Process for producing optically active 4-hydroxy-2-ketoglutaric acids

Abstract: The object of the present invention is to provide a method for producing an optically active 4-hydroxy-2-ketoglutaric acid which is industrially applicable and advantageous. The present invention provides a process for producing an optically active 4-hydroxy-2-ketoglutaric acid, which comprises allowing glyoxylic acid and pyruvic acid to coexist with a biocatalyst which has an activity of forming the optically active 4-hydroxy-2-ketoglutaric acid from pyruvic acid and glyoxylic acid in an aqueous medium to convert glyoxylic acid into the optically active 4-hydroxy-2-ketoglutaric acid and recovering the optically active 4-hydroxy-2-ketoglutaric acid from the aqueous medium.

Description: The present invention relates to a process for producing an optically active 4-hydroxy-2-ketoglutaric acid in which a biocatalyst having an activity for formation of the optically active 4-hydroxy-2-ketoglutaric acid from pyruvic acid and glyoxylic acid is used to produce the optically active 4-hydroxy-2-ketoglutaric acid from pyruvic acid or a compound capable of being converted into pyruvic acid and glyoxylic acid. The optically active 4-hydroxy-2-ketoglutaric acid is useful as a starting material for synthesizing a medicament. As conventional methods for producing the optically active 4-hydroxy-2-ketoglutaric acid, chemical deamination of threo- and erythro-4-hydroxy-L-glutamic acid to obtain L- and D-4-hydroxy-2-ketoglutaric acid, respectively (J. Biol. Chem., 288, 3660 (1963)) and resolution of DL-4-hydroxy-2-ketoglutaric acid synthesized from oxaloacetic acid and glyoxylic acid into D- and L-form (Methods in Enzymology, 17 part B, 253) are known. It is also possible to enzymatically synthesize 4-hydroxy-2-ketoglutaric acid from pyruvic acid and glyoxylic acid by using ketohydroxy-2-glutaric acid aldolase present in animals and E.coli (J. Chem. Soc. Perkin. Trans., 1, 1085 (1992)). However, no microorganism other than E.coli has previously been known to produce an enzyme with an activity of forming 4-hydroxy-2-ketoglutaric acid from glyoxylic acid and pyruvic acid. These conventional methods are insufficient for industrial application due to the following points: (1) the starting materials are expensive; (2) resolution step of DL-form is required; (3) yield is low, etc. Consequently, there exists a need for development of a method for producing optically active 4-hydroxy-2-ketoglutaric acid which is industrially applicable and advantageous. An object of the present invention is therefore to provide a process for producing optically active 4-hydroxy-2-ketoglutaric acid which is industrially applicable and advantageous. According to the present invention, there is provided a process for producing an optically active D-4-hydroxy-2-ketoglutaric acid, which comprises: (a) allowing glyoxylic acid and pyruvic acid to coexist with a biocatalyst which is selected from cells, a culture and processed cells of a microorganism belonging to the genus cellvibrio or Bacillus having an activity of forming the optically active D-4-hydroxy-2-ketoglutaric acid from pyruvic acid and glyoxylic acid in an aqueous medium, thereby to convert the glyoxylic acid and pyruvic acid into the optically active D-4-hydroxy-2-ketoglutaric acid; and (b) recovering the optically active D-4-hydroxy-2-ketoglutaric acid from the aqueous medium. The D-4-hydroxy-2-ketoglutaric acid thus obtained can be converted into 4-hydroxyproline. A carbapenem antibiotic can then be synthesised from the 4-hydroxyproline. The present invention will be described in detail below. The biocatalysts used in the present invention are cells, a culture and processed cells of a microorganism which is capable of forming D-4-hydroxy-2-ketoglutaric acid from pyruvic acid and glyoxylic acid. The "processed cells of microorganism" include treated cells such as dried cells, lyophilized cells, surfactant-treated cells, enzymatically treated cells, ultrasonically treated cells, mechanically ground cells and solvent-treated cells; protein-containing fractions of the cells; immobilized products of processed or processed cells; and the like. Any microorganism which has the ability to form the optically active 4-hydroxy-2-ketoglutaric acid from pyruvic acid and glyoxylic acid can be employed. Examples of the microorganisms having such activity are those belonging to the genus Cellvibrio or Bacillus . Specifically, the microorganisms may include Cellvibrio gilvus ATCC 13127 strain, Bacillus sp. OC187 strain, Bacillus sp. S16 strain, etc. ATCC 13127 and OC187 strains can be deprived of their activities of formation of L-4-hydroxy-2-ketoglutaric acid by heat-treating them at a temperature of 60 to 90°C for a period of time of 5 minutes to 2 hours. Alternatively, such inactivated strains can be obtained by mutagenizing microorganisms, which belong to the genus Cellvibrio or Bacillus and have the ability to form D-4-hydroxy-2-ketoglutaric acid from pyruvic acid and glyoxylic acid, by a conventional mutagenesis technique, for example, with N-methyl-N'-nitro-N-nitrosoguanidine (NTG), and then screening the resulting mutants for the inactivation in the formation of L-4-hydroxy-2-ketoglutaric acid. S16 strain is a mutant derived from OC187 and which lacks the activity for formation of L-4-hydroxy-2-ketoglutaric acid. Bacillus sp. OC187 strain has been isolated by the present inventors from soil in Machida city, Tokyo, Japan. The bacteriological properties of the strain OC187 are shown in Tables 1-1 to 1-6 below. (A) Morphological properties Morphological properties Cell morphology rod Cell size 0.8-1.0 × 3.0-4.0 µm Cell polymorphism not observed Cell motility observed Position of flagella peripheral Spore observed Spore morphology ellipse Position of spore central (B) Cultural Characteristics in Various Media Cultural Characteristics in Various Media Bouillon-Agar Medium (Meat extract) growth Abundant surface smooth color light pink gloss none diffusible pigments negative Bouillon-liquid Medium (Meat extract) surface growth not observed turbidity positive Bouillon-Gelatin Medium Liquefaction of gelatin negative Litmus milk reaction acid coagulation positive liquefaction negative (C) Physiological Properties (D) Other Properties Other Properties 1. Degradation of esculin + 2. Degradation of malonic acid - 3. Degradation of arginine + 4. Decarboxylation of lysine - 5. Decarboxylation of ornithine - 6. Deamination of phenylalanine - 7. Resistance to sodium chloride viable at 10% + : Positive, - : Negative (E) Chemotaxonomic Properties Chemotaxonomic Properties 1 ○ Base composition of DNA (G + C mol%) 45.9 2 ○ Cellular lipid · major quinone MK-7 3 ○ Diamino acid composition of cell wall peptidoglycan meso-A 2 pm The strain having the bacteriological properties mentioned above was classified according to Bergey's Manual of Systematic Bacteriology, Vol.2 (1986). As a result, the strain was classified to the genus Bacillus and was designated as the Bacillus sp. OC187. Bacillus sp. OC187 and S16 strains were deposited with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology in Japan on February 24, 1994 as FERM BP-4646 and FERM BP-4647, respectively, under the Budapest Treaty. A culture of Bacillus sp. OC187 (FERM BP-4646) and Bacillus sp. S16 (FERM BP-4647) also form part of the invention Culturing of the microorganisms used in the present invention can be carried out by using a conventional synthetic or natural medium. When a bacterium of the genus Cellvibrio or Vibrio is used to synthesize D-4-hydroxy-2-ketoglutaric acid, D-galactonic acid is usable as a carbon source. Various nitrogen-containing organic compounds are usable as nitrogen source, such as ammonia; ammonium salts, e.g., ammonium sulfate, ammonium chloride, ammonium acetate and, ammonium phosphte; urea; nitrate salts, as well as peptone, meat extract, yeast extract, corn steep liquor, and the like, so long as they can be assimilated by the microorganism used. Potassium dihydrogen phosphate, potassium hydrogen phosphate, ammonium sulfate, ammonium chloride, sodium chloride, magnesium sulfate, ferrous sulfate, manganese sulfate and the like can be used as inorganic salts, so long as they can be assimilated by the microorganism used. In addition to these salts, trace elements such as calcium, zinc, boron, copper, cobalt, molybdenum, and the like may be added to the culture medium. If necessary, the culture medium may contain a vitamin such as thiamin and biotin, an amino acid such as glutamic acid and aspartic acid, and/or a nucleic acid-related compound such as adenine and guanine. The optically active 4-hydroxy-2-ketoglutaric acid can be synthesized from pyruvic acid or a compound capable of being converted into pyruvic acid, and glyoxylic acid, by using either a method wherein the substrates are added to the culture in which the microorganism is growing or a method wherein the substrates are applied to the culture in which growth of the microorganism has been completed, to the cells isolated from the culture, or to the processed cells of microorganism. When the former method is employed, pyruvic acid and glyoxylic acid can each be added to the culture medium either at the starting point of or in the course of the microorganism growth in a concentration range of 1 to 200 g/l, preferably 20 to 200 g/l. The period of time required for the culture will vary depending upon the microorganism used. However, it takes about 10 to 100 hours. When the latter method is employed, it can be carried out by reacting 1 to 200 g/l, preferably 20 to 200 g/l of pyruvic acid, with 1 to 200 g/l, preferably 20 to 200 g/l of glyoxylic acid, in the presence of 0.1 to 200 g/l, preferably 5 to 100 g/l of cells of the microorganism or processed product thereof (in the latter case, calculated in amount prior to treatment) in an aqueous medium at a pH value of 3 to 11, preferably 5 to 9, at a temperature of 15 to 60 °C, preferably 25 to 45 °C. The reaction time is about 30 minutes to about 80 hours. Examples of the aqueous medium usable in the present invention include, for example, phosphate buffer and saline. If necessary, a surfactant such as Triton X-100 (Nacalai Tesque, Inc.) and Nymeen (Nippon oil & fats CO., LTD.) and an organic solvent such as toluene and xylene may also be added in the aqueous medium in an amount of 0.1 to 20 g/l. Any compound capable of being converted into pyruvic acid by the biocatalyst which has an activity of forming the optically acitve 4-hydroxy-2-ketoglutaric acid from pyruvic acid and glyoxylic acid, can be used instead of pyruvic acid. Examples of the compounds are saccharides such as glucose, fructose and maltose; glycerol; lactic acid, ammonium lactate and the like. Isolation of the optically active 4-hydroxy-2-ketoglutaric acid can be carried out by the conventional method used for isolation of an organic acid from an aqueous medium. For example, the reaction supernatant may be separated from the cells or processed product thereof by centrifugation and applied to either or both of an ion exchange resin and a membrane. The optically active 4-hydroxy-2-ketoglutaric acid obtainable by the present invention is useful as a material for synthesizing a medicament. For example, starting from 4-hydroxy-2-ketoglutaric acid, carbapenem antibiotics can be synthesized via 4-hydroxyproline. Conversion of 4-hydroxy-2-ketoglutaric acid into 4-hydroxyproline can be carried out according to the method described in Japanese Unexamined Patent Publication No. 266995/91. The carbapenem antibiotics can be obtained by synthesizing N-p-nitrobenzyloxycarbonyl-3-mercapto-pyrrolidine from 4-hydroxyproline according to the method described in Heterocycles, 24 , 1331 (1986) and then treating the intermediate according to the method described in Japanese Unexamined Patent Publication No. 32879/83. By the present invention, the optically active 4-hydroxy-2-ketoglutaric acid which is useful as a material for synthesizing a medicament can advantageously be obtained on industrial scale. DESCRIPTION OF THE PREFFERRED EMBODIMENTS The present invention is described in more detail by the following examples. However, the Examples are not intended to restrict the scope of the present invention. Example 1 Cellvibrio gilvus ATCC 13127 strain was inoculated into 3 ml of L medium (containing 10 g of Bactopeptone, 5 g of yeast extract and 10 g of NaCl in 1 liter of water and being adjusted to pH 7.0), and then cultured for 16 hours at 30 °C with shaking. Then, 2 ml of the culture was then inoculated into 20 ml of GNMS medium having the composition shown in Table 2 below. Composition of GNMS medium (amounts per liter) Calcium D-galactonate 10 g KH 2 PO 4 2 g (NH 4 ) 2 SO 4 2 g FeSO 4 • 7H 2 O 5 mg MnSO 4 • 7H 2 O 2 mg MgSO 4 • 7H 2 O 0.5 g CaCl 2 10 mg Yeast extract 1 g Peptone 1 g pH 7.0 Two 10 ml aliquots of this medium were put into two test tubes separately, and cultured for 20 hours. After cultivation, one test tube of the two was subjected to heat treatment at 80°C for 1 hour and the other was maintained at 30°C. In each case, the cells were collected by centrifugation, and resuspended in 1 ml of sterilized reactant solution (a) (containing 3 g of KH 2 PO 4 , 6 g of NaH 2 PO 4 , 1 g of NH 4 Cl, 0.16 g of MgSO 4 •7H 2 O, 5 g of NaCl, 11 mg of CaCl 2 , 100 mmol sodium pyruvate and 100 mmol glyoxylic acid in 1 liter of purified water and being adjusted to pH 7.0 with NaOH), and the suspension was shaken in a 2059 tube (Falcon Co.) at 37 °C for 5 hours. After the completion of reaction, the cells were removed from the suspension by centrifugation, and the amount of 4-hydroxy-2-ketoglutaric acid formed in the supernatant was determined by HPLC using SUMICHIRAL OA-5000 column (Sumitomo Chemical Co.). The results are shown in Table 3. In this determination, standard compounds for D- and L-4-hydroxy-2-ketoglutaric acid were prepared according to the methods described in J. Biol. Chem., 237, 476 (1962) and J. Biol. Chem., 238, 3660 (1963), respectively. Heat treatment Yield of 4-hydroxy-2-ketoglutaric acid (mM) D-form L-form not done 32.0 8.0 done 30.0 0.0 Example 2 Cellvibrio gilvus ATCC 13127 strain was inoculated into 9 ml of L medium and cultured for 16 hours at 30 °C with shaking. Whole amount of this culture was then inoculated into 300 ml of GNMS medium in 2 liter volume Erlenmeyer flask and cultured for 20 hours at 30°C with shaking. After heat treatment of this culture for 1 hour at 80 °C, the cells were collected by centrifugation, and suspended in 60 ml of the reactant solution (a) in 300 ml volume beaker, and the suspension was stirred while maintaining pH at 6.5 with 1 N sulfuric acid. After 6 hours, 3 ml of 2 M sodium pyruvate and 3 ml of 2 M sodium glyoxylate were added. After 24 hours from the starting point of this reaction, the cells were removed from the suspension by centrifugation, and the amount of 4-hydroxy-2-ketoglutaric acid formed in the supernatant was determined by HPLC. The amount of D-4-hydroxy-2-ketoglutaric acid was determined to be 125 mM and L-form was not detected. The above supernatant (50 ml) was passed through a column filled with Diaion PA 416 (Mitsubishi Kasei, Corp.), followed by elution by an exponential gradient of 0-6 M formic acid. Fractions containing 4-hydroxy-2-ketoglutaric acid which was eluted with 6-8 column volumes eluent were combined and concentrated under reduced pressure. Afterward, the concentrate was provided with 0.01 M Ca(OH) 2 and adjusted to pH 7.5 therewith. Acetone was added to the resulting solution to precipitate calcium salt of D-4-hydroxy-2-ketoglutaric acid. Thus, 0.5 g of calcium salt of D-4-hydroxy-2-ketoglutaric acid was obtained. Example 3 This example was carried out in the same manner as Example 1 except that Bacillus sp. OC187 strain was used instead of Cellvibrio gilvus ATCC 13127 strain. The results are shown in Table 5 (see Example 4). Example 4 Bacillus sp. OC187 strain was cultured in L medium for 6 hours at 30°C and the cells were collected. After washing with 0.05 M Tris-maleate buffer (pH6.0), the cells were suspended in the same buffer at about 1 x 10 9 cells/ml. To this suspension was added NTG to the final concentration of 600 mg/l and the suspension was maintained at 30°C for 20 minutes to mutagenize the cells. The mutagenized cells were smeared on MG agar medium having the composition shown in Table 4. Composition of MG agar medium (amounts per liter) KH 2 PO 4 2 g (NH 4 ) 2 SO 4 2 g FeSO 4 • 7H 2 O 5 mg MnSO 4 • 7H 2 O 2 mg MgSO 4 • 7H 2 O 0.5 g CaCl 2 10 mg Yeast extract 1 g Peptone 1 g Calcium D-galactonate 5 g Agar 20 g pH 7.0 The colonies formed were transferred onto L agar medium (L medium supplemented with 2 % of agar), and evaluation of the strains for their productivity of 4-hydroxy-2-ketoglutaric acid was carried out by using the same procedures as those described in Example 1 without the heat treatment. Analysis of 4-hydroxy-2-ketoglutaric acid contained in the supernatant after centrifugation, which was conducted in the same manner as in Example 1, showed the presence of a mutant, S 16, which produces only D-4-hydroxy-2-ketoglutaric acid without L-4-hydroxy-2-ketoglutaric acid. The analytical result of reaction supernatant for S 16 is shown in Table 5. Yield of 4-hydroxy-2-ketoglutaric acid (mM) Strains Heat treatment D-form L-form OC 187 not done 28.8 7.7 OC 187 done 27.6 0.0 S 16 not done 27.8 0.0

Claims: A process for producing an optically active D-4-hydroxy-2-ketoglutaric acid, which comprises: (a) allowing glyoxylic acid and pyruvic acid to coexist with a biocatalyst which is selected from cells, a culture and processed cells of a microorganism belonging to the genus Cellvibrio or Bacillus having an activity of forming the optically active D-4-hydroxy-2-ketoglutaric acid from pyruvic acid and glyoxylic acid in an aqueous medium, thereby to convert the glyoxylic acid and pyruvic acid into the optically active D-4-hydroxy-2-ketoglutaric acid; and (b) recovering the optically active D-4-hydroxy-2-ketoglutaric acid from the aqueous medium. A process according to claim 1, wherein pyruvic acid is provided from a compound capable of being converted into pyruvic acid by the biocatalyst. A process according to claim 2, wherein the compound is selected from glucose, fructose, maltose, glycerol, lactic acid and ammonium lactate. A process according to any one of the preceding claims wherein the microorganism is Bacillus sp. OC187 (FERM BP-4646) or Bacillus sp. S16 (FERM BP-4647). A process according to any one of the preceding claims, further comprising converting the D-4-hydroxy-2-ketoglutaric acid thus obtained into 4-hydroxyproline. A process according to claim 5, further comprising synthesising a carbapenem antibiotic from the 4-hydroxyproline. A culture of Bacillus sp. OC187 (FERM BP-4646). A culture of Bacillus sp. S16 (FERM BP-4647).

Inventors: HASHIMOTO SHINICHI, KATSUMATA RYOICHI, OCHIAI KEIKO, HASHIMOTO, SHINICHI, KATSUMATA, RYOICHI, OCHIAI, KEIKO
Applicants: KYOWA HAKKO KOGYO KK, KYOWA HAKKO KOGYO CO., LTD.
Application year: 1995
Main classification: C12P7
IPC codes: C12P41, C12R1, C12N1, C12P7