| PATENT NUMBER | This data is not available for free |
| PATENT GRANT DATE | April 2, 2002 |
| PATENT TITLE |
Method for producing optically active compound |
| PATENT ABSTRACT | The present invention provides a method for industrially advantageously producing (S)-4-hydroxy-2-ketoglutaric acid and for producing compounds which are formed by biosynthesis from the precursor (S)-4-hydroxy-2-ketoglutaric acid, for example, for producing the compounds (2S,4S)-4-hydroxy-L-glutamic acid and (2S,4S)-4-hydroxy-L-proline, using a recombinant microorganism carrying a recombinant DNA harboring the DNA fragment encoding 4(S)-4-hydroxy-2-ketoglutaric acid aldolase gene |
| PATENT INVENTORS | This data is not available for free |
| PATENT ASSIGNEE | This data is not available for free |
| PATENT FILE DATE | January 19, 2001 |
| PATENT FOREIGN APPLICATION PRIORITY DATA | This data is not available for free |
| PATENT REFERENCES CITED |
Cancer Research, 48, 2483-2491, 1988. J.Mol. Biol. 16, 118-133 (1966). Biochimicha Biophys. Acta. 72, 619-629 (1963). Methods in Enzymology, 272-285 (1971). Methods in Enzymology, 632-637 (1971). Tetrahedron Letters 28, 1277-1280 (1987). Stryer, Biochemistry, 3.sup.rd Edition, p 388-389, 1988. Maloy et al, J. Bacteriol, 1982, vol. 149, p 173-180. Meloche et al., Biochem Biophys Res Comm, 1975, vol. 65, p 1033-1039. |
| PATENT PARENT CASE TEXT | This data is not available for free |
| PATENT CLAIMS |
What is claimed is: 1. A method for producing (2S,4S)-4-hydroxy-L-proline, comprising: (a) reacting a biocatalyst, having activity to convert (S)-4-hydroxy-2-ketoglutaric acid into (2S,4S)-4-hydroxy-L-proline, with (S)-4-hydroxy-2-ketoglutaric acid in an aqueous medium, and (b) collecting (2S,4S)-4-hydroxy-L-proline generated in the aqueous medium. 2. A method according to claim 1, wherein the biocatalyst is a culture or cells or treated products of a microorganism. 3. A method according to claim 2, wherein the microorganism belongs to genus Escherichia or Corynebacterium. 4. A method according to claim 2, wherein the microorganism is a microorganism having resistance to proline analogs. 5. A method according to claim 2, wherein the microorganism is a microorganism having glutamic acid requirement. 6. Recombinant plasmid pKSR101. 7. A biologically pure culture of Escherichia coli FERM BP-5919. 8. A biologically pure culture of Escherichia coli FERM BP-5920. 9. A biologically pure culture of Escherichia coli FERM BP-5921. 10. A biologically pure culture of Escherichia coli FERM BP-5922. 11. A biologically pure culture of Escherichia coli FERM BP-5923. 12. A biologically pure culture of Escherichia coli FERM BP-6382 |
| PATENT DESCRIPTION |
BACKGROUND OF THE INVENTION FIELD OF THE INVENTION The present invention relates to a method for producing (S)-4-hydroxy-2-ketoglutaric acid and to methods for producing compounds which can be formed from a precursor (S)-4-hydroxy-2-ketoglutaric acid, e.g., compounds such as (2S,4S)-4-hydroxy-L-glutamic acid and (2S,4S)-4-hydroxy-L-proline. (2S,4S)-4-hydroxy-L-proline has biological activities including anti-tumor cell activity [Cancer Res.48., 2483(1988)] and anti-mast cell activity (Japanese Unexamined Patent Publication No. 63-218621). (S)-4-hydroxy-2-ketoglutaric acid and (2S,4S)-4-hydroxy-L-glutamic acid are useful for the production of (2S,4S)-4-hydroxy-L-proline. As a conventional method for producing (S)-4-hydroxy-2-ketoglutaric acid, a number of methods have been known, including a chemical deamination of threo-4-hydroxy-L-glutamic acid [Methods in Enzymology, 17 part B, 275]. The present inventors previously disclosed a method for producing (S)-4-hydroxy-2-ketoglutaric acid (Japanese Unexamined Patent Publication No. 7-289284), comprising allowing (e.g., providing) a biocatalyst, having activity to generate (S)-4-hydroxy-2-ketoglutaric acid from pyruvic acid, to act on glyoxylic acid and pyruvic acid or a compound capable of being converted into pyruvic acid through the action of the biocatalyst. Compared with the methods conventionally known, the method is far more industrially advantageous., but the method is disadvantageous in that the accumulation of (S)-4-hydroxy-2-ketoglutaric acid is less if inexpensive glucose is used as the substrate, and that expensive pyruvic acid should necessarily be used as the substrate so as to yield an accumulation level of (S)-4-hydroxy-2-ketoglutaric acid above 20 mM. The following conventional methods for producing (2S,4S)-4-hydroxy-L-glutamic acid have been known; a method comprising allowing glutamate dehydrogenase to act on chemically synthesized DL-4-hydroxy-2-ketoglutaric acid in the presence of ammonia and NADPH and separating the resulting 4(R)- and 4(S)-4-hydroxy-glutamic acid by ion exchange chromatography; a method comprising extracting (2S,4S)-4-hydroxy-L-glutamic acid from a plant (Phlox decussata) [Methods in Enzymology, 17 part B, 277]; and a method comprising allowing transaminase to act on L-4-hydroxy-2-ketoglutaric acid and cysteine sulfinic acid [Tetrahedron Letters, 28, 1277 (1987)]. The present inventors have previously disclosed a method for producing (2S,4S)-4-hydroxy-L-glutamic acid, comprising allowing (e.g., providing) a biocatalyst, having activity to generate (2S,4S)-4-hydroxy-L-glutamic acid from pyruvic acid and glyoxylic acid in the presence of an amino group donor, to act on glyoxylic acid and pyruvic acid or a compound capable of being converted into pyruvic acid (Japanese Unexamined Patent Publication No. 8-80198). The method is industrially advantageous in that only the 4(S) form can be produced; however, the method is laborious and disadvantageous in that the method further requires a step of converting (S)-4-hydroxy-L-ketoglutamic acid into (2S,4S)-4-hydroxy-L-glutamic acid by adding another bacterium to (S)-4-hydroxy-L-ketoglutamic acid after the step of synthesis of (S)-4-hydroxy-L-ketoglutamic acid so as to produce a great amount of (2S,4S)-4-hydroxy-L-glutamic acid by the method. As a conventional method for producing (2S,4S)-4-hydroxy-L-proline, the following methods have been known; a method comprising culturing a microorganism of genus Helicoceras or Acrocylindrium and extracting proline from the culture (Japanese Unexamined Patent Publication No. 5-111388); and a method comprising allowing (e.g., providing) a microorganism, having activity to convert 4-hydroxy-2-ketoglutaric acid into 4-hydroxy-L-proline, to act on 4-hydroxy-2-ketoglutaric acid (Japanese Unexamined Patent Publication No. 3-266996); and the like. However, the industrial application of these methods is difficult, because the yield of the former method is low and the latter method requires laborious procedures for separation and purification of the simultaneously generated 4(S) form and 4(R) form. SUMMARY OF THE INVENTION An object of the present invention is to provide a method for industrially advantageously producing (S)-4-hydroxy-2-ketoglutaric acid and compounds produced from the precursor (S)-4-hydroxy-2-ketoglutamic acid, for example (2S,4S)-4-hydroxy-L-glutamic acid and (2S,4S)-4-hydroxy-L-proline. The present invention relates to a method for producing an optically active compound, comprising allowing (e.g., providing) a recombinant microorganism, carrying recombinant DNA including a DNA fragment encoding (S)-4-hydroxy-2-ketoglutarate aldolase (abbreviated as "KAL gene" hereinbelow), to act on sugar and glyoxylic acid in the presence or absence of an amino group donor in an aqueous medium and collecting optically active (S)-4-hydroxy-2-ketoglutaric acid generated in the aqueous medium or a compound produced from the precursor (S)-4-hydroxy-2-ketoglutaric acid (abbreviated as "4(S)KHG" hereinbelow). BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 depicts plasmid pKSR101 and a restriction map of the plasmid; FIG. 2 depicts plasmid pKSR601 and a restriction map of the plasmid; FIG. 3 depicts the construction process of plasmid pKSR125 and a restriction map of the plasmid; and FIG. 4 depicts plasmid pKSR50 and a restriction map of the plasmid. DETAILED DESCRIPTION OF THE INVENTION The present invention relates to a method for producing (S)-4-hydroxy-2-ketoglutaric acid (4(S)KHG) or to a method for producing a compound which can be formed from the precursor 4(S)KHG. The compound, which can be produced from the precursor (S)-4-hydroxy-2-ketoglutaric acid, includes (2S,4S)-4-hydroxy-L-glutamic acid [abbreviated as "4(S)HG" hereinbelow], (2S,4S)-4-hydroxy-L-proline [abbreviated as "4(S)HYP" hereinbelow], (S)-4-hydroxy-L-glutamine, (S)-4-hydroxy-L-arigine, (S)-4-hydroxy-L-ornithine, and the like. (S)-4-hydroxy-L-glutamine, (S)-4-hydroxy-L-arigine, (S)-4-hydroxy-L-ornithine are useful as a feed additive for animals. According to the present invention, the compound can be formed directly, using the microorganism carrying the recombinant DNA harboring the KAL gene (i.e., the precursor 4(S)KHG need not be formed as an intermediate). Or, the microorganism can be used as a biocatalyst to convert the precursor 4(S)KHG to the compound. By referring to the compound from a precursor 4(S)KHG herein, we mean either technique for forming the compound. The method for producing 4(S)KHG, 4(S)HG and 4(S)HYP using a microorganism carrying a recombinant DNA harboring the KAL gene is described below. The KAL gene includes such gene derived from microorganisms of genus Escherichia, Pseudomonas, Paracoccus, Providencia, Rhizobium or Morganella; the KAL gene is preferably the gene from genus Escherichia. The method for recovering the KAL gene from, for example, genus Escherichia is now specifically described. From a microorganism having activity of 4-hydroxy-2-ketoglutarate aldolase, for example E. coli strain W3110 (ATCC 14948), the chromosomal DNA is prepared by a conventional method [Biochim. Biophys. Acta., 72, 619 (1963)]. Based on the nucleotide sequence published in a reference [R. V. Patil and E. E. Dekker, J. Bacteriol. 174, 102 (1992)], an oligonucleotide primer is synthesized. Subsequently, polymerase chain reaction (abbreviated as "PCR" hereinbelow) [R. F. Saiki et al., Science 230, 1350(1985)] is conducted on a template of the resulting chromosomal DNA to obtain the above gene. To introduce the KAL gene into a host, for example, Escherichia coli, any vector may be used, including phage vector, plasmid vector and the like, as long as the vector can be autonomously replicated or can incorporate the gene into the chromosome of a host microorganism. Vectors suitable for a Escherichia coil host include pBR322, pUC119, pACY184 and pTrS33 (Japanese Unexamined Patent Publication No. 2-227075) carrying trp promoter. A vector suitable for a host of a microorganism of genus Corynebacterium includes a vector from pCG1. A recombinant DNA from the KAL gene and a vector DNA can be prepared together with various recombinant mixtures, by digesting the two DNAs in vitro with restriction enzymes having the same restriction site, and subjecting the digested products to ligation with DNA ligase. Using the resulting recombinant mixture, the host microorganism is transformed and a transformant strain having activity to catalyze the reaction to generate 4(S)KHG from pyruvic acid and glyoxylic acid is selected, whereby the recombinant DNA can be obtained from the strain. Such recombinant DNA specifically includes pKSR101, pKSR125 and pKSR601. Transformation can be carried out according to known methods, for example, molecular cloning as described in Molecular Cloning, T. Maniatis et al., Cold Spring Harbor Laboratory, 1982. A recombinant microorganism carrying a recombinant DNA harboring the KAL gene can be prepared, by incorporating a DNA fragment carrying the genetic information into the vector DNA to prepare a recombinant DNA, and subsequently transforming a host microorganism with the resulting recombinant DNA. As such host microorganism, any microorganism may be usable, as long as the microorganism can incorporate the recombinant DNA and can express enzyme activity to catalyze the reaction to generate 4(S)KHG from pyruvic acid and glyoxylic acid on the basis of the genetic information. The microorganism may include, for example, microorganisms of genus Escherichia or Corynebacterium. More specifically, the microorganism includes for example strain ATCC 33625 of Escherichia coli K-12, Corynebacterium glutamicum ATCC13032, and Corynebacterium acetoacidophilum FERM P-4962. To produce 4(S)KHG, 4(S)HG or 4(S)HYP using the recombinant microorganism carrying the recombinant DNA harboring the KAL gene, a microorganism having at least one property of possessing a lipoate requirement or possessing a reduction or loss of malic acid synthase activity is preferably used as the host microorganism. As such microorganism, any microorganism capable of incorporating the recombinant DNA and expressing the enzyme activity to catalyze the reaction to generate 4(S)KHG from pyruvic acid and glyoxylic acid on the basis of the genetic information may be used, including for example microorganisms of genus Escherichia or Corynebacterium. More specifically, the microorganism includes for example strain ATCC 33625 of Escherichia coli K-12, Corynebacterium glutamicum ATCC 13032, and Corynebacterium acetoacidophilum FERM P-4962. More specifically, an Escherichia coli K-12 sub-strain NHK40 [lipoate requirement (lip), 4KAL deletion (eda)] may be used. The strain NHK40 was deposited with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology in Japan on Apr. 16, 1997 as FERM BP-5919 under the Budapest Treaty. To produce 4(S)HG, a microorganism in which phosphoenolpyruvate carboxylase activity is deleted is preferably used as the host microorganism. As such microorganism, any microorganism capable of incorporating the recombinant DNA-and expressing the enzyme activity to catalyze the reaction to generate 4(S)KHG from pyruvic acid and glyoxylic acid on the basis of the genetic information may be used, including for example microorganisms of genus Escherichia or Corynebacterium. More specifically, the microorganism includes, for example, strain ATCC 33625 of Escherichia coli K-12, Corynebacterium glutamicum strain ATCC 13032, and Corynebacterium acetoacidophilum FERM P-4962. More specifically, an Escherichia coli K-12 sub-strain NHK46 [lip, eda, malic acid synthase deletion (glc), phosphoenolpyruvate carboxylate deletion (ppc)] may be used. The Escherichia coli NHK46 was deposited with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology in Japan on Apr. 16, 1997 as FERM BP-5920 under the Budapest Treaty. To produce 4(S)HYP, alternatively, a microorganism having at least one property of a lipoate requirement, the reduction or deletion of malic acid synthase activity and deletion of phosphoenolpyruvate carboxylase activity and being resistant to proline analogs is more preferably used. Such proline analogs include azetidine-2-carboxylic acid, 3,4-dehydroproline and thioproline. As such microorganism, any microorganism capable of incorporating the recombinant DNA and expressing the enzyme activity to catalyze the reaction to generate 4(S)KHG from pyruvic acid and glyoxylic acid on the basis of the genetic information may be used, including for example microorganisms of genus Escherichia or Corynebacterium. More specifically, the microorganism includes strain ATCC 33625 of Escherichia coli K-12, Corynebacterium glutamicum strain ATCC 13032, and Corynebacterium acetoacidophilum FERM P-4962. More specifically, an Escherichia coli K-12 sub-strain NHK47 [having lip, eda., glc, ppc, and anti-azetidine-2-carboxylate resistance] is mentioned. The Escherichia coli strain NHK47 was deposited with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology in Japan on Apr. 16, 1997 as FERM BP-5921 under the Budapest Treaty. The various deletion strains or resistant strains mentioned above may be strains of wild type having the properties described above, or may be obtained by subjecting their parent strains with no such properties to conventional mutation process such as treatment with mutation agents for example N-methyl-N'-nitro-N-nitrosoguanidine (NTG), UV irradiation or .gamma. irradiation, coating the resulting strains on an appropriate agar plate medium, harvesting a grown mutant strain, and selecting a strain with the deletion or reduction of the objective enzyme activity compared with the parent strains or harvesting a strain more resistant to the analogs than the parent strains. Transducing the deletion mutation (transduction) from a strain with the objective deletion or resistance mutation into a desirable strain, using phage P1, allows recovery of various deletion mutant strains and resistance mutant strains for strains of the Escherichia coli K-12 [J. H. Miller, Experiments in Molecular Genetics, Cold Spring Harbor, Laboratory (1972)]. The microorganism to be used in accordance with the present invention can be cultured by conventional culturing procedures. The culture medium to be used for such culturing may be any natural medium or any synthetic medium, as long as the medium contains carbon source, nitrogen source, inorganic salts and the like, which can be assimilated by the microorganism to be used, whereby the microorganism can be cultured efficiently. Any carbon source which can be assimilated by the microorganism to be used may be usable, including sugars such as glucose, fructose, sucrose, maltose, starch, starch hydrolysate, and molasses; organic acids such as acetic acid, lactic acid and gluconic acid; or alcohols such as ethanol and propanol. Any nitrogen source which can be assimilated by the microorganism may be usable, including inorganic salts such as ammonia, ammonium sulfate, ammonium chloride, and ammonium phosphate; ammonium salts of organic acids, peptone, casein hydrolysate, meat extract, yeast extract, corn steep liquor, soy bean bran, soy bean bran hydrolysate, various fermentation bacteria and digestion products of the bacteria. Any inorganic salt which can be assimilated by the microorganism may be usable, including potassium phosphate, ammonium sulfate, ammonium chloride, sodium chloride, magnesium sulfate, ferrous sulfate and manganese sulfate. Additionally, salts of calcium, zinc, boron, copper, cobalt and molybdenum may be added as trace elements. If necessary, the culture medium may contain vitamins such as for example thiamin and biotin, amino acids such as glutamic acid and aspartic acid, and nucleic acidrelated substances such as adenine and guanine. Culturing is carried out under aerobic conditions, by agitation culture or submerged aeration agitation culture. The culturing is carried out at preferably 20 to 45.degree. C. for 10 to 96 hours at pH 5.0 to 9.0. The pH is adjusted with inorganic or organic acids, alkaline solutions, urea, calcium carbonate, and ammonia. The culture thus produced may be used as it is for the objective reaction; in the alternative, the culture may be treated, and the resulting treated product may be subjected to the subsequent reaction. The treated product includes the forms of condensate and dried product, freeze-dried product, surfactant-treated product, organic solvent-treated product, thermally treated product, enzymatically treated product, ultrasonication-treated product and mechanical disruption-treated product of the culture, and immobilized products of the bacteria or treated products of the bacteria. Examples of the aqueous medium to be used in the present invention include water; buffers such as phosphate, carbonate, acetate, borate, citrate, and Tris; and aqueous solutions containing organic solvents including alcohols such as methanol and ethanol; esters such as ethyl acetate; ketones such as acetone; and amides such as acetamide. If necessary, furthermore, surfactants such as Triton X-100 (Nacalai Tesque, Inc.) and Nonion HS204 (NOF Corporation), as well as organic solvents such as toluene and xylene, may be added at about 0.1 to 20 g/liter into the medium. The amino group donor to be used in accordance with the present invention includes ammonia; inorganic ammonium salts such as ammonium sulfate, ammonium chloride, and urea; and various amino acids such as glutamic acid. The concentration of the amino group donor is 0.1 to 100 g/liter, preferably 1 to 50 g/liter. The concentration for production of 4(S)KHG by allowing the recombinant microorganism carrying the recombinant DNA harboring the KAL gene to act on sugar and glyoxylic acid, is generally 5 to 100 g/liter. The concentrations of sugar and glyoxylic acid are both 1 to 200 g/liter, preferably 20 to 200 g/liter. Any sugar which can be assimilated by the recombinant strain may be usable, including glucose, fructose, sucrose, maltose, starch, starch hydrolysate and molasses The reaction is carried out at 15 to 80.degree. C., preferably 25 to 60.degree. C., at a pH of 3 to 11, preferably a pH of 5 to 9, for 1 to 96 hours. In the above process, 4(S)KHG may be prepared by adding glyoxylic acid at the concentration mentioned above, at the starting point or in the course of the culturing of a microorganism carrying the recombinant DNA harboring the KAL gene. Sugar may be added in advance as the culture substrate or may be added together with glyoxylic acid. The resulting 4(S)KHG may be isolated and purified by conventional purification processes of organic acids. From the reaction supernatant from which solids are removed by centrifuge, for example, 4(S)KHG can be isolated and purified by a process by means of ion exchange resin and membrane process in combination. As the sugar used for producing 4(S)HG or 4(S)HYP by allowing the recombinant microorganism carrying the recombinant DNA harboring the KAL gene to act on sugar and glyoxylic acid in an aqueous medium in the presence of an amino group donor, any sugar which can be assimilated by the recombinant strain may be used, including glucose, fructose, sucrose, maltose, starch, starch hydrolysate, and molasses. The bacterial concentration for the reaction is generally 5 to 100 g/l. The concentrations of sugar and glyoxylic acid are both 1 to 200 g/l, preferably 10 to 200 g/l. The reaction is carried out at 15 to 80.degree. C., preferably 25 to 60.degree. C. at a pH of 3 to 11, preferably a pH of 5 to 9, for 1 to 96 hours. In the process, 4(S)HG or 4(S)HYP may be prepared by adding glyoxylic acid at the concentration mentioned above at the starting point of or in the course of the culturing of a microorganism carrying the recombinant DNA harboring the KAL gene. Additionally, 4(S)HYP may also be produced by adding a biocatalyst having activity to convert 4(S)KHG into 4(S)HYP, with 4(S)KHG in the presence of an amino group donor to an aqueous medium. An example of the use of 4(S)KHG for producing 4(S)HYP include isolated and purified 4(S)KHG, a crude sample thereof which contains no 4(R)KHG or 4(R)HG therein, and a reaction solution containing 4(S)KHG formed through the reaction of a biocatalyst. The concentration of 4(S)KHG is 1 to 200 g/liter, preferably 20 to 200 g/liter. Examples of the biocatalyst having activity of converting 4(S)KHG into 4(S)HYP in the presence of the amino group donor include cells, a culture and processed cells of microorganisms having activity of converting 4(S)KHG into 4(S)HYP. Such microorganisms include microorganisms of genus Escherichia and Corynebacterium. More specifically, the microorganisms include strain ATCC 33625 of Escherichia coli K-12, which is prepared by modifying proBA gene (encoding proB and proA) coding for the enzyme of proline synthesis in Escherichia coli and then preparing plasmid pKSR25 carrying the resulting mutant proBA gene with reduced feed back inhibition, and thereafter introducing the plasmid into an Escherichia coli strain. More preferably, a mutant strain with a glutamic acid requirement is mentioned. Such a mutant strain can be prepared by subjecting its parent strain to conventional mutagenesis technique, for example, N-methyl-N'-nitro-N-nitrosoguanidine (NTG), UV irradiation or .gamma. irradiation, coating the resulting strains on an appropriate agar plate medium, harvesting a grown mutant strain, and selecting a strain with glutamic acid requirement for the growth. In a case of a microorganism of Escherichia coli K-12, furthermore, a deletion mutant strain can also be produced by transduction. Such a microorganism includes NHK3/pKSR25 strain, which is prepared by first obtaining an isocitrate dehydrogenase deletion mutation (icd) of strain ATCC 33625 of Escherichia coli K-12 to obtain strain NHK3, and subsequently introducing pKSR25 into strain NHK3; such a microorganism also includes strain (NHK3/pKSR25+pKSR50), with plasmid pKSR50 additionally containing glutamate dehydrogenase and glucose-6-phosphate dehydrogenase having been introduced therein. A host microorganism with a glutamic acid requirement and with resistance to azetidine-2-carboxylic acid and proline analogs such as 3,4-dehydroproline and thioproline is more advantageously used. Such microorganism can be obtained by subjecting its parent strain to mutagenesis and transduction; additionally, the microorganism can be obtained by introducing a plasmid having proline analog resistance into the parent strain. More specifically, Escherichia coli strain NHK23/pKSR25+pKSR50 is mentioned. Escherichia coli strains NHK3/pKSR25+pKSR50 and Escherichia coli strain NHK23/pKSR25+pKSR50 were deposited with the National Institute of Bioscience and Human-Technology, Agency of Industrial Science and Technology in Japan on Apr. 16, 1997 as FERM BP-5922 and BP-5923, respectively, under the Budapest Treaty. The concentration of the biocatalyst to be used for the reaction is generally 5 to 100 g/liter. The reaction is carried out at 15 to 80.degree. C., preferably 25 to 60.degree. C. at a pH of 3 to 11, preferably a pH of 5 to 9, for 1 to 96 hours. 4(S)HYP is produced by adding 4(S)KHG at the starting point of or in the course of culturing of a microorganism having activity of converting 4(S)KHG into 4(S)HYP in the presence of the amino group donor. 4(S)HG or 4(S)HYP thus produced can be isolated by conventional purification methods for amino acids. By a combination of an ion exchange resin and a membrane process, for example, 4(S)HG or 4(S)HYP can be isolated from a reaction supernatant from which solids are preliminarily removed-by centrifugation. EXAMPLES The present invention will now be described in more detail in the following examples. Unless otherwise specified, the general procedures for recombinant DNA were according to the method described in Molecular Cloning, A Laboratory Manual, T. Maniatis et al., Cold Spring Harbor Laboratory, 1982. |
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