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United States Patent (19) 11 Patent Number: 4,994,382 Ameyama Et Al

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United States Patent (19) 11 Patent Number: 4,994,382 Ameyama Et Al United States Patent (19) 11 Patent Number: 4,994,382 Ameyama et al. 45 Date of Patent: Feb. 19, 1991 54 PROCESS FOR PRODUCTION OF rolo-Quinorine Quinone'; Agric. Biol. Chem., vol. 48, PYRROLO-QUINOLINE QUINONE No. 2, Feb. 1984, pp. 561-565. Shimao et al., "Pyrroloquinoline Quinone as an Essen (75) Inventors: Minoru Ameyama; Osao Adachi, both tial Growth Factor for a Poly(vinyl alchol)-Degrading of Yamaguchi, Japan Symbiont, Pseudomonas sp VM15C"; Agric. Biol. 73 Assignee: Ube Industries, Ltd., Yamaguchi, Chem., vol. 48, No. 11, (Nov., 1984), pp. 2873-2876. Japan DeBeer et al.; "The Prosthetic Group of Methylamine Dehydrogenase From Pseudomonas AMI"; Biochimica (21) Appl. No.: 357,668 et Biophysica Acta, vol. 622, (1980), pp. 370-374. Chemical Abstracts, vol. 100, No. 21, (1984), p. 496, 22 Filed: May 26, 1989 Abstract No. 173083r, Ameyama et al. Chemical Abstracts, vol. 99, No 15, (1983), p. 287, Ab Related U.S. Application Data stract No. 118240a, J. A. Duine et al. 63 Continuation of Ser. No. 739,046, May 29, 1985, aban Patent Abstracts of Japan, vol. 8, No. 323, (1984). doned. E. J. Corey et al., J. Am. Chen. Soc. 1981, 103, 5599-5600. 30 Foreign Application Priority Data Primary Examiner-Herbert J. Lilling May 29, 1984 JP Japan ................................ 59-1074.06 Attorney, Agent, or Firm-Finnegan, Henderson, 51) Int. Cl......................... C12N 1/20; C12P 17/18; Farabow, Garrett & Dunner C12P 17/16; C12R 1/38 57 ABSTRACT 52 U.S. C. .................................... 435/119; 435/118; There are disclosed a new process for production of 435/133; 435/247; 435/252.34; 435/253.3; pyrrolo-quinoline quinone, comprising culturing a bac 435/822; 435/874 terium belonging to the genus Paracoccus, Protamino 58 Field of Search ............... 435/119, 118, 133, 247, bacter or Pseudomonas and capable of producing pyr 435/822,874, 253.3, 252.34 rolo-quinoline quinone in a culture medium to produce 56 References Cited the pyrrolo-quinoline quinone in the cultured broth, and recovering the pyrrolo-quinoline quinone from the cul U.S. PATENT DOCUMENTS tured broth; and a new process for production of pyr 4,37,843 3/1982 MacLennan et al. ............... 435/253 rolo-quinoline quinone, comprising culturing a bacte rium belonging to the genus Paracoccus, Protaminobac FOREIGN PATENT DOCUMENTS ter or Pseudomonas and capable of producing the pyr 206471 4/1986 European Pat. Off. ............ 435/18 rolo-quinoline quinone in a culture medium to form cultured cells, separating the cells from the cultured OTHER PUBLICATIONS broth, resuspending the separated cells in a reaction Duine et al., “The Prosthetic Group of Methanol Dehy medium containing precursors of the pyrrolo-quinoline drogenase'; Biochem. J., vol. 187, (1980), pp. 221-226. quinone, incubating the reaction medium to produce the Drieg et al., Bergey's Manual of Systematic Bacteriology, pyrrolo-quinoline quinone, and recovering the pyrrolo vol. 1, Williams and Wilkins, Baltimore, Md., (1984); quinoline quinone from the reaction medium. pp. 399, 400. Ameyama et al., “Microbial Production of Pyr 2 Claims, 2 Drawing Sheets U.S. Patent Feb. 19, 1991 Sheet 1 of 2 4,994,382 Fig. I O.OO O.O5 O O 2 4 6 8 O 12 14 x O M PQQ U.S. Patent Feb. 19, 1991 Sheet 2 of 2 4,994,382 s Fi 9. 2 TIME (hour) 4,994,382 1 2 product isomers derived from the reactions, and pro PROCESS FOR PRODUCTION OF vide a rather low yield of PQQ. PYRROLO-QUINOLINE QUINONE To overcome the above-mentioned disadvantages of the chemical synthetic processes, there has been pres This application is a continuation of application Ser. 5 ented a biological process for the production of PQQ No. 06/739,046, filed May 29, 1985. wherein cells of a microorganism are cultured in a me dium to accumulate PQQ and related compounds, and BACKGROUND OF THE INVENTION the accumulated PQQ is recovered by, for example, 1. Field of the Invention extraction with solvents (see Japanese Unexamined The present invention relates to a process for the 10 Patent Application (Kokai) No. 59.113896 published on microbial production of pyrrolo-quinoline quinone June 30, 1984), or simple chromatographic method on (hereinafter referred to as PQQ). ionexchangers (see, for example, Agri Biol. Chem. Vol. 2. Description of the Related Art PQQ has the fol 48,561-565 (1984)). lowing formula: Therefore, a new process for the production of PQQ, 5 which is more practical and economical, is desired. COOH (I) SUMMARY OF THE INVENTION The present invention provides a new process for the production of PQQ comprising, culturing a bacterium belonging to the genus Paracoccus, Protaminobacter or Pseudomonas and capable of producing pyrrolo-quino line quinone in a culture medium to produce the pyr rolo-quinoline quinone in the cultured broth, and recov ering the pyrrolo-quinoline quinone from the cultured and can be reversibly reduced to a reduced type PQQ 25 broth. (PQQH2) having the following formula: There is also provided a process for the production of PQQ comprising culturing the above-mentioned bacte rium in a culture medium, to form cultured cells, sepao COOH (II) rating the cells from the cultured broth, resuspending 30 the separated cells in a reaction medium containing precursors of the pyrrolo-quinoline quinone, incubating the reaction medium to produce the pyrrolo-quinoline quinone, and recovering the pyrrolo-quinoline quinone 35 from the reaction medium. BRIEF DESCRIPTION OF THE DRAWINGS On the basis of this property, PQQ has an ability to FIG. 1 is a calibration chart showing the relationship convert apo-type quinoenzymes to the holo-type between an amount of PQQ and a difference of absorp thereof. For example, PQQ acts as a coenzyme for tion per minute at 600 nm as measured by using a mem methanol dehydrogenase in methanol-utilizing bacteria, brane of 0.16 mg as protein (AOD600/min./0.16 mg). alcohol dehydrogenase, aldehyde dehydrogenase, glyc FIG. 2 is a graph showing a profile of the culturing of erol dehydrogenase, glucose dehydrogenase, or the like Paracoccus denitrificans in Example 1. in acetic acid bacteria. PQQ is also physiologically in DESCRIPTION OF THE PREFERRED portant as a coenzyme for copper containing amine 45 EMBODIMENTS oxidase of animal, plant or microbial origin, amine de hydrogenase or choline dehydrogenase, or other vari The following bacteria are typically used in the pres ous kinds of oxidoreductases which are inhibited by ent invention: carbonyl reagents. (1) Paracoccus denitrificans (IFO 13301) Moreover, PQQ may be a very important substance 50 (2) Protaminobacter ruber (IFO 3708) having vitamin actions because it acts as a coenzyme for (3) Pseudomonas A1-2 (FERM P-7599) important enzymes as described above, taking as an (4) Pseudomonas P1-1 (FERM P-7598) analogy the fact that coenzymes for other oxidoreduc (5) Pseudomonas P2-2 (FERM P-7600) tases and transferases, such as thiamine pyrrophosphate, (6) Pseudomonas P2-3 (FERM P-7597) nicotinamide adenine dinucleotide, nicotinamide ade 55 (7) Pseudomonas N1-1 (FERM P-7596) nine dinucleotide phosphate, pyridoxal phosphate, fla The above-mentioned bacteria (1) and (2) have been vin adenine dinucleotide, and flavin mononucleotide deposited at the Institute for Fermentation, Osaka have to be taken in as vitamin, such as vitamin B1, nico (IFO) in Japan, and can be freely supplied to the public. tinic acid, vitamin B6, and vitamin B2 respectively. On The bacteria (3) to (7) were isolated by the present the basis of the above-mentioned physiological impor invention, and deposited at the Fermentation Research tance, PQQ is useful for pharmaceutical purposes. Institute (FRI) in Japan, on April 27, 1984. The bacteri Conventionally, for the production of PQQ, chemical um(7) was transferred to the international deposition synthetic processes are known (see, for example, J. Ann. under the Budapest Treaty on the International Recog Chem. Soc. Vol. 103, 5599-5600 (1981)). However, nition of the Deposit of Microorganisms for the Pur these chemical synthetic processes have various disad 65 poses of Patent Procedure on Apr. 25, 1985, and given vantages. For example, the processes are time-consum the number FERM BP-775. ing due to the necessity for multi-reaction steps, require The properties of the newly isolated bacteria are set complicated operations to remove many kinds of by forth in the following tables. 4,994,382 3 4. may be added step by step or continuously during cul TABLE 1 turing. The nitrogen source includes organic nitrogen Growth of Microorganisms on Various Carbon Sources sources such as amino acids, nucleic acids, protein hy Microorganism drolyzates, yeast extract, corn steep liquor, and inor Carbon Source Al-2 P1-1 P2-2 P2-3 N1. 5 ganic nitrogen sources such as ammonium salts, ammo Methanol -- - -- ---- -- ---- Formaldehyde - - -- - nia water, gaseous ammonia, and nitrates. The above Sodium formate - --- mentioned nitrogen sources may be used alone or in Ethanoi -- ----' -- -- --- * combination. The concentration of nitrogen source Acetaldehyde - - -- varies according to the kind of nitrogen source used, Sodium acetate -- -- * -- * -- --" IO Methylamine -- -- -- and is preferably within 0.05% to 0.5% by weight. D-Glucose ---- -- ---- ---- -- The culturing is preferably carried out under the Glycerol ---- ---- -H -- -- aerobic condition which is accomplished by aeration D-Fructose ---- - -- -- -- Malate -- - - - and agitation of the medium in a fermentor, or by shak D-Mannitol -- -- - - - -- -- ---- ing a culture flask containing the medium. The tempera Lactose ---- -- ---- -- 15 ture for culturing is generally 0° C. to 40” C., preferably Saccharose -r ----' -- ---- -- D-Galactose -- -- -- -- - 20 C. to 35° C. The pH of the medium is generally 2 to L-Arabinose -- -- -- -- - 9, preferably 5.5 to 8.0. The culturing time is generally D-Xylose -- - --- -- -- 20 to 150 hours, preferably 50 to 100 hours. In the pres Polypepton" ---- -- ---- ent embodiment, PQQ is accumulated in the cultured "Production of fluorescent pigments 20 broth.
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