- monoester represented by Formula (II'): vide a highly productive method for preparing malonic acid monoesters which are useful as intermediates in HOOCCH2COOR' (II') the synthesis of various chemical products, medicines, agricultural chemicals, etc. 45 wherein R' is alkenyl, aryl, aralkyl or C3-20 [0007] The present inventors have found that a alkyl, comprising treating a cyanoacetic acid ester malonic acid monoester is produced selectively when a represented by Formula (I'): cyanoacetic acid ester is treated with a culture, cells or a product from treated cells of a microorganism having NCCH2COOR' (I') nitrilase activity; according to that method, a highly pure 50 malonic acid monoester can be prepared without side wherein R' is as defined in Formula (II'), with reactions such as hydrolysis of ester bonds. Thus, the a culture, cells or a product from treated cells of a present invention has been achieved. microorganism having nitrilase activity to thereby [0008] The present invention includes the following hydrolyze the cyanoacetic acid ester. inventions. 55 (9) The method of (8) above, wherein the cyanoacetic acid ester is continuously added to the (1) A method for preparing a malonic acid reaction solution while maintaining the concentra- monoester represented by Formula (II): tion of the cyanoacetic acid ester in the solution in

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the range from 0.01 to 10% by weight during the cyanoacetate. hydrolysis. [0018] The microorganism to be used in the invention is not particularly limited as long as it belongs to the [0009] Hereinbelow, the present invention will be genus Corynebacterium, Gordona or Rhodococcus described in detail. 5and has nitrilase activity. Specific examples of the [0010] The alkyl represented by R in Formula (I) or microorganism include Corynebacterium nitrilophilus (II) may be of either a straight-chain or branched-chain ATCC 21419, Gordona terrae MA-1 (FERM BP-4535) structure. The number of carbon atoms in this alkyl is 1- and Rhodococcus rhodochrous ATCC 33025. 20, preferably 1-10 and more preferably 2-6. Specific [0019] Among these microorganisms, Gordona examples of this alkyl include methyl, ethyl, n-propyl, 10 terrae MA-1 has been deposited at the National Insti- isopropyl, n-butyl, sec-butyl, tert-butyl, isobutyl, n- tute of Bioscience and Human-Technology, Agency of pentyl, isopentyl, hexyl, heptyl, octyl, 2-ethylhexyl, Industrial Science and Technology, 1-3, Higashi 1- decyl, dodecyl, tetradecyl, hexadecyl, octadecyl and chome, Tsukuba-shi, Ibaraki-ken, Japan, under the eicosyl. above-indicated accession number. Corynebacterium [0011] The alkyl represented by R' in Formula (I') or 15 nitrilophilus and Rhodococcus rhodochrous are availa- (II') may be of either a straight-chain or branched-chain ble from depositories such as American Type Culture structure. The number of carbon atoms in this alkyl is 3- Collection (ATCC), 12301 Parklawn Drive, Rockville, 20, preferably 3-10 and more preferably 3-6. Specific Maryland 20852, U.S.A. examples of this alkyl include n-propyl, isopropyl, n- [0020] When a cyanoacetic acid ester represented butyl, sec-butyl, tert-butyl, isobutyl, n-pentyl, isopentyl, 20 by Formula (I) wherein R is alkenyl, aryl, aralkyl or C3-20 hexyl, heptyl, octyl, 2-ethylhexyl, decyl, dodecyl, tetra- alkyl [i.e. a cyanoacetic acid ester represented by For- decyl, hexadecyl, octadecyl and eicosyl. mula (I')] is used as the substrate, the microorganism to [0012] The alkenyl represented by R may be of be used is not particularly limited as long as it has nitri- either a straight-chain or branched-chain structure. The lase activity. In addition to the above-described microor- number of carbon atoms in this alkenyl is 2-20, prefera- 25 ganisms, a microorganism belonging to the genus bly 2-6. Specific examples of this alkenyl include vinyl, Pseudomonas, Brevibacterium, Nocardia, allyl, crotyl (2-butenyl) and isopropenyl (1-methylvinyl). Arthrobacter, Bacillus, Escherichia, Micrococcus, [0013] The alkenyl represented by R' may be of Streptomyces, Aeromonas, Mycoplana, Cellulomonas, either a straight-chain or branched-chain structure. The Erwinia or Candida , for example, and having nitrilase number of carbon atoms in this alkenyl is 3-20, prefera- 30 activity may be used in the invention. bly 3-6. Specific examples of this alkenyl include allyl, [0021] More specifically, Pseudomonas synxanta crotyl (2-butenyl) and isopropenyl (1-methylvinyl). IAM 12356, Brevibacterium acetylicum IAM 1790, [0014] As the aryl represented by R or R', an aro- Nocardia asteroides IFO 3384, Arthrobacter oxydans matic hydrocarbon group such as phenyl, 1-naphthyl, 2- IFO 12138, Bacillus subtills ATCC 21697, Escherichia naphthyl; an aromatic hetrocyclic group such as furyl, 35 coli IFO 3301, Micrococcus luteus ATCC 383, Strepto- thienyl, pyrrolyl, oxazolyl, isoxazolyl, thiazolyl, isothia- myces griseus IFO 3355, Aeromonas punctata IFO zolyl, imidazolyl, pyrazolyl, pyridyl, pyrimidinyl, pyridazi- 13288, Mycoplana dimorpha ATCC 4297, Cellulo- nyl, pyrazinyl, quinolyl, isoquinolyl; or the like may be monas fimi IAM 12107, Erwinia herbicola IFO 12686 or enumerated. Candida quilliermondii IFO 0566 may be enumerated. [0015] As the aralkyl represented by R or R', ben- 40 Of these, microorganisms with ATCC numbers are zyl, 1-naphthylmethyl, 2-naphthylmethyl, phenethyl (2- available from American Type Culture Collection, 12301 phenylethyl), 1-phenylethyl, phenylpropyl, phenylbutyl, Parklawn Drive, Rockville, Maryland 20852, U.S.A.; phenylpentyl, phenylhexyl, methylbenzyl, methylphene- those with IAM numbers are available from IAM Culture thyl, dimethylbenzyl, dimethylphenethyl, trimethylben- Collection, Center for Cellular and Molecular Research, zyl, ethylbenzyl, diethylbenzyl or the like may be 45 Institute of Molecular and Cellular Biosciences, The enumerated. University of Tokyo, 1-1, Yayoi 1-chome, Bunkyo-ku, [0016] Among the cyanoacetic acid esters repre- Tokyo, Japan; and those with IFO numbers are available sented by Formula (I), representative compounds are, from Institute for Fermentation, Osaka, 17-85, Jusohon- for example, methyl cyanoacetate, ethyl cyanoacetate, machi 2-chome, Yodogawa-ku, Osaka-shi, Osaka, n-propyl cyanoacetate, isopropyl cyanoacetate, n-butyl 50 Japan. cyanoacetate, tert-butyl cyanoacetate, 2-ethylhexyl [0022] The microorganism may be cultured in either cyanoacetate, allyl cyanoacetate and benzyl cyanoace- a liquid medium or solid medium. A medium is used tate. which appropriately contains carbon sources and nitro- [0017] Among the cyanoacetic acid esters repre- gen sources usually assimilable to microorganisms, sented by Formula (I'), representative compounds are, 55 vitamins, minerals, and the like. It is also possible to add for example, n-propyl cyanoacetate, isopropyl cyanoac- to the medium a small amount of a nitrile or lactam com- etate, n-butyl cyanoacetate, tert-butyl cyanoacetate, 2- pound in order to improve the hydrolyzing ability of the ethylhexyl cyanoacetate, allyl cyanoacetate and benzyl microorganism. The nitrile compound is a C1-12 straight-

3 56EP 1 008 655 A1 chain or branched-chain aliphatic or aromatic nitrile. For deactivation of the enzyme by the substrate, the sub- example, propionitrile, isovaleronitrile, hexanenitrile, strate is added in such a manner that the substrate con- acrylonitrile, adiponitrile, benzonitrile, 2-pyridinecarbon- centration in the reaction solution is maintained itrile or the like may be enumerated. As the lactam com- preferably at 0.01-10% by weight, more preferably at pound, γ-butyrolactam, δ-valerolactam or ε-caprolactam 50.1-5% by weight. may be enumerated, for example. The cultivation is car- [0026] After completion of the reaction, the microor- ried out at a temperature and a pH level at which the ganism cells used as a catalyst are removed by centrif- microorganism can grow. Preferably, the cultivation is ugation, filtration or the like. Then, it is possible to carried out under the optimum cultivation conditions for recover the unreacted cyanoacetic acid ester by extract- the microorganism strain used. In order to promote the 10 ing the resultant solution with a solvent such as hexane, growth of the microorganism, aeration-agitation may be ethyl acetate, etc. After the pH of the extraction residue employed. is adjusted to 1-2 with an acid such as sulfuric acid, [0023] In the present invention, a culture obtained hydrochloric acid, etc., the residue is extracted with a by culturing the above-mentioned microorganism with solvent such as hexane, ethyl acetate, etc. to thereby nitrilase activity in a medium may be used without any 15 obtain a malonic acid monoester, the reaction product. treatment. Alternatively, cells of the microorganism har- vested from the culture by centrifugation or other opera- BEST MODES FOR CARRYING OUT THE INVEN- tion may be used. Further, a product from treated cells TION of the microorganism may also be used. Specific examples of the product from treated cells include cells 20 [0027] Hereinbelow, the present invention will be treated with acetone, toluene, etc.; disrupted cells; a cell described more specifically with reference to the follow- free extract obtained from disrupted cells; and a crude ing Examples. However, the scope of the present inven- or purified enzyme separated from cells. It is possible to tion is not limited to these Examples. recycle such cells or product after the reaction by using such cells or product after entrapping/immobilizing them 25 [Example 1] in a cross-linked acrylamide gel or the like or immobilizing them in a solid carrier such as an ion exchange [0028] Gordana terrae MA-1 (FERM BP-4535) was resin, diatom earth, etc. physically or chemically. inoculated into 3 ml of sterilized LB medium (1% [0024] In the present invention, preferably, a polypeptone, 0.5% yeast extract, 0.5% NaCl) and cul- malonic acid monoester may be prepared by the follow- 30 tured at 30°C for 24 hrs under shaking. One milliliter of ing procedures. Briefly, a cyanoacetic acid ester (a sub- the resultant cell culture liquid was inoculated into 100 strate) is added to a reaction medium to thereby ml of the following sterilized medium A and cultured at dissolve or suspend the ester. A culture, etc. of the 30°C for 48 hrs. microorganism which will work as a catalyst is added to the reaction medium before or after the addition of the 35 Medium A (pH 7.2) substrate. Then, a hydrolysis reaction is performed while controlling the reaction temperature and, if neces- [0029] sary, the pH of the reaction solution. As the reaction medium, deionized water or buffer may be used, for example. The reaction temperature is usually 0-70 °C, 40 preferably 10-35°C. A temperature at which the nitrilase Glycerol 1.0% activity of the microorganism cells, etc. is enhanced may be selected for the reaction. The pH of the reaction Isovaleronitrile 0.2% solution depends on the optimum pH of the enzyme of Yeast extract 0.02% the microorganism used. Generally, it is preferable to 45 KH PO 0.2% perform the reaction at pH 6-9 because side-reactions 2 4 resulting from the chemical hydrolysis can be inhibited NaCl 0.1% at such pH levels. The concentration of the cells or prod- MgSO g 7H O 0.02% uct from treated cells in the reaction solution is usually 4 3 g 0.01 to 5% by weight in dry weight. The substrate con- 50 FeSO4 7H2O 10 ppm centration in the reaction solution is not particularly lim- g CoCl2 4H2O 10 ppm ited as long as it falls within the range from 0.01 to 70% g by weight. Preferably, the substrate concentration is CaCl2 2H2O 1 ppm within the range from 0.1 to 15% by weight. g MnCl2 4H2O 7 ppm [0025] Further, it is possible to accumulate the 55 malonic acid monoester at a high concentration by continuously adding the cyanoacetic acid ester during the [0030] After completion of the cultivation, the cul- hydrolysis reaction. At that time, in order to minimize the ture liquid was centrifuged. The total volume of the

4 78EP 1 008 655 A1 resultant cells were washed with deionized water and [Example 7] then suspended in 100 ml of 50 mM phosphate buffer (pH 7.0). The turbidity of this cell suspension was OD630 [0036] Mono-tert-butyl malonate (1.05 g) was = 5.5. To this cell suspension, 1.00 g of ethyl cyanoace- obtained (yield: 92.5%) in the same manner as in Exam- tate was added as a substrate and reacted at 30°C for 1 5ple 6 except that tert-butyl cyanoacetate was used as hr. The resultant reaction solution was analyzed by high the substrate. performance liquid chromatography (HPLC; column: TSKgel ODS-120A (Tosoh Corp.), 4.6 mm I.D. x 25 cm; [Example 8] mobile phase: 5% acetonitrile, 95% water, 0.1% phosphoric acid; ﬂow rate: 0.5 ml/min; detection: UV 220 10 [0037] Monoallyl malonate (1.02 g) was obtained nm). As a result, it was found that the whole substrate (yield: 87.2%) in the same manner as in Example 6 had been converted to monoethyl malonate. After com- except that allyl cyanoacetate was used as the sub- pletion of the reaction, cells were removed by centrifu- strate. gation. Then, 2N HCl was added to the resultant solution to adjust the pH to 2.0. Thereafter, monoethyl 15 [Example 9] malonate, the reaction product, was extracted from the solution with ethyl acetate. Anhydrous sodium sulfate [0038] Mono-2-ethylhexyl malonate (1.01 g) was was added to the resultant organic layer for dehydration, obtained (yield: 91.8%) in the same manner as in Exam- and the solvent was removed by distillation. As a result, ple 6 except that 2-ethylhexyl cyanoacetate was used 1.05 g of monoethyl malonate was obtained (yield: 20 as the substrate. 89.9%). [Example 10] [Example 2] [0039] A reaction was performed in the same man- [0031] Monoethyl malonate (1.07 g) was obtained 25 ner as in Example 6 except that benzyl cyanoacetate (yield: 91.6%) in the same manner as in Example 1 was used as the substrate. After completion of the except that Corynebacterium nitrilophilus ATCC 21419 enzyme reaction, 10% of the benzyl cyanoacetate was was used as the microorganism. unreacted. The unreacted benzyl cyanoacetate was extracted with ethyl acetate for removal. After this [Example 3] 30 extraction, 2N HCl was added to the resultant aqueous layer to adjust the pH to 2.0. Then, monobenzyl [0032] Monomethyl malonate (0.97 g) was obtained malonate, the reaction product, was extracted with ethyl (yield: 81.4%) in the same manner as in Example 1 acetate. Anhydrous sodium sulfate was added to the except that methyl cyanoacetate was used as the sub- resultant organic layer for dehydration, and the solvent strate. 35 was removed by distillation. As a result, 0.89 g of monobenzyl malonate was obtained (yield: 80.3%). [Example 4] [Examples 11-19] [0033] Mono-n-propyl malonate (1.05 g) was obtained (yield: 91.3%) in the same manner as in Exam- 40 [0040] To a cell suspension prepared in the same ple 1 except that n-propyl cyanoacetate was used as the manner as in Example 1, ethyl cyanoacetate was added substrate. to give a concentration of 5-50% by weight and reacted at 25°C for 20 hrs. After completion of the reaction, the [Example 5] yield was determined by liquid chromatography. The 45 results are shown in Table 1. [0034] Monoisopropyl malonate (1.02 g) was obtained (yield; 88.7%) in the same manner as in Exam- ple 1 except that isopropyl cyanoacetate was used as Table 1 the substrate. Example Substrate Concentration Yield (%) 50 (% by weight) [Example 6] 11 5 100 [0035] Mono-n-butyl malonate (1.06 g) was 12 10 100 obtained (yield: 93.4%) in the same manner as in Exam- 13 15 84.1 ple 1 except that n-butyl cyanoacetate was used as the 55 substrate. In the HPLC analysis, 40% acetonitrile, 60% 14 20 59.6 water and 0.1% phosphoric acid were used as the 15 25 48.3 mobile phase.

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Table 1 (continued) = 5.6. To this cell suspension, 1.00 g of n-propyl cyanoacetate was added as a substrate and reacted at 30°C Example Substrate Concentration Yield (%) for 1 hr. The resultant reaction solution was analyzed by (% by weight) high performance liquid chromatography (HPLC; col- 16 30 37.3 5umn: TSKgel ODS-120A (Tosoh Corp.), 4.6 mm I.D. x 17 35 29.7 25 cm; mobile phase: 5% acetonitrile, 95% water, 0.1% phosphoric acid; ﬂow rate: 0.5 ml/min; detection: UV 18 40 25.3 220 nm). As a result, it was found that the whole sub- 19 50 19.6 strate had been converted to mono-n-propyl malonate. 10 After completion of the reaction, cells were removed by centrifugation. Then, 2N HCl was added to the resultant [Example 20] solution to adjust the pH to 2.0. Thereafter, mono-n-propyl malonate, the reaction product, was extracted from [0041] To 100 ml of a cell suspension prepared in the solution with ethyl acetate. Anhydrous sodium sul- the same manner as in Example 1, 5 g of ethyl cyanoa- 15 fate was added to the resultant organic layer for dehy- cetate was added and reacted at 25°C. Thereafter, 30 g dration, and the solvent was removed by distillation. As of ethyl cyanoacetate was further added to the reaction a result, 1.06 g of mono-n-propyl malonate was solution in portions while measuring the substrate con- obtained (yield: 89.9%). centration in the solution so that it did not exceed 5% by weight. After 30 hrs, the yield was 100%. 20 [Example 22]

[Example 21] [0045] Monoisopropyl malonate (1.01 g) was obtained (yield: 88.6%) in the same manner as in Exam- [0042] Rhodococcus rhodochrous ATCC 33025 ple 21 except that isopropyl cyanoacetate was used as was inoculated into 3 ml of sterilized LB medium (1% 25 the substrate. polypeptone, 0.5% yeast extract, 0.5% NaCl) and cultured at 30°C for 24 hrs under shaking. One milliliter of [Example 23] the resultant cell culture liquid was inoculated into 100 ml of the following sterilized medium A and cultured at [0046] Mono-n-butyl malonate (1.05 g) was 30°C for 48 hrs. 30 obtained (yield: 93.3%) in the same manner as in Exam- ple 21 except that n-butyl cyanoacetate was used as the Medium A (pH 7.2) substrate. In the HPLC analysis, 40% acetonitrile, 60% water and 0.1% phosphoric acid were used as the [0043] mobile phase. 35 [Example 24]

Glycerol 1.0% [0047] Mono-tert-butyl malonate (1.04 g) was obtained (yield: 92.4%) in the same manner as in Exam- Isovaleronitrile 0.2% 40 ple 23 except that tert-butyl cyanoacetate was used as Yeast extract 0.02% the substrate. KH PO 0.2% 2 4 [Example 25] NaCl 0.1% 45 [0048] Monoallyl malonate (1.04 g) was obtained MgSO g 7H O 0.02% 4 2 (yield: 87.1%) in the same manner as in Example 23 g FeSO4 7H2O 10 ppm except that allyl cyanoacetate was used as the sub- g strate. CoCl2 4H2O 10 ppm g CaCl2 2H2O 1 ppm 50 [Example 26] g MnCl2 4H2O 7 ppm [0049] Mono-2-ethylhexyl malonate (1.00 g) was obtained (yield: 91.7%) in the same manner as in Exam- [0044] After completion of the cultivation, the cul- ple 23 except that 2-ethylhexyl cyanoacetate was used ture liquid was centrifuged. The total volume of the 55 as the substrate. resultant cells were washed with deionized water and then suspended in 100 ml of 50 mM phosphate buffer (pH 7.0). The turbidity of this cell suspension was OD630

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[Example 27] intermediates in the synthesis of various chemical products, medicines, agricultural chemicals and the like with [0050] A reaction was performed in the same man- high productivity. ner as in Example 23 except that benzyl cyanoacetate was used as the substrate. After completion of the 5 Claims enzyme reaction, 10% of the benzyl cyanoacetate was unreacted. The unreacted benzyl cyanoacetate was 1. A method for preparing a malonic acid monoester extracted with ethyl acetate for removal. After this represented by Formula (II): extraction, 2N HCl was added to the resultant aqueous layer to adjust the pH to 2.0. Then, monobenzyl 10 HOOCCH2COOR (II) malonate, the reaction product, was extracted with ethyl acetate. Anhydrous sodium sulfate was added to the wherein R is alkenyl, aryl, aralkyl or C1-20 resultant organic layer for dehydration, and the solvent alkyl, comprising treating a cyanoacetic acid ester was removed by distillation. As a result, 0.88 g of represented by Formula (I): monobenzyl malonate was obtained (yield: 80.2%). 15 NCCH2COOR (I) [Examples 28-36] wherein R is as deﬁned in Formula (II), with [0051] To a cell suspension prepared in the same a culture, cells or a product from treated cells of a manner as in Example 21, n-propyl cyanoacetate was 20 microorganism belonging to the genus added to give a concentration of 5-50% by weight and Corynebacterium, Gordona or Rhodococcus and reacted at 25°C for 20 hrs. After completion of the reac- having nitrilase activity to thereby hydrolyze said tion, the yield was determined by liquid chromatography. cyanoacetic acid ester. The results are shown in Table 2. 25 2. The method according to claim 1, wherein said cyanoacetic acid ester is continuously added to the Table 2 reaction solution while maintaining the concentra- Example Substrate Concentration Yield (%) tion of said cyanoacetic acid ester in said solution in (% by weight) the range from 0.01 to 10% by weight during the 30 hydrolysis. 28 5 100

29 10 100 3. The method according to claim 1, wherein said C1- alkyl represented by R is C alkyl and said 30 15 85.1 20 3-20 microorganism having nitrilase activity is a microor- 31 20 57.6 35 ganism belonging to the genus Rhodococcus. 32 25 44.3 4. The method according to claim 3, wherein said 33 30 32.3 cyanoacetic acid ester is continuously added to the 34 35 26.7 reaction solution while maintaining the concentra- 40 tion of said cyanoacetic acid ester in said solution in 35 40 20.3 the range from 0.01 to 10% by weight during the 36 50 15.6 hydrolysis.

5. The method according to claim 1, wherein said [Example 37] 45 microorganism having nitrilase activity is Coryne- bacterium nitrilophilus ATCC 21419. [0052] To 100 ml of a cell suspension prepared in the same manner as in Example 21, 5 g of n-propyl 6. The method according to claim 1, wherein said cyanoacetate was added and reacted at 25°C. Thereaf- microorganism having nitrilase activity is Gordona ter, 20 g of n-propyl cyanoacetate was further added to 50 terrae MA-1 (FERM BP-4535). the reaction solution in portions while measuring the substrate concentration in the solution so that it did not 7. The method according to claim 1, wherein said exceed 5% by weight. After 30 hrs, the yield was 100%. microorganism having nitrilase activity is Rhodo- coccus rhodochrous ATCC 33025. INDUSTRIAL APPLICABILITY 55 8. A method for preparing a malonic acid monoester [0053] According to the present invention, it is pos- represented by Formula (II'): sible to prepare malonic acid monoesters useful as

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HOOCCH2COOR' (II')

wherein R' is alkenyl, aryl, aralkyl or C3-20 alkyl, comprising treating a cyanoacetic acid ester represented by Formula (I'): 5

NCCH2COOR' (I')

wherein R' is as deﬁned in Formula (II'), with a culture, cells or a product from treated cells of a 10 microorganism having nitrilase activity to thereby hydrolyze said cyanoacetic acid ester.

9. The method according to claim 8, wherein said cyanoacetic acid ester is continuously added to the 15 reaction solution while maintaining the concentration of said cyanoacetic acid ester in said solution in the range from 0.01 to 10% by weight during the hydrolysis. 20

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