Europäisches Patentamt *EP001482055A1* (19) European Patent Office Office européen des brevets (11) EP 1 482 055 A1 (12) EUROPEAN PATENT APPLICATION (43) Date of publication: (51) Int Cl.7: C12P 13/02, C08G 69/28 01.12.2004 Bulletin 2004/49 (21) Application number: 04010711.2 (22) Date of filing: 05.05.2004 (84) Designated Contracting States: • Endo, Shuichi AT BE BG CH CY CZ DE DK EE ES FI FR GB GR Kawasaki-shi Kanagawa (JP) HU IE IT LI LU MC NL PL PT RO SE SI SK TR • Mori, Yukiko Designated Extension States: Kawasaki-shi Kanagawa (JP) AL HR LT LV MK • Totsuka, Kazuhiko Kawasaki-shi Kanagawa (JP) (30) Priority: 26.05.2003 JP 2003147688 • Hirao, Yoshinori Kawasaki-shi Kanagawa (JP) (71) Applicant: Ajinomoto Co., Inc. Tokyo (JP) (74) Representative: Strehl Schübel-Hopf & Partner Maximilianstrasse 54 (72) Inventors: 80538 München (DE) • Nishi, Kiyohiko Kawasaki-shi Kanagawa (JP) (54) Method for producing cadaverine dicarboxylate and its use for the production of nylon (57) Cadaverine dicarboxylate is produced by per- of the solution at a level sufficient for the enzymatic de- forming an enzymatic decarboxylation reaction of a carboxylation reaction to occur, for example, 4.0 to 8.0. lysine solution while adding a dicarboxylic acid contain- ing 4 to 10 carbons to the lysine solution to maintain pH EP 1 482 055 A1 Printed by Jouve, 75001 PARIS (FR) EP 1 482 055 A1 Description BACKGROUND OF THE INVENTION 5 Field of the Invention [0001] The present invention relates to a method for producing cadaverine dicarboxylate. Cadaverine dicarboxylate can be used as a raw material for producing nylon. 10 Description of the Related Art [0002] Naphtha, a fossil material, is a major raw material in the production of plastics. Disposal of plastics which are not recycled has recently become a environmental concern due to the release of carbon dioxide when disposed of by burning, etc.. Accordingly, with the goal of preventing global warming and encouraging a recycling society, it is strongly 15 desirable to replace raw materials for producing plastics with those derived from biomass. [0003] Polylactic acid is known as a plastic produced using biomass as a raw material. A method for producing polylactic acid includes first extracting starch or sugar from a plant, then producing lactic acid by fermentation using the extracted starch or sugar as a carbon source, and then chemically polymerizing the resulting lactic acid. Polylactic acid is expected to be used in various industrial products, including container packages, garments, and others. How- 20 ever, since polylactic acid has a melting point of about 190°C, it is not suitable for high temperature uses. [0004] Plastics which have high heat resistance include nylon, particularly polyamide. An example of a widely-used nylon is nylon-66, which is produced by polymerizing hexamethylenediamine, which is a diamine containing 6 carbons, and adipic acid, which is a dicarboxylic acid containing 6 carbons, at a molar ratio of 1:1. Since nylon-66 has a melting point of 250°C or higher, it is a plastic material which is able to withstand high temperature conditions. 25 [0005] The aforementioned hexamethylenediamine is produced using benzene, propylene, or butadiene and can be obtained from the raw material naphtha. However, production methods from biomass are unknown. On the other hand, pentamethylenediamine containing 5 carbons, also known as cadaverine, is known to be produced from lysine, an amino acid, using lysine decarboxylase (hereinafter "LDC") (Enzyme Handbook, 1st ed., p. 636, Asakura Shoten). Therefore, if nylon is produced using a pentamethylenediamine containing 5 carbons as a raw material instead of 30 hexamethylenediamine containing 6 carbons, it is then possible to provide a plastic material produced using a raw material derived from biomass and usable under high temperature conditions. [0006] LDC is known to exist in bacteria such as Bacterium cadaveris (Soda K. et al., Biochem. Biophys. Res. Com., Vol. 34, pp.34-39, 1969) and Escherichia coli (E. coli) (Sabo D.L. et al., Biochemistry, Vol. 13, pp.662-670, 1974) and plants such as Lathyrus sativus (Ramakrishna S. et al., Phytochemistry, Vol. 15, pp.83-86, 1976). LDC can be extracted 35 from these organisms and used for the production of cadaverine. Furthermore, the sequence of the LDC gene (cadA) of E. coli is known (Watson N. et al., Journal of Bacteriology, Vol. 174, pp.530-540, 1992; Meng S.Y. et al. Journal of Bacteriology, Vol. 174, pp.2659-2669, 1992). Furthermore, a method of producing cadaverine by culturing a host in which enzymatic activity of LDC or the lysine-cadaverine antiporter is amplified using such an LDC gene or the like has been suggested (Japanese Patent Laid-open (Kokai) No. 2002-223770), and a method of producing cadaverine 40 by allowing LDC derived from a recombinant cell in which the intracellular activity of LDC is amplified to act on lysine has also been suggested (Japanese Patent Laid-open No. 2002-223771). [0007] However, if LDC is allowed to act on lysine, carbon dioxide is released by the decarboxylation reaction of LDC, and hence the pH is raised by the production of cadaverine during the reaction. Therefore, to prevent the rise of pH and maintain the optimal pH for the enzymatic reaction, it is necessary to perform the reaction in a buffer of a high 45 concentration, or successively add an acid to the reaction system to neutralize the alkalinity (Japanese Patent Laid- open (Kokai) Nos. 2002-223770 and 2002-223771). In general, inorganic acids such as hydrochloric acid, sulfuric acid and phosphoric acid or organic acids such as acetic acid are often used for neutralization of pH during an enzymatic reaction. When alkalinity is neutralized with these acids, the cadaverine which is obtained from the reaction mixture is in the form of a salt such as cadaverine hydrochloride, cadaverine sulfate, cadaverine phosphate and cadaverine 50 acetate. [0008] Known methods for producing nylons include condensation polymerization of a dicarboxylic acid dihalide and a diamine in the presence of a base. Alternatively, a method of heating a salt or a lower condensate, which has formed from a dicarboxylic acid and a diamine under melting conditions, to polycondense is known.(Ise, N. et al., Shinkobun- shikagakujoron, p.22, Kagakudojin, 1995) When cadaverine is obtained by the enzymatic reaction and then polymer- 55 ized with a dicarboxylic acid by either method, free cadaverine must be re-prepared from a salt of cadaverine. Therefore, the process becomes complicated and is no longer economical. [0009] Furthermore, a method for producing lysine by fermentation is known, and includes culturing a bacterium in a medium containing adipic acid, succinic acid, fumaric acid or a salt thereof as a main component, and maintained 2 EP 1 482 055 A1 at pH 7.5 to 8.2 with ammonium hydroxide (Japanese Patent Laid-open No. 49-126891). In this method, the bacterium is sub-cultured to allow proliferation thereof and maintain dynamic equilibrium of cells, and then the cells are cultured with changing a part of the medium conditions or culture conditions to perform fermentation with a shifted equilibrium of substance metabolism and thereby accumulate lysine in the medium at a high concentration. 5 SUMMARY OF THE INVENTION [0010] An object of the present invention is to provide a method for economically producing cadaverine as a diamine to be used as a raw material for production of nylon in a form easily and most efficiently used in the polymerization. 10 [0011] It is a further object of the present invention to provide a method for producing a cadaverine dicarboxylate comprising subjecting a lysine solution to an enzymatic decarboxylation reaction and maintaining the pH of said solution at a level sufficient for said reaction to occur by adding dicarboxylic acid to said solution. [0012] It is a further object of the present invention to provide the method as described above, wherein pH of the solution is maintained at about 4.0 to 8.0. 15 [0013] It is a further object of the present invention to provide the method as described above, wherein the dicarboxylic acid contains 4 to 10 carbons. [0014] It is a further object of the present invention to provide the method as described above, wherein the dicarboxylic acid is adipic acid. [0015] It is a further object of the present invention to provide the method as described above, wherein the enzymatic 20 decarboxylation reaction is performed using lysine decarboxylase, a cell producing lysine decarboxylase or a processed product of the cell producing lysine decarboxylase. [0016] It is a further object of the present invention to provide the method as described above, wherein the cell is modified to have increased lysine decarboxylase activity. [0017] It is a further object of the present invention to provide the method as described above, wherein the cell is 25 recombinant. [0018] It is still a further object of the present invention to provide the method as described above wherein the cell is modified to have increased copy number of a gene encoding lysine decarboxylase. [0019] It is a still further object of the present invention to provide the method as described above, wherein the cell is modified by modifying an expression regulatory sequence of a gene encoding lysine decarboxylase. 30 [0020] It is a still further object of the present invention to provide the method as described above, wherein the expression of the gene encoding lysine decarboxylase is enhanced. [0021] It is a still further object of the present invention to provide the method as described above, wherein the cell is an Escherichia coli cell. [0022] It is a still further object of the present invention to provide the method as described above, wherein the gene 35 encoding lysine decarboxylase is a cadA gene.