EP0000560B1 - Process for the preparation of optically active alpha-hydroxycarboxylic acids - Google Patents

Process for the preparation of optically active alpha-hydroxycarboxylic acids Download PDF

Info

Publication number
EP0000560B1
EP0000560B1 EP78100464A EP78100464A EP0000560B1 EP 0000560 B1 EP0000560 B1 EP 0000560B1 EP 78100464 A EP78100464 A EP 78100464A EP 78100464 A EP78100464 A EP 78100464A EP 0000560 B1 EP0000560 B1 EP 0000560B1
Authority
EP
European Patent Office
Prior art keywords
acid
nrrl
hydroxy
ferm
hydroxycarboxylic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired
Application number
EP78100464A
Other languages
German (de)
French (fr)
Other versions
EP0000560A1 (en
Inventor
Yasuji Suhara
Hiromi Maruyama
Toyoaki Sawada
Mayumi Ogawa
Kazuteru Yokose
Morio Fujiu
Kimihiro Watanabe
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
F Hoffmann La Roche AG
Original Assignee
F Hoffmann La Roche AG
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by F Hoffmann La Roche AG filed Critical F Hoffmann La Roche AG
Publication of EP0000560A1 publication Critical patent/EP0000560A1/en
Application granted granted Critical
Publication of EP0000560B1 publication Critical patent/EP0000560B1/en
Expired legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
    • C07C51/347—Preparation of carboxylic acids or their salts, halides or anhydrides by reactions not involving formation of carboxyl groups
    • C07C51/367—Preparation of carboxylic acids or their salts, halides or anhydrides by reactions not involving formation of carboxyl groups by introduction of functional groups containing oxygen only in singly bound form
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C51/00—Preparation of carboxylic acids or their salts, halides or anhydrides
    • C07C51/347—Preparation of carboxylic acids or their salts, halides or anhydrides by reactions not involving formation of carboxyl groups
    • C07C51/377—Preparation of carboxylic acids or their salts, halides or anhydrides by reactions not involving formation of carboxyl groups by splitting-off hydrogen or functional groups; by hydrogenolysis of functional groups
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
    • C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
    • C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
    • C07D213/54—Radicals substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D213/55—Acids; Esters
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/10—Nitrogen as only ring hetero atom
    • C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P41/00—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture
    • C12P41/002—Processes using enzymes or microorganisms to separate optical isomers from a racemic mixture by oxidation/reduction reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/42—Hydroxy-carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
    • C12P7/6409—Fatty acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/07—Bacillus
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/15—Corynebacterium
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/38—Pseudomonas
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/465—Streptomyces
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/832—Bacillus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/832—Bacillus
    • Y10S435/837—Bacillus megaterium
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/843—Corynebacterium
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/874—Pseudomonas
    • Y10S435/876—Pseudomonas fluorescens
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/874—Pseudomonas
    • Y10S435/877—Pseudomonas putida
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10S—TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10S435/00—Chemistry: molecular biology and microbiology
    • Y10S435/8215—Microorganisms
    • Y10S435/822—Microorganisms using bacteria or actinomycetales
    • Y10S435/886—Streptomyces

Definitions

  • the present invention relates to a new process for the production of optically active ⁇ -hydroxycarboxylic acids, in particular a process for the production of optically pure D- or L- ⁇ -hydroxycarboxylic acids.
  • a D La-hydroxycarboxylic acid of the general formula in which R represents a branched or straight-chain alkyl radical having 1-13 C atoms or a pyridyl radical, or a salt thereof by means of enantiospecific dehydrogenase activity having microorganisms belonging to the Genera Streptomyces, Pseudomonas or Bacillus * or to the Coryneform group asymmetrically dehydrated to the a-ketocarboxylic acid or a salt thereof, releasing the acid from a salt obtained and finally the undehydrated enantiomer of a-Hydroxycarboxylic acid and optionally the a-keto carboxylic acid isolated from the reaction medium.
  • the a-ketocarboxylic acid obtained is reduced to a-hydroxycarboxylic acid in a manner known per se and this is returned to the process as the starting material.
  • racemic ⁇ -hydroxycarboxylic acids of formula I are 2-hydroxypropionic acid, 2-hydroxybutyric acid, 2-hydroxyvaleric acid, 2-hydroxy-3-methylbutyric acid, 2-hydroxycaproic acid, 2-hydroxy-4-methylvaleric acid, 2-hydroxy-3,3 -dimethylbutyric acid, 2-hydroxyheptylic acid, 2-hydroxy-4-methylcaproic acid, 2-hydroxycaprylic acid, 2-hydroxypelargonic acid, 2-hydroxydecanoic acid, 2-hydroxyundecanoic acid, 2-hydroxydodecanoic acid, 2-hydroxytridecanoic acid, 2-hydroxytetradecanoic acid, 2-hydroxypanoic acid -3-pyridylacetic acid and the like.
  • microorganisms which can be used in the process according to the invention include the microorganisms which have enantiospecific dehydrase activity and which belong to the Genera Streptomyces, Pseudomona or Bacillus or to the Coryneform group.
  • enantiospecific dehydrase activity means the ability to selectively convert one of the enantiomers of a D, La-hydroxycarboxylic acid of formula I or a salt thereof into the corresponding a-ketocarboxylic acid or a salt thereof by asymmetric dehydrogenation, while the other enantiomer in the form of an optically active a-hydroxycarboxylic acid or a salt thereof
  • the microorganism can be used in the form of the fermentation broth or in the form of an extract thereof.
  • Preferred strains which can be used in the method according to the invention are the following bacteria and actinomycetes isolated from soil samples from different regions of Japan, and strains and variants thereof which are related to these.
  • the table below shows the taxonomic characteristics of such bacteria and the location. All bacteria mentioned in this table have been deposited in the Fermentation Research Institute, Agency of Industrial Science and Technology, Chiba, Japan under the FERM-P numbers given. Corresponding cultures are also deposited in the USA: the NRRL numbers refer to cultures deposited in the United States Department of Agriculture, Northern Utilization Research and Development Division, Peoria, Illinois, the ATCC numbers refer to cultures that are in the American Type Culture Collection, Rockville, Maryland.
  • a variant of Bacillu megaterium that forms an acidic acid from glucose is a variant of Bacillu megaterium that forms an acidic acid from glucose.
  • Bacillus species somewhat similar to B. circulans and B. firmus. However, the strain differs from the former strain by the morphology of the spores and from the latter strain by the anaerobic growth.
  • the different genera of the Coryneform group all differ from the genus Arthrobacter due to the acid production from glucose, fructose and other sugars, from the genus Kuruthia due to the lack of motility, from the genus Microbacterium in that all these strains are found when heated in skimmed milk 72 ° C did not survive for 15 minutes, and from the Cellulomonas genus due to the inability to utilize cellulose.
  • strain No. 3657 is very closely related to Brevibacterium albidum. Despite some differences in the acid production from D-mannose, sucrose, inositol and glycerin, the strain FERM-P No. 3657 (NRRL B-1 1088) identified as B. albidum due to the analogy between all properties.
  • the strain FERM-P No. 3659 (NRRL 11090) can be regarded as a nitrate non-reducing variant of Ps. Putida.
  • the FERM-P No. strain is among various Pseudomonas strains. 3170 (ATCC 31302) most closely related to Ps. Cepacia. Due to the differences in gelatin liquefaction, casein hydrolysis, growth at 42 ° C and nitrate reduction, the strain cannot be considered identical to Ps. Cepacia.
  • This strain isolated from a soil sample from Uwajima-shi, Ehime Pref., Japan, develops well on various ISP agar media for actionomycetes and forms a well-developed aerial mycelium with spore-forming hyphae.
  • the spores are in the form of chains of about 50 spores per chain occur, are cylindrical, have a spiky surface and a size of 0.4-0.8 x 0.8 ⁇ 1.4 ⁇ . The formation of any other specific organ was not observed.
  • the growth (vegetative mycelium) is slightly brownish gray to light brown and the aerial mycelium is slightly gray to gray.
  • the soluble pigment is brownish to reddish. The formation of melanin-like pigment was not observed.
  • This strain isolated from a soil sample from Suwa-shi, Nagano Pref., Japan, also develops well on various ISP agar media. A well-developed vegetative mycelium forms a relatively long, straight or curved aerial mycelium. Neither vortex nor spiral formation is observed.
  • the spores, in chains of more than 50 spores per chain, are cylindrical, have a smooth surface and a size of 0.5-0.7 x 0.8-1.2 ⁇ .
  • the growth is pale yellowish brown to pale yellow and the aerial mycelium is white. A yellowish soluble pigment is formed. No melanin-like pigment was observed.
  • strains FERM-P No. 3160 (NRRL 11083) and FERM-P No. 3660 (NRRL 11091) are typical Streptomyces species.
  • the asymmetrical dehydrogenation according to the invention can be carried out, for example, by bringing the culture broth or cultured cells of the microorganisms together with a DL- ⁇ -hydroxycarboxylic acid or a salt thereof.
  • the culture broth can be prepared by inoculating the microorganism with an appropriate medium.
  • the culture medium can contain, for example, meat extract, yeast extract, peptone, corn steep liquor, other commonly sent natural substances or mixtures thereof, as well as saccharides or other carbon sources, or organic or inorganic compounds containing nitrogen, such as amino acids or nitric acid.
  • the pH of the culture medium can be adjusted to 7 by adding suitable salts, such as sodium phosphate or sodium chloride or another metal salt.
  • Submerged cultures shake cultures or stationary cultures can be used.
  • the cultivation is preferably carried out under aerobic conditions.
  • the cultivation temperature is generally in the range of 20-40 ° C, preferably in the range of 25-35 ° C.
  • the cultivation expediently takes 20-80 hours.
  • the growth of the strains reaches the stationary phase.
  • a DL-a-hydroxycarabonic acid of the general formula I or a salt thereof can be added as a substrate to the culture broth in which the growth of the strain has reached the stationary phase.
  • the concentration of the substrate is expediently 1-200 mg / ml, preferably 3-120 mg / ml.
  • the asymmetric dehydrogenation can be carried out under the conditions described above by continuing the submerged culture, the shaking cultures or the stationary culture.
  • the reaction time depends, among other things, on the species and strain of the microorganism used, the composition of the medium, the nature and the concentration of the substrate. In general, however, a reaction time of 70-360 hours is sufficient.
  • the end of the reaction can be determined by measuring the amount of reaction product by means of gas chromatographic or colorimetric methods, as described below.
  • the asymmetric dehydrogenation can also be carried out under the conditions described above by adding the substrate to the culture medium and then inoculating with the microorganisms.
  • the asymmetric dehydrogenation can also be carried out by contacting treated cell substance with DL-a-hydroxycarboxylic acids or salts thereof.
  • treated cell substance refers to all materials that have been obtained by treatment of the microorganisms and are capable of maintaining or increasing the enantiospecific dehydrogenase activity. Such materials are, for example, the mycelium or cells which have been isolated from the culture broth and washed, or lyophilized powder thereof; cell-free extract obtained from the cultivated cells or from the mycelium in a manner known per se; or purified or partially purified dehydrogenase preparations which have been obtained in a manner known per se by purification from said cell-free extracts.
  • the asymmetrical dehydrogenation can be carried out in an aqueous solution, for example a buffer solution or a fresh medium.
  • the pH of the reaction solution is usually between 7 and 8.5, in particular around 8.
  • the reaction temperature is expediently 20-60 ° C., in particular 25-50 ° C.
  • the end of this reaction can be determined by measuring the amount and the optical purity of the remaining ⁇ -hydroxycarboxylic acid and the amount of the ketocarboxylic acid formed in the culture broth or the reaction mixture by means of gas chromatography or colorimetric methods.
  • the asymmetric dehydrogenation according to the invention selectively converts one of the enantiomers of DL- ⁇ -hydroxycarboxylic acid of the general formula I or a salt thereof into the corresponding ( 1- ketocarboxylic acid or a salt thereof, while the other enantiomer in the form of the optically active a-hydroxycarboxylic acid or of a salt thereof, whether the remaining optically active substance is in D or L form depends on the nature of the microorganism used.
  • reaction products are obtained in the form of salts, the latter can be converted into the free optically active a-hydroxycarboxylic acid and a-ketocarboxylic acids by adding an acid.
  • optically active ⁇ -hydroxycarboxylic acids and the ⁇ -ketocarboxylic acids can be isolated from the reaction solution in a manner known per se, for example due to differences in solubility, by chromatographic fractionation using an ion exchange resin or silica gel or fractional distillation.
  • the compounds can also be treated with hydrazines in a manner known per se, the a-ketocarboxylic acids being selectively converted into the hydrazones, which can be separated off by customary franking methods.
  • optically pure D- or L-a-hydroxycarboxylic acids and a-ketocarboxylic acids are easily obtained in high yield.
  • the a-ketocarboxylic acid obtained is hydrogenated in a manner known per se and the hydrogenation product is returned as starting material to the asymmetric dehydrogenation.
  • the hydrogenation can be carried out in a manner known per se, preferably using a catalyst such as Raney nickel. If water is used as the solvent in the catalytic hydrogenation, the catalyst is filtered off and the filtrate is concentrated and the concentrate can then be used directly for the asymmetric dehydrogenation.
  • a-hydroxycarboxylic acids obtained by the process according to the invention can be used as intermediates for the production of pharmaceutical products, for example penicillins (cf. US Pat. No. 3839322 and the reissue thereof No. 29003 and US Pat. Nos. 3956323 and 3957758), amino acids and the like, and also can be used as biochemical reagents.
  • D- and L-lactic acid can be mentioned as important representatives of the said a-hydroxycarboxylic acids.
  • aqueous media of the following composition:
  • the amount of remaining a-hydroxycarboxylic acid stated in the examples, its optical purity and the amount of a-ketocarboxylic acid obtained can be determined by the following methods.
  • the retention times of the I.S. and the derivatives of a-hydroxycarboxylic acids are 4.4, 6.6 and 7.8 minutes, respectively, under the above conditions.
  • the areas belonging to the peaks are converted into concentration values using the calibration curve.
  • a baffled 500 ml Erlenmeyer flask with 90 ml of medium A was inoculated with Bacillus freudenreichii NRS137KH20B (FERM-P No. 3169, ATCC 31301) from the slant culture.
  • the incubation was carried out under the conditions described in Example 1 for 24 hours.
  • 3.17 g of sodium DL-2-hydroxy-4-methylvalerianate was added and fermentation was continued for 82 hours.
  • the substrate remaining in the fermentation broth contained 15.2 mg / ml free L-hydroxy acid, optical purity 100%.
  • the fermentation was therefore stopped and the fermentation solutions were combined and centrifuged.
  • the supernatant was acidified to pH 1.5 with 38 ml of 50% sulfuric acid and extracted three times with 1 liter of diethyl ether.
  • the combined ether extracts were washed three times with 10 ml of saturated sodium chloride solution and evaporated under reduced pressure.
  • 100 ml of toluene was added to the residue, and the mixture was concentrated under reduced pressure to remove water.
  • the yellowish brown, oily residue was dissolved in 400 ml of petroleum ether and the solution was kept in the refrigerator overnight.
  • Example 3 The mother liquor and washings from Example 3 containing 4-methyl-2-oxovaleric acid and a small amount of D-2-hydroxy-4-methylvaleric acid were combined and transferred to a separatory funnel. Then 1N NaOH was added with shaking until the pH of the aqueous layers reached 7.0 (addition of 299 ml of 1N NaOH). The aqueous layer was separated and concentrated to about 150 ml under reduced pressure. 100 ml of distilled water containing Raney nickel T4 (prepared from 36 g of a nickel / aluminum alloy containing 42% nickel by the method of Nishimura, Bull. Chem. Soc. Japan, 32, 61-64, 1959) were added to the residue .
  • Raney nickel T4 prepared from 36 g of a nickel / aluminum alloy containing 42% nickel by the method of Nishimura, Bull. Chem. Soc. Japan, 32, 61-64, 1959
  • the mixture was hydrogenated at room temperature under normal pressure until hydrogen uptake ceased.
  • the catalyst was filtered off.
  • the filtrate was acidified with 25 ml of 50% sulfuric acid and then extracted veirmal with 500 ml of ethyl ether.
  • the ether extracts were combined, washed with 10 ml of saturated sodium chloride solution and concentrated under reduced pressure. 100 ml of toluene were added to the residue and the mixture was again concentrated under reduced pressure to a light yellow oil. 160 ml of petroleum ether was added for the purpose of crystallization.
  • DL-2-hydroxy-4-methylvaleric acid was dissolved in medium A to a concentration of 3.0 mg / ml and the pH was adjusted to 7.0 by adding 5N NaOH.
  • a baffled Erlenmeyer flask containing 100 ml Medium A was filled with one of the strains FERM-P No. 3166, (NRRL B-11086), FERM-P No. 3657 (NRRL B-11088), FERM-P No. 3658 (NRRL 11089), FERM-P No. 3167 (ATCC 31300), FERM-P No. 3659 (NRRL 11090) and FERM-P No. 3170 (ATCC 31302) from a 2-10 day old agar slant culture and inoculated for 24-72 hours under the same conditions as in Example 2.
  • the culture broth was centrifuged for 10 minutes with cooling and the separated cells were washed twice with 100 ml of phosphate buffer (pH 7.0) containing 0.85% NaCl and then suspended in 100 ml of this buffer.
  • the cell suspension was mixed in 5 ml portions with DL-a-hydroxycarboxylic acid so that the concentration was 20 mg / ml.
  • the reaction mixture was incubated at 27 ° C for 20 minutes.
  • the activity of each organism for the different substrates was determined by the above-described spectrophotometric analysis of the corresponding 2-oxocarboxylic acid formed. The results are shown in the table below, in which reactivity is expressed as relative reactivity to the substrate DL-2-hydroxy-4-methylvaleric acid (100).
  • each strain with 2-hydroxy-4-methylvaleric acid is: 5.8; 6.0; 0.95; 5.5; 3.5 and 0.54 nmol / min / mg protein with the strains FERM-No. 3166, 3657, 3658, 3167, 3170 and 3659, respectively.
  • a baffled Erlenmeyer flask containing 100 ml of Medium A was inoculated with Brevibacterium albidum NRS-130KH20B (FERM-P No. 3657, NRRL B-11088) from the slant culture and cultured under the same conditions as in Example 2 for 24 hours.
  • the culture broth (90 ml) was added to 22 ml of sterilized 50% glycerol, suspended and stored at -196 ° C in liquid nitrogen.
  • a baffled Erlenmeyer flask containing 100 ml of Medium B was inoculated by adding 2 ml of the above thawed cell suspension and cultured under identical conditions for 24 hours. 8.8 g of sodium DL-2-hydroxy-4-methyl valerianat was added as substrate and the fermentation was continued. 72 and 120 hours after addition of the substrate, the remaining amounts of the substrate in the broth, the optical purity (expressed in% of the D-enantiomer) and the amount of 4-methyl-2-oxo-valeric acid obtained were determined by the methods described above . The results are shown in the following table:
  • a 1 liter glass fermentor with 300 ml of medium B was sterilized, then with 6 ml of the cell suspension no. 3657 inoculated and cultivated for 16 hours at 27 ° C under aerobic conditions (air flow 0.5 1 / min, 300 revolutions / minute).
  • 24.6 g of sodium 2-hydroxy-4-methylvalerianate were added as the substrate and the pH of the culture was adjusted to 8.5 by adding 2N HCl.
  • the fermentation was continued at pH 8.5 under the same conditions (air flow 0.3 l / min, 300 revolutions / minute).
  • the analysis of the substrate, 72 and 120 hours after substrate addition, is given in the following table:
  • a baffled 500 ml Erlenmeyer flask with 100 ml of medium B was inoculated with 1 ml of the cell suspension of B. albidum NRS-130KH20B (FERM-P No. 3657, NRRL B-11088) prepared according to Example 8 and added for 24 hours Cultivated at 27 ° C with stirring and the conditions described in Example 2. Then 1.48 g of the sodium salt of DL-a-hydroxy-3-pyridylacetic acid was added and the fermentation was continued for 120 hours.
  • the culture broth (pH 9.2) was centrifuged and passed through a column with 71 ml of Dowex-1x4 (acetate form, 200-400 mesh).
  • the anion exchanger was then washed with water and eluted with a mixture of 2N acetic acid / methanol (1: 1 v / v).
  • the eluate was checked by thin layer chromatography (plate: silica gel, solvent: ethyl acetate-acetic acid-water-methanol, 10: 2: 1: 2, v / v; detection: UV rays).
  • the fractions containing UV absorption spots (Rf 0.12) were combined and concentrated under reduced pressure. The residue was dissolved in 20 ml of water and lyophilized to 793 mg powder.
  • a baffled 500 ml Erlenmeyer flask containing 100 ml of medium B was inoculated with 2 ml of the cell suspension of B. albidum NRS-130KH20B (FERM-No. 3657, NRRL B-11088) prepared according to Example 8 and 24 hours cultivated at 27 ° C with shaking (180 movements / minute).
  • the culture broth was centrifuged for 10 minutes, the secreted cells were washed three times with 100 ml of 0.82% sodium chloride containing 0.02M phosphate buffer (pH 7.5) and then suspended in 100 ml of the same buffer.
  • the hydrazone mother liquor was washed twice with 50 m! Extracted diethyl ether.
  • the combined ether extracts were washed with a small amount of water and the pH of the aqueous layer was adjusted to 7.0 with shaking and addition of 1N NaOH.
  • the washed cell preparation prepared from 100 ml of the culture of B. albidum NRS-130KH20B (FERM-P No. 3657, NRRL B-1 1088) prepared according to Example 11, was washed again with distilled water and lyophilized, whereby 478 mg of lyophilisate ( dry cell powder).
  • the dry cell powder was suspended in 100 ml of 0.02 M phosphate buffer (pH 7.5).
  • the mixture was mixed with 2.34 g of sodium DL-2-hydroxy-4-methylvalerianate and, after adjusting the pH to 7.5, shaken at 27 ° C. (180 movements / minute). After shaking for 48 and 72 hours, the substrate remaining in the buffer, the optical purity and the amount of 4-methyl-2-oxovaleric acid obtained were determined by the method described above. The results are shown in the following table:

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Engineering & Computer Science (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Genetics & Genomics (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • General Chemical & Material Sciences (AREA)
  • Oil, Petroleum & Natural Gas (AREA)
  • Medicinal Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Virology (AREA)
  • Biomedical Technology (AREA)
  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
  • Enzymes And Modification Thereof (AREA)

Description

Die vorliegende Erfindung betrifft ein neues Verfahren zur Herstellung von optisch aktiven α-Hydoxycarbonsäuren, insbesondere ein Verfahren zur Herstellung von optisch reinen D- oder L-α-Hydroxycarbonsäuren.The present invention relates to a new process for the production of optically active α-hydroxycarboxylic acids, in particular a process for the production of optically pure D- or L-α-hydroxycarboxylic acids.

Die Herstellung von optisch aktiven a-Hydroxycarbonsäuren wurde bisher durch Racematspaltung von D,L-a-Hydroxycarbonsäuren mit Hilfsmitteln, wie optisch aktiven Aminen, durchgeführt. Dieses bekannte Verfahren hat sich jedoch als unwirtschaftlich erwiesen, insbesondere weil die Hilfsmittel .kostspielig sind und die Auftrennung kompliziert ist.The production of optically active a-hydroxycarboxylic acids has hitherto been carried out by resolution resolution of D, L-a-hydroxycarboxylic acids with auxiliaries such as optically active amines. However, this known method has proven to be uneconomical, in particular because the auxiliaries are expensive and the separation is complicated.

Nach dem erfindungsgemässen Verfahren ist es nun möglich, optisch reine α-Hydroxycarbonsäuren mit hoher Ausbeute in sehr einfacher Weise zu erhalten. Dieses Verfahren ist dadurch gekennzeichnet, dass man eine D,L-a-Hydroxycarbonsäure der allgemeinen Formel

Figure imgb0001
worin R einen verzweigten oder geradkettigen Alkylrest mit 1-13 C-Atomen oder einen Pyridylrest darstellt,
oder ein Salz davon mittels enantiospezifische Dehydrogenaseaktivität aufweisenden, zu den Genera Streptomyces, Pseudomonas oder Bacillus *oder zur Coryneformgruppe gehörenden Mikroorganismen asymmetrisch zur a-Ketocarbonsäure bzw. zu einem Salz davon dehydriert, aus einem erhaltenen Salz die Säure freisetzt und schliesslich das nicht dehydrierte Enantiomere der a-Hydroxycarbonsäure und gegebenenfalls die a-Ketocarbonsäure aus dem Reaktionsmedium isoliert.According to the process according to the invention, it is now possible to obtain optically pure α-hydroxycarboxylic acids in a very simple manner and in high yield. This process is characterized in that a D, La-hydroxycarboxylic acid of the general formula
Figure imgb0001
 in which R represents a branched or straight-chain alkyl radical having 1-13 C atoms or a pyridyl radical,
or a salt thereof by means of enantiospecific dehydrogenase activity having microorganisms belonging to the Genera Streptomyces, Pseudomonas or Bacillus * or to the Coryneform group asymmetrically dehydrated to the a-ketocarboxylic acid or a salt thereof, releasing the acid from a salt obtained and finally the undehydrated enantiomer of a-Hydroxycarboxylic acid and optionally the a-keto carboxylic acid isolated from the reaction medium.

Gemäss einer bevorzugten Ausführungsform des erfindungsgemässen Verfahrens wird die erhaltene a-Ketocarbonsäure in an sich bekannter Weise zur a-Hydroxycarbonsäure reduziert und diese als Ausgangsmaterial in das Verfahren zurückgeführt.According to a preferred embodiment of the process according to the invention, the a-ketocarboxylic acid obtained is reduced to a-hydroxycarboxylic acid in a manner known per se and this is returned to the process as the starting material.

Typische Beispiele von racemischen a-Hydroxycarbonsäuren der Formel I sind 2-Hydroxypropionsäure, 2-Hydroxybuttersäure, 2-Hydroxyvaleriansäure, 2-Hydroxy-3-methylbuttersäure, 2-Hydroxycapronsäure, 2-Hydroxy-4-methylvaleriansäure, 2-Hydroxy-3,3-dimethylbuttersäure, 2-Hydroxyheptylsäure, 2-Hydroxy-4-methylcapronsäure, 2-Hydroxycaprylsäure, 2-Hydroxypelargonsäure, 2-Hydroxydecansäure, 2-Hydroxyundecansäure, 2-Hydroxydodecansäure, 2-Hydroxytridecansäure, 2-Hydroxytetradecansäure, 2-Hydroxypentadecansäure, a-Hydroxy-3-pyridylessigsäure und dgl.Typical examples of racemic α-hydroxycarboxylic acids of formula I are 2-hydroxypropionic acid, 2-hydroxybutyric acid, 2-hydroxyvaleric acid, 2-hydroxy-3-methylbutyric acid, 2-hydroxycaproic acid, 2-hydroxy-4-methylvaleric acid, 2-hydroxy-3,3 -dimethylbutyric acid, 2-hydroxyheptylic acid, 2-hydroxy-4-methylcaproic acid, 2-hydroxycaprylic acid, 2-hydroxypelargonic acid, 2-hydroxydecanoic acid, 2-hydroxyundecanoic acid, 2-hydroxydodecanoic acid, 2-hydroxytridecanoic acid, 2-hydroxytetradecanoic acid, 2-hydroxypanoic acid -3-pyridylacetic acid and the like.

Die im erfindungsgemässen Verfahren verwendbaren Mikroorganismen umfassen die zu den Genera Streptomyces, Pseudomona oder Bacillus oder zur Coryneformgruppe gehörenden, enantiospezifische Dehydraseaktivität aufweisenden Mikroorganismen. Im Rahmen der vorliegenden Erfindung bedeutet der Ausdruck "enantiospezifische Dehydraseaktivität die Fähigkeit, eines der Enantiomeren einer D,L-a-Hydroxycarbonsäure der Formel I oder eines Salzes davon durch asymmetrische Dehydrierung selektiv in die entsprechende a-Ketocarbonsäure oder ein Salz davon überzuführen, während das andere Enantiomere in Form einer optisch aktiven a-Hydroxycarbonsäure oder eines Salzes davon übrig bleibt. Der Mikroorganismus kann in Form der Gärbrühe oder in Form eines Extraktes davon verwendet werden.The microorganisms which can be used in the process according to the invention include the microorganisms which have enantiospecific dehydrase activity and which belong to the Genera Streptomyces, Pseudomona or Bacillus or to the Coryneform group. In the context of the present invention, the term "enantiospecific dehydrase activity means the ability to selectively convert one of the enantiomers of a D, La-hydroxycarboxylic acid of formula I or a salt thereof into the corresponding a-ketocarboxylic acid or a salt thereof by asymmetric dehydrogenation, while the other enantiomer in the form of an optically active a-hydroxycarboxylic acid or a salt thereof The microorganism can be used in the form of the fermentation broth or in the form of an extract thereof.

Bevorzugte im erfindungsgemässen Verfahren verwendbare Stämme sind die folgenden, aus Bodenproben von verschiedenen Gegenden von Japan isolierten Bakterien und Actinomyceten, sowie mit diesen verwandte Stämme und Varianten davon. In der nachstehenden Tabelle sind die taxonomischen Merkmale solcher Bakterien sowie der Fundort angegeben. Alle in dieser Tabelle erwähnten Bakterien sind im Fermentation Research Institute, Agency of Industrial Science and Technology, Chiba, Japan unter den angegebenen FERM-P-Nummern deponiert worden. Entsprechende Kulturen sind ebenfalls in USA deponiert: die NRRL-Nummern beziehen sich auf in United States Department für Landwirtschaft, Northern Utilization Research and Development Division, Peoria, Illinois, deponierte Kulturen, die ATCC-Nummern auf Kulturen, welche in American Type Culture Collection, Rockville, Maryland, deponiert wurden.Preferred strains which can be used in the method according to the invention are the following bacteria and actinomycetes isolated from soil samples from different regions of Japan, and strains and variants thereof which are related to these. The table below shows the taxonomic characteristics of such bacteria and the location. All bacteria mentioned in this table have been deposited in the Fermentation Research Institute, Agency of Industrial Science and Technology, Chiba, Japan under the FERM-P numbers given. Corresponding cultures are also deposited in the USA: the NRRL numbers refer to cultures deposited in the United States Department of Agriculture, Northern Utilization Research and Development Division, Peoria, Illinois, the ATCC numbers refer to cultures that are in the American Type Culture Collection, Rockville, Maryland.

A-1) Morphologische Eigenschaften und Kultureigenschaften von grampositiven, Sporen bildenden StäbchenA-1) Morphological and cultural properties of gram-positive, spore-forming rods

Figure imgb0002
Figure imgb0002

A-2) Physiologische Eigenschaften von grampositiven, Sporen bildenden StäbchenA-2) Physiological properties of gram-positive, spore-forming rods

Figure imgb0003
Figure imgb0003
Figure imgb0004
Figure imgb0004
Figure imgb0005
Figure imgb0005
Figure imgb0006
Figure imgb0006
Figure imgb0007
Figure imgb0007
Figure imgb0008
Figure imgb0008
Figure imgb0009
Figure imgb0009

Aus diesen Resultaten geht hervor, dass man diese Stämme anhand von Bergey's Manual of Determinative Bacteriology (8th Ed., 1974; 7th Ed., 1957); Riichi Sakazaki, "Identification of Medical Bacteria", Kindai Shuppan, 1971; und Kazuhiko Yamada and Kazuo Komagata., J. Gen. Appl. Microbiol., 18, 417, 1972 wie folgt identifizieren kann:These results indicate that these strains can be identified using Bergey's Manual of Determinative Bacteriology (8th Ed., 1974; 7th Ed., 1957); Riichi Sakazaki, Identification of Medical Bacteria, Kindai Shuppan, 1971; and Kazuhiko Yamada and Kazuo Komagata., J. Gen. Appl. Microbiol., 18, 417, 1972 can identify as follows:

1) Stamm FERM-P No. 3164 (NRRL B-11084):1) FERM-P No. 3164 (NRRL B-11084):

Eine aus Glucose anaerob säurebildende Variante von Bacillu megaterium.A variant of Bacillu megaterium that forms an acidic acid from glucose.

2) Stamm FERM-P No. 3165 (NRRL 11085):2) strain FERM-P No. 3165 (NRRL 11085):

Bacillus species, mit gewisser Aehnlichkeit mit B. circulans und B. firmus. Der Stamm unterscheidet sich jedoch vom ersteren Stamm durch die Morphologie der Sporen und vom letzteren Stamm durch das anaerobe Wachstum.Bacillus species, somewhat similar to B. circulans and B. firmus. However, the strain differs from the former strain by the morphology of the spores and from the latter strain by the anaerobic growth.

3) Stamm FERM-P No. 3169 (ATCC 31301):3) FERM-P No. 3169 (ATCC 31301):

Als Bacillus freudenreichii identifiziert.Identified as Bacillus freudenreichii.

4) Stämme FERM-P No. 3166 (NRRL B-1 1086), FERM-P No. 3167 (ATCC 31300), FERM-P No. 3657 (NRRL B-11088) und FERM-P No. 3168 (NRRL 11087):4) Strains FERM-P No. 3166 (NRRL B-1 1086), FERM-P No. 3167 (ATCC 31300), FERM-P No. 3657 (NRRL B-11088) and FERM-P No. 3168 (NRRL 11087):

Diese vier Stämme sind aerob bis faultativ anaerob, grampositiv, nichtbeweglich, nicht-sporenbildend und nicht-säurefest, gerade bis leicht gekrümmte Stäbchen in der ersten Wachstumsphase, und kugelförmig bis eiförmig nach mehr als 1 Tag Wäshstum. Diese Eigenschaften zeigen, dass alle diese Stämme der sogenannten Coryneform-Gruppe angehören. Bekanntlich (siehe z.B. Yamada, Komagata, J. Gen. Appl. Microbiol., 18, 417, 1972) ist es schwierig, für diese Gruppe die Familie und Gattung anzugeben. Jedoch ist die folgende Indentifizierung anhand von Bergey's Manual möglich. Die verschiedenen Gattungen der Coryneform-Gruppe unterscheiden sich alle von der Gattung Arthrobacter durch die Säureproduktion aus Glucose, Fructose und anderen Zuckern, von der Gattung Kuruthia durch die fehlende Motilität, von der Gattung Microbacterium dadurch, dass alle diese Stämme beim Erhitzen in abgerahmter Milch auf 72°C während 15 Minuten nicht überleben, und von der Gattung Cellulomonas durch die Unfähigkeit, Cellulose zu verwerten.These four strains are aerobic to rotative anaerobic, gram-positive, immobile, non-spore-forming and non-acid-proof, straight to slightly curved rods in the first growth phase, and spherical to ovoid after more than 1 day of growth. These characteristics show that all these strains belong to the so-called Coryneform group. As is well known (see e.g. Yamada, Komagata, J. Gen. Appl. Microbiol., 18, 417, 1972), it is difficult to specify the family and genus for this group. However, the following identification based on Bergey's Manual is possible. The different genera of the Coryneform group all differ from the genus Arthrobacter due to the acid production from glucose, fructose and other sugars, from the genus Kuruthia due to the lack of motility, from the genus Microbacterium in that all these strains are found when heated in skimmed milk 72 ° C did not survive for 15 minutes, and from the Cellulomonas genus due to the inability to utilize cellulose.

Stamm Ferm-P No. 3168 (NRRL B-11087):Strain Ferm-P No. 3168 (NRRL B-11087):

Aus den in der Tabelle angegebenen Eigenschaften dieses Stammes geht hervor, dass er mit Corynebacterium callunae, Brevibacterium lactofermentum, B. vitaruman und C. hydrocarboclastus sehr nah verwandt ist. Die drei letzten Stämme unterscheiden sich jedoch vom Stamm No. 3168 durch ihre pleomorphen Eigenschaften und durch Granulabildung. Trotz des Unterschiedes in der Verwetung von Zuckern und in der Säureproduktion ist der Stamm FERM-P No. 3168 (NRRL B-1 1087) am nächsten mit C. hydrocarboclastus verwandt und wurde daher als eine Variante von C. hydrocarboclastus identifiziert.The properties of this strain shown in the table show that it is very closely related to Corynebacterium callunae, Brevibacterium lactofermentum, B. vitaruman and C. hydrocarboclastus. The last three strains differ from strain No. 3168 due to its pleomorphic properties and granule formation. Despite the difference in sugar usage and acid production, the strain FERM-P No. 3168 (NRRL B-1 1087) most closely related to C. hydrocarboclastus and was therefore identified as a variant of C. hydrocarboclastus.

Stamm FERM-P No. 3657 (NRRL B-11088):Strain FERM-P No. 3657 (NRRL B-11088):

Die Eigenschaften dieses Stammes, nämlich fehlende Bildung von Granula, Säureproduktion, hydrolytische Aktivitäten gegen Gelatine und Casein, positive DN-ase, negative Urease und Fehlen von Wachstum in 10% NaCI lassen darauf schliessen, dass der Stamm No. 3657 mit Brevibacterium albidum sehr nah verwandt ist. Trotz einiger Unterschiede in der Säureproduktion aus D-Mannose, Saccharose, Inosit und Glycerin wurde der Stamm FERM-P No. 3657 (NRRL B-1 1088) auf Grund der Analogie zwischen allen anderen Eigenschaften als B. albidum identifiziert.The properties of this strain, namely the lack of granule formation, acid production, hydrolytic activities against gelatin and casein, positive DN-ase, negative urease and lack of growth in 10% NaCI suggest that strain No. 3657 is very closely related to Brevibacterium albidum. Despite some differences in the acid production from D-mannose, sucrose, inositol and glycerin, the strain FERM-P No. 3657 (NRRL B-1 1088) identified as B. albidum due to the analogy between all properties.

Stämme FERM-P No. 3166 (NRRL B-11086) und FERM-P No. 3167 (ATCC 31300):Strains FERM-P No. 3166 (NRRL B-11086) and FERM-P No. 3167 (ATCC 31300):

Diese beiden Stämme sind sich sehr ähnlich und nicht voneinander unterscheidbar. Unter den in Bergey's Manual (7. und 8. Ausgabe) und Komagata et al. (J. Gen Appl. Microbiol., 18, 399, 1972) beschriebenen Arten wurde Brevibacterium tegmenticota am nächsten verwandt gefunden. Da sich jedoch diese Stämme von B. tegmenticola durch die Grösse der Zelle, die Reaktionen in Lackmusmilch und die Säureproduktion aus Maltose und Saccharose unterscheiden, kann hier nicht von Identität gesprochen werden. Es scheint daher zweckmässig, diese Stämme als neue Arten, die eine gewisse Aehnlichkeit mit B. tegmenticola haben, anzusehen.These two strains are very similar and indistinguishable from each other. Among those described in Bergey's Manual (7th and 8th editions) and Komagata et al. (J. Gen Appl. Microbiol., 18, 399, 1972) Brevibacterium tegmenticota was found to be most closely related. However, since these strains of B. tegmenticola differ in the size of the cell, the reactions in litmus milk and the acid production from maltose and sucrose, we cannot speak of identity here. It therefore seems appropriate to consider these strains as new species which are somewhat similar to B. tegmenticola.

5) Stamm FERM-P No. 3658 (NRRL 11089):5) strain FERM-P No. 3658 (NRRL 11089):

Identifiziert als Pseudomonas fluorescens.Identified as Pseudomonas fluorescens.

6) Stämme FERM-P No. 3172 (ATCC 31303) und FERM-P No. 3659 (NRRL 11090):6) Strains FERM-P No. 3172 (ATCC 31303) and FERM-P No. 3659 (NRRL 11090):

Identifiziert als Pseudomonas putida. Es wurden einige Unterschiede zwischen den Eigenschaften der beiden Stämme, wie z.B. der Nitratreduktion, festgestellt. Da man feststellen konnte, dass die Nitratreduktion bei 16-85% der Stämme dieser Art positiv war, kann der Stamm FERM-P No. 3659 (NRRL 11090) als eine Nitrat nicht-reduzierende Variante von Ps. putida angesehen werden.Identified as Pseudomonas putida. There were some differences between the characteristics of the two strains, e.g. the nitrate reduction. Since it was found that the nitrate reduction was positive in 16-85% of these strains, the strain FERM-P No. 3659 (NRRL 11090) can be regarded as a nitrate non-reducing variant of Ps. Putida.

7) Stamm FERM-P No. 3170 (ATCC 31302):7) strain FERM-P No. 3170 (ATCC 31302):

Pseudomonas species. Unter verschiedenen Pseudomonas-Stämmen ist der Stamm FERM-P No. 3170 (ATCC 31302) am nächsten mit Ps. cepacia verwandt. Auf Grund der Unterschiede in der Gelatineverflüssigung, der Caseinhydrolyse, des Wachstums bei 42°C und der Nitratreduktion kann der Stamm nicht als identisch mit Ps. cepacia angesehen werden.Pseudomonas species. The FERM-P No. strain is among various Pseudomonas strains. 3170 (ATCC 31302) most closely related to Ps. Cepacia. Due to the differences in gelatin liquefaction, casein hydrolysis, growth at 42 ° C and nitrate reduction, the strain cannot be considered identical to Ps. Cepacia.

Die isolierten Actinomyceten haben folgende Wachtumseigenschaften:The isolated Actinomycetes have the following growth characteristics: 1) Stamm FERM-P No. 3160 (NRS-79KH-1A, NRRL 11083):1) FERM-P No. 3160 (NRS-79KH-1A, NRRL 11083):

Dieser aus einer Bodenprobe von Uwajima-shi, Ehime Pref., Japan, isolierte Stamm entwickelt sich gut auf verschiedenen ISP-Agarmedien für Actionomyceten und bildet ein gutentwickeltes Luftmycel mit sporenbildenden Hyphen. Die Sporen, die in Form von Ketten von etwa 50 Sporen pro Kette vorkommen, sind zylindrisch, haben eine stachlige Oberfläche und eine Grösse von 0,4-0,8 x 0,8~1,4 µ. Die Bildung von irgendeinem anderen spezifischen Organ wurde nicht beobachtet.This strain, isolated from a soil sample from Uwajima-shi, Ehime Pref., Japan, develops well on various ISP agar media for actionomycetes and forms a well-developed aerial mycelium with spore-forming hyphae. The spores are in the form of chains of about 50 spores per chain occur, are cylindrical, have a spiky surface and a size of 0.4-0.8 x 0.8 ~ 1.4 µ. The formation of any other specific organ was not observed.

Auf den meisten getesteten Agarmedien ist das Wachstum (vegetatives Mycel) leicht bräunlichgrau bis hellbraun und das Luftmycel ist leicht grau bis grau. Das lösliche Pigment ist bräunlich bis rötlich. Die Bildung von Melanin-artigem Pigment wurde nicht beobachtet.On most agar media tested, the growth (vegetative mycelium) is slightly brownish gray to light brown and the aerial mycelium is slightly gray to gray. The soluble pigment is brownish to reddish. The formation of melanin-like pigment was not observed.

2) FERM-P No. 3600 (NRS-125KH-27A, NRRL 11091):2) FERM-P No. 3600 (NRS-125KH-27A, NRRL 11091):

Dieser aus einer Bodenprobe von Suwa-shi, Nagano Pref., Japan isolierte Stamm entwickelt sich auch gut auf verschiedenen ISP-Agarmedien. Aus einem gut entwickelten vegetativen Mycel bildet sich ein relativ langes, gerades oder gekrümmtes Luftmycel. Weder Wirbel- noch Spiralbildungen werden beobachtet. Die Sporen, in Ketten von mehr als 50 Sporen pro Kette, sind zylindrisch, haben eine glatte Oberfläche und eine Grösse von 0,5-0,7 x 0,8-1,2 µ.This strain, isolated from a soil sample from Suwa-shi, Nagano Pref., Japan, also develops well on various ISP agar media. A well-developed vegetative mycelium forms a relatively long, straight or curved aerial mycelium. Neither vortex nor spiral formation is observed. The spores, in chains of more than 50 spores per chain, are cylindrical, have a smooth surface and a size of 0.5-0.7 x 0.8-1.2 µ.

Das Wachstum ist schwach gelblich-braun bis schwach gelb und das Luftmycel ist weiss. Es bildet sich ein gelbliches lösliches Pigment. Es wurde kein Melanin-artiges Pigment beobachtet.The growth is pale yellowish brown to pale yellow and the aerial mycelium is white. A yellowish soluble pigment is formed. No melanin-like pigment was observed.

Aus den obigen Eigenschaften geht klar hervor, dass die Stämme FERM-P No. 3160 (NRRL 11083) und FERM-P No. 3660 (NRRL 11091) typische Streptomyces Species sind.It is clear from the above properties that the strains FERM-P No. 3160 (NRRL 11083) and FERM-P No. 3660 (NRRL 11091) are typical Streptomyces species.

Die erfindungsgemässe asymmetrische Dehydrierung kann beispielsweise dadurch durchgeführt werden, dass man die Kulturbrühe oder kultivierte Zellen der Mikroorganismen mit einer DL-α-Hydroxycarbonsäure oder einem Salz hiervon zusammenbringt. Die Kulturbrühe kann durch Beimpfen eines zweckmässigen Mediums mit dem Mikroorganismus hergestellt werden. Das Kulturmedium kann beispielsweise Fleichextrakt, Hefeextrakt, Pepton, Maisquellwasser, weitere üblicherweise versendete Natursubstanzen oder Gemische davon, sowie Saccharide oder andere Kohlenstoffquellen, oder organische oder anorganische, Stickstoff enthaltende Verbindungen, wie Aminosäuren oder Salpetersäure, enthalten. Nötigenfalls kann das pH des Kulturmediums durch Zugabe von geeigneten Salzen, wie Natriumphosphat oder Natriumchlorid oder eines anderen Metallsalzes auf 7 gestellt werden. Es können Submerskulturen, Schüttelkulturen oder stationäre Kulturen verwendet werden. Die Kultivierung wird jedoch vorzugsweise unter aeroben Bedingungen durchgeführt. Die Kultivierungstemperatur liegt im allgemeinen im Bereich von 20-40°C, vorzugsweise im Bereich von 25-35°C. Die Kultivierung dauert zweckmässig 20-80 Stunden. Unter den oben beschriebenen Kultivierungsbedingungen erreicht das Wachstum det Stämme die stationäre Phase. Eine DL-a-Hydroxycarabonsäure der allgemeinen Formel I oder ein Salz hiervon kann als Substrat der Kulturbrühe zugesetzt werden, in der das Wachstum des Stammes die stationäre Phase erreicht hat. Die Konzentration des Substrats ist zweckmässig 1-200 mg/mi, vorzugsweise 3-120 mg/mi. Die asymmetrische Dehydrierung kann unter den oben beschriebenen Bedingungen durch Fortsetzung der Submerskultur, der Schüttelkulturen oder der stationären Kultur durchgeführt werden. Die Reaktionszeit hängt unter anderem von der Spezies und dem Stamm des verwendeten Mikroorganismus, der Zusammensetzung des Mediums, - der Natur und der Konzentration des Substrats ab. Im allgemeinen genügt jedoch eine Reaktionszeit von 70-360 Stunden. Das Ende der Reaktion kann durch Messung der Menge an Reaktionsprodukt mittels, gas-chromatographischen oder kolorimetrischen Methoden, wie weiter unten beschrieben, bestimmt werden.The asymmetrical dehydrogenation according to the invention can be carried out, for example, by bringing the culture broth or cultured cells of the microorganisms together with a DL-α-hydroxycarboxylic acid or a salt thereof. The culture broth can be prepared by inoculating the microorganism with an appropriate medium. The culture medium can contain, for example, meat extract, yeast extract, peptone, corn steep liquor, other commonly sent natural substances or mixtures thereof, as well as saccharides or other carbon sources, or organic or inorganic compounds containing nitrogen, such as amino acids or nitric acid. If necessary, the pH of the culture medium can be adjusted to 7 by adding suitable salts, such as sodium phosphate or sodium chloride or another metal salt. Submerged cultures, shake cultures or stationary cultures can be used. However, the cultivation is preferably carried out under aerobic conditions. The cultivation temperature is generally in the range of 20-40 ° C, preferably in the range of 25-35 ° C. The cultivation expediently takes 20-80 hours. Under the cultivation conditions described above, the growth of the strains reaches the stationary phase. A DL-a-hydroxycarabonic acid of the general formula I or a salt thereof can be added as a substrate to the culture broth in which the growth of the strain has reached the stationary phase. The concentration of the substrate is expediently 1-200 mg / ml, preferably 3-120 mg / ml. The asymmetric dehydrogenation can be carried out under the conditions described above by continuing the submerged culture, the shaking cultures or the stationary culture. The reaction time depends, among other things, on the species and strain of the microorganism used, the composition of the medium, the nature and the concentration of the substrate. In general, however, a reaction time of 70-360 hours is sufficient. The end of the reaction can be determined by measuring the amount of reaction product by means of gas chromatographic or colorimetric methods, as described below.

Die asymmetrische Dehydrierung kann unter den oben beschriebenen Bedingungen auch durch Zusatz des Substrats zum Kulturmedium und darauffolgendes Beimpfen mit den Mikroorganismen vorgenommen werden.The asymmetric dehydrogenation can also be carried out under the conditions described above by adding the substrate to the culture medium and then inoculating with the microorganisms.

Die asymmetrische Dehydrierung kann ferner durch Zusammenbringen behandelter Zellsubstanz mit DL-a-Hydroxycarbonsäuren oder Salzen hiervon durchgeführt werden. Der Ausdruck "behandelte Zellsubstanz" bezeichnet alle Materialien, die durch Behandlung der Mikroorganismen erhalten wurden und fähig sind, die enantiospezifische Dehydrogenaseaktivität beizubehalten oder zu erhöhen. Solche Materialien sind beispielsweise das Mycel oder Zellen, die aus der Kulturbrühe isoliert und gewaschen wurden, oder lyophilisierte Pulver davon; in an sich bekannter Weise aus den kultivierten Zellen oder aus dem Mycel erhaltener zellfreier Extrakt; oder gereinigte oder teilweise gereinigte Dehydrogenasepräparate, welche in an sich bekannter Weise durch Reinigung aus den besagten zellfreien Extrakten erhalten wurden. Falls eine solche behandelte Zellsubstanz verwendet wird, kann die asymmetrische Dehydrierung in einer wässrigen Lösung, beispielsweise einer Pufferlösung oder einem frischem Medium, durchgeführt werden. Das pH der Reaktionslösung liegt gewöhnlich zwischen 7 und 8,5, insbesondere bei etwa 8. Die Reaktionstemperatur liegt zweckmässig bei 20-60°C, insbesondere bei 25-50°C. Das Ende dieser Reaktion kann durch Messung der Menge und der optischen Reinheit der zurückbleibenden α-Hydroxycarbonsäure sowie der Menge der in der Kulturbrühe oder dem Reaktionsgemisch gebildeten Ketocarbonsäure mittels gaschromatographischer oder kolorimetrischer Methoden bestimmt werden.The asymmetric dehydrogenation can also be carried out by contacting treated cell substance with DL-a-hydroxycarboxylic acids or salts thereof. The term "treated cell substance" refers to all materials that have been obtained by treatment of the microorganisms and are capable of maintaining or increasing the enantiospecific dehydrogenase activity. Such materials are, for example, the mycelium or cells which have been isolated from the culture broth and washed, or lyophilized powder thereof; cell-free extract obtained from the cultivated cells or from the mycelium in a manner known per se; or purified or partially purified dehydrogenase preparations which have been obtained in a manner known per se by purification from said cell-free extracts. If such a treated cell substance is used, the asymmetrical dehydrogenation can be carried out in an aqueous solution, for example a buffer solution or a fresh medium. The pH of the reaction solution is usually between 7 and 8.5, in particular around 8. The reaction temperature is expediently 20-60 ° C., in particular 25-50 ° C. The end of this reaction can be determined by measuring the amount and the optical purity of the remaining α-hydroxycarboxylic acid and the amount of the ketocarboxylic acid formed in the culture broth or the reaction mixture by means of gas chromatography or colorimetric methods.

Durch die erfindungsgemässe asymmetrische Dehydrierung wird eines der Enantiomeren der DL-α-Hydroxycarbonsäure der allgemeinen Formel I oder ein Salz hiervon selektiv in die entsprechende (1-Ketocarbonsäure oder ein Salz hiervon übergeführt, während das andere Enantiomere in Form der optisch aktiven a-Hydroxycarbonsäure oder eines Salzes davon zurückbleibt. Ob die zurückbleibende optisch aktive Substanz in D-oder L-Form vorliegt, hängt von der Natur des verwendeten Mikroorganismus ab.The asymmetric dehydrogenation according to the invention selectively converts one of the enantiomers of DL-α-hydroxycarboxylic acid of the general formula I or a salt thereof into the corresponding ( 1- ketocarboxylic acid or a salt thereof, while the other enantiomer in the form of the optically active a-hydroxycarboxylic acid or of a salt thereof, whether the remaining optically active substance is in D or L form depends on the nature of the microorganism used.

Wünscht man die D-Form, können folgende Mikroorganismen verwendet werden:

Figure imgb0010
If you want the D-shape, the following microorganisms can be used:
Figure imgb0010

Wünscht man die L-Form, können folgende Mikroorganismen verwendet werden:

Figure imgb0011
If you want the L-shape, the following microorganisms can be used:
Figure imgb0011

Falls die Reaktionsprodukte in Form von Salzen erhalten werden, können letztere durch Zusatz einer Säure in die freien optisch aktiven a-Hydroxycarbonsäure und a-Ketocarbonsäuren übergeführt werden.If the reaction products are obtained in the form of salts, the latter can be converted into the free optically active a-hydroxycarboxylic acid and a-ketocarboxylic acids by adding an acid.

Die optisch aktiven a-Hydroxycarbonsäuren und die a-Ketocarbonsäuren können in an sich bekannter Weise, beispielsweise auf Grund von Löslichkeitsunterschieden, durch chromatographische Fraktionierung mittels eines Ionenaustauscherharzes oder Silicagel oder fraktionierte Destillation, aus der Reaktionslösung isoliert werden. Die Verbindungen können auch mit Hydrazinen in an sich bekannter Weise behandelt werden, wobei die a-Ketocarbonsäuren selektiv in die Hydrazone übergeführt werden, welche durch übliche Franktionierungsmethoden abgetrennt werden können.The optically active α-hydroxycarboxylic acids and the α-ketocarboxylic acids can be isolated from the reaction solution in a manner known per se, for example due to differences in solubility, by chromatographic fractionation using an ion exchange resin or silica gel or fractional distillation. The compounds can also be treated with hydrazines in a manner known per se, the a-ketocarboxylic acids being selectively converted into the hydrazones, which can be separated off by customary franking methods.

Nach dem erfindungsgemässen Verfahren werden optisch reine D- oder L-a-Hydroxycarbonsäuren und a-Ketocarbonsäuren mit hoher Ausbeute in einfacher Weise erhalten.According to the process of the invention, optically pure D- or L-a-hydroxycarboxylic acids and a-ketocarboxylic acids are easily obtained in high yield.

In einer bevorzugten Ausführungsform des erfindungsgemässen Verfahrens wird die erhaltene a-Ketocarbonsäure in an sich bekannter Weise hydriert und das Hydrierungsprodukt als Ausgangsmaterial in die asymmetrische Dehydrierung zurückgeführt. Die Hydrierung kann in an sich bekannter Weise, vorzugsweise mittels eines Katalysators, wie Raney-Nickel, durchgeführt werden. Falls bei der katalytischen Hydrierung Wasser als Lösungsmittel verwendet wird, wird der Katalysator abfiltriert und das Filtrat konzentriert und das Konzentrat kann dann direkt für die asymmetrische Dehydrierung verwendet werden.In a preferred embodiment of the process according to the invention, the a-ketocarboxylic acid obtained is hydrogenated in a manner known per se and the hydrogenation product is returned as starting material to the asymmetric dehydrogenation. The hydrogenation can be carried out in a manner known per se, preferably using a catalyst such as Raney nickel. If water is used as the solvent in the catalytic hydrogenation, the catalyst is filtered off and the filtrate is concentrated and the concentrate can then be used directly for the asymmetric dehydrogenation.

Die nach dem erfindungsgemässen Verfahren erhaltenen a-Hydroxycarbonsäuren können als Zwischenprodukte für die Herstellung von pharmazeutischen Produkten, beispielsweise von Penicillinen (vgl. die US Patentschrift 3839322 und die Reissue davon Nr. 29003 sowie die US Patentschriften 3956323 und 3957758), Aminosäuren und dergleichen, sowie als biochemische Reagenzien verwendet werden. Als wichtige Vertreter der besagten a-Hydroxycarbonsäuren können die D- und die L-Milchsäure genannt werden.The a-hydroxycarboxylic acids obtained by the process according to the invention can be used as intermediates for the production of pharmaceutical products, for example penicillins (cf. US Pat. No. 3839322 and the reissue thereof No. 29003 and US Pat. Nos. 3956323 and 3957758), amino acids and the like, and also can be used as biochemical reagents. D- and L-lactic acid can be mentioned as important representatives of the said a-hydroxycarboxylic acids.

Die in den nachstehenden Beispielen verwendeten Medien sind wässrige Medien folgender Zusammensetzung:

Figure imgb0012
The media used in the examples below are aqueous media of the following composition:
Figure imgb0012

Die in den Beispielen angegebene Menge an verbleibender a-Hydroxycarbonsäure, deren optische Reinheit und die Menge an erhaltener a-Ketocarbonsäure kann durch folgende Methoden bestimmt werden.The amount of remaining a-hydroxycarboxylic acid stated in the examples, its optical purity and the amount of a-ketocarboxylic acid obtained can be determined by the following methods.

1) Quantitative Bestimmung der 2-Hydroxycarbonsäuren in den Fermentationsbrühen:1) Quantitative determination of the 2-hydroxycarboxylic acids in the fermentation broth:

2,0 ml Fermentationsbrühefiltrat werden in einem 1,6 g Ammoniumsulfat enthaltendem 10 ml Zentrifugenglas aufgenommen, mit 10%iger Schwefelsäure angesäuert und dann mit 2,0 ml Aethylacetat extrahiert. 1,5 ml des Aethylacetatextraktes werden unter vermindertem Druck eingedampft. Dem Rückstand werden 2 ml Toluol zugesetzt und das Gemisch wird erneut zur Trockene eingedampft. Der Rückstand wird in 0,2 ml Pyridin gelöst und 0,1 ml Benzoylchlorid werden zugesetzt. Nach 20 Minuten bei Raumtemperatur wird das Gemisch im Eisbad abgekühlt, mit 1,0 ml eines 0,4% Diäthylterephthalat (interner Standard, 1.S.) enthaltenden 5:1-Gemisches von Toluol und n-Propanol behandelt und 20 Minuten bei Raumtemperatur belassen. Nach Zusatz von 2,0 ml Wasser wird das Gemsich geschüttelt und dann 5 Minuten unter 3000 Umdrehungen pro Minute zentrifugiert Die Toluolschicht wird über wasserfreiem Natriumsulfat getrocknet und ein Teil der Lösung (gewöhnlich 2 µl) wird unter den folgenden Bedingungen durch Gaschromatrographie analysiert:2.0 ml of fermentation broth filtrate are taken up in a 10 ml centrifuge glass containing 1.6 g of ammonium sulfate, acidified with 10% strength sulfuric acid and then extracted with 2.0 ml of ethyl acetate. 1.5 ml of the ethyl acetate extract are evaporated under reduced pressure. 2 ml of toluene are added to the residue and the mixture is again evaporated to dryness. The residue is dissolved in 0.2 ml of pyridine and 0.1 ml of benzoyl chloride are added. After 20 minutes at room temperature, the mixture is cooled in an ice bath with 1.0 ml of a 0.4% Treated 5: 1 mixture of toluene and n-propanol containing diethyl terephthalate (internal standard, 1.S.) and left for 20 minutes at room temperature. After adding 2.0 ml of water, the mixture is shaken and then centrifuged for 5 minutes at 3000 revolutions per minute. The toluene layer is dried over anhydrous sodium sulfate and a part of the solution (usually 2 μl) is analyzed by gas chromatography under the following conditions:

Figure imgb0013
Figure imgb0013

Die Retentionszeiten des I.S. und der Derivate der a-Hydroxycarbonsäuren, z.B. der α-Nydroxy-γ-methylvaleriansäure und der a-Hydroxyhexansäure, sind unter den obigen Bedingungen 4,4, 6,6 bzw. 7,8 Minuten. Die den Peaks zugehörigen Flächen werden mittels der Eichkurve in Konzentrationswerte umgerechnet.The retention times of the I.S. and the derivatives of a-hydroxycarboxylic acids, e.g. α-hydroxy-γ-methylvaleric acid and a-hydroxyhexanoic acid are 4.4, 6.6 and 7.8 minutes, respectively, under the above conditions. The areas belonging to the peaks are converted into concentration values using the calibration curve.

2) Bestimmung der optischen Reinheit der α-Hydroxycarbonsäuren in Fermentationsbrühen:2) Determination of the optical purity of the α-hydroxycarboxylic acids in fermentation broths:

7 ml des Fermentationsbrühefiltrats werden mit 1 ml 10%iger Schwefelsäure angesäuert und mit 5 ml Aethylacetat extrahiert. 4 ml Teilmengen des Aethylacetatextrakts werden unter vermindertem Druck zur Trockene eingedampft. Der Rückstand wird mit 2 ml 4% HCI enthaltendem n-Propanol behandelt. Nach 3 Stunden bei Raumtemperatur wird das Gemisch unter vermindertem Druck eingedampft und die letzten Spuren von n-Propanol werden mittels Toluol entfernt. Dem Rückstand werden 0,2 ml L-(-)-Methyloxycarbonylchloridlösung (hergestellt nach der Methode von J. W. Westley, J. Org. Chem. 33, 3798, 1968) und 0,2 ml Pyridin zugesetzt. Nach 30 Minuten bei Raumtemperatur wird das Gemisch mit 2 ml kaltem Wasser behandelt. Dann werden 0,8 ml Toluol zugesetzt. Das Gemisch wird geschüttelt und zentrifugiert. Nach Trocknen mit Natriumsulfat wird ein Teil der Toluolphasen unter den gleichen Bedingungen wie für die quantitative Bestimmung der a-Hydroxycarbonsäuren durch Gaschromatographie analysiert. Beispiele von Retentionszeiten für einige aus α-Hydroxycarbonsäuren abgeleitete diastereomere Carbonate sind aus der nachstehenden Tabelle ersichtlich. Die optische Reinheit kann mittels folgender Gleichung ermittelt werden:

  • AL: Fläche des Peaks des L-Enantiomers
  • AD: (Fläche des Peaks des D-Enantiomers)/k
  • k: Verhältnis zwischen den Detektorangaben der Diastereomeren (LD/LL)
    Figure imgb0014
    Figure imgb0015
7 ml of the fermentation broth filtrate are acidified with 1 ml of 10% sulfuric acid and extracted with 5 ml of ethyl acetate. 4 ml portions of the ethyl acetate extract are evaporated to dryness under reduced pressure. The residue is treated with 2 ml of 4% HCI containing n-propanol. After 3 hours at room temperature the mixture is evaporated under reduced pressure and the last traces of n-propanol are removed using toluene. 0.2 ml of L - (-) - methyloxycarbonyl chloride solution (prepared by the method of JW Westley, J. Org. Chem. 33, 3798, 1968) and 0.2 ml of pyridine are added to the residue. After 30 minutes at room temperature, the mixture is treated with 2 ml of cold water. Then 0.8 ml of toluene are added. The mixture is shaken and centrifuged. After drying with sodium sulfate, some of the toluene phases are analyzed by gas chromatography under the same conditions as for the quantitative determination of the a-hydroxycarboxylic acids. Examples of retention times for some diastereomeric carbonates derived from α-hydroxy carboxylic acids are shown in the table below. The optical purity can be determined using the following equation:
  • AL: area of the peak of the L enantiomer
  • AD: (area of the peak of the D enantiomer) / k
  • k: ratio between the detector data of the diastereomers (LD / LL)
    Figure imgb0014
    Figure imgb0015

3) Quantitative Bestimmung der a-Ketocarbonsäuren3) Quantitative determination of the a-ketocarboxylic acids

Es wird die kolorimetrische Methode von H. Katsuki et al., Anal. Biochem. 43, 349 (1971) verwendet.The colorimetric method of H. Katsuki et al., Anal. Biochem. 43, 349 (1971).

Beispiel 1example 1

Vier mit Schikane versehene 500 ml Erlenmeyer-Kolben mit je 100 ml des Mediums A wurden im Autoklav behandelt und dann mit Bacillus sp. NRS-112KH25B (FERM-P No. 3165, NRRL 11085) aus der Schrägkultur beimpft. Die Inkubation wurde 26 Stunden bei 27°C unter Schütteln (180 Bewegungen pro Minute) durchgeführt. Danach wurden jedem Kolben 3,54 g Natrium-DL-2-hydroxy-4-methylvalerianat zugesetzt und die Fermentation wurde 84 Stunden bei 27°C unter Schütteln fotgeführt. Mittels der oben beschriebenen gaschromatographischen Methode wurde gefunden, dass das in der Kulturbrühe zurückbleibende Substrat 15,5 mg/ml freie D-Hydroxysäure (optische Reinheit: 100%) enthielt.Four baffled 500 ml Erlenmeyer flasks with 100 ml each of medium A were treated in the autoclave and then treated with Bacillus sp. Inoculated NRS-112KH25B (FERM-P No. 3165, NRRL 11085) from the slant culture. The incubation was carried out at 27 ° C. with shaking (180 movements per minute) for 26 hours. Thereafter, 3.54 g of sodium DL-2-hydroxy-4-methylvalerianate was added to each flask and the fermentation was photaged for 84 hours at 27 ° C with shaking. Using the gas chromatographic method described above, it was found that the substrate remaining in the culture broth contained 15.5 mg / ml of free D-hydroxy acid (optical purity: 100%).

Die so erhaltene Gärbrühe (390 ml) wurde mit 50 ml 20%iger Schwefelsäure angesäuert und mit 300 ml Aethyjacetat extrahiert. Die wässrigen Schichten wurden nochmals mit 100 ml Aethylacetat extrahiert. Die vereinigten Aethylacetatextrakte wurden über wasserfreiem Natriumsulfat getrocknet und unter vermindertem Druck eingeengt. Nach Kristallisation des Rückstandes aus Diäthyläther-Petroläther erhielt man 4,95 g D-2-Hydroxy-4-methylvaleriansäure, Schmelzpunkt 79,6°C,

Figure imgb0016
(c=2, 1 N NaOH), durch Massenspektrometrie mit hoher Auflösung ermittelte Bruttoformel: C6H12O3.The fermentation broth thus obtained (390 ml) was acidified with 50 ml of 20% sulfuric acid and extracted with 300 ml of ethyl acetate. The aqueous layers were extracted again with 100 ml of ethyl acetate. The combined ethyl acetate extracts were dried over anhydrous sodium sulfate and concentrated under reduced pressure. After crystallization of the residue from diethyl ether-petroleum ether, 4.95 g of D-2-hydroxy-4-methylvaleric acid were obtained, melting point 79.6 ° C.
Figure imgb0016
 (c = 2.1 N NaOH), gross formula determined by mass spectrometry with high resolution: C 6 H 12 O 3 .

Die Mutterlauge wurde unter vermindertem Druck eingeengt und der gelbliche braune, ölige Rückstand der fraktionierten Destillation unterworfen, wobei man 3,6 g 4-Methyl-2-oxo-valeriansäure in Form eines farblosen Oels erhielt, Siedepunkt 65-75°C (10 mmHg), durch Massensprektrometrie mit hoher Auflösung ermittelte Bruttoformel: C6H10O3.The mother liquor was concentrated under reduced pressure and the yellowish brown, oily residue was subjected to fractional distillation, giving 3.6 g of 4-methyl-2-oxo-valeric acid in the form of a colorless oil, boiling point 65-75 ° C (10 mmHg ), gross formula determined by mass spectrometry with high resolution: C 6 H 10 O 3 .

Beispiel 2Example 2

Ein mit Schikane versehener 500 ml Erlenmeyer-Kolben mit 90 ml des Mediums A wurde mit Bacillus freudenreichii NRS137KH20B (FERM-P No. 3169, ATCC 31301) von der Schrägkultur beimpft. Die Inkubation wurde unter den in Beispiel 1 beschriebenen Bedingungen 24 Stunden durchgeführt. Danach wurden 3,17 g Natrium-DL-2-hydroxy-4-methylvalerianat zugesetzt und die Gärung wurde 82 Stunden fortgesetezt. Das in der Fermentationsbrühe zurückbleibende Substrat enthielt 15,2 mg/mi freie L-Hydroxysäure, optische Reinheit 100%. Die so erhaltene Gärlösung wurde dann in zu Beispiel 1 analoger Weise behandelt und man erhielt 1,18 g kristalline L-2-Hydroxy-4-methyl- valeriansäure, Schmelzpunkt 79,1 °C,

Figure imgb0017
(c=2, 1 N NaOH) und 0,7 g 4-Methyl-2- oxovaleriansäure, Sidepunkt 65-75°C (10 mmHg).A baffled 500 ml Erlenmeyer flask with 90 ml of medium A was inoculated with Bacillus freudenreichii NRS137KH20B (FERM-P No. 3169, ATCC 31301) from the slant culture. The incubation was carried out under the conditions described in Example 1 for 24 hours. Then 3.17 g of sodium DL-2-hydroxy-4-methylvalerianate was added and fermentation was continued for 82 hours. The substrate remaining in the fermentation broth contained 15.2 mg / ml free L-hydroxy acid, optical purity 100%. The fermentation solution thus obtained was then treated in a manner analogous to Example 1 and 1.18 g of crystalline L-2-hydroxy-4-methylvaleric acid were obtained, melting point 79.1 ° C.
Figure imgb0017
 (c = 2.1 N NaOH) and 0.7 g of 4-methyl-2-oxovaleric acid, side point 65-75 ° C (10 mmHg).

Beispiel 3Example 3

Zehn mit Schikane versehene 500 ml Erlenmeyer=Kolben, ententhaltend je 100 ml des Mediums B, wurden mit Coryneform-Stamm NRS-130KH20B (FERM-P No. 3657, NRRL B-1 1088) von der Schräqkultur beimpft und 24 Stunden unter den gleichen Bedingun wie in Beispiel 1 bebrütet. Jedem Kolben wurden als Substrat 8,19 g Natrium-DL-2-hydroxy-4-methylvalerianat zugesetzt und die Gärung wurde 120 Stunden unter den gleichen Bedingungen fortgesetzt. Nach der oben beschriebenen Methode wurde gefunder dass 120 Stunden nach Zusatz des Substrats 35 mg/ml 4-Methyl-2-oxo- valeriansäure und 37 mg/ml reine D-Hydroxysäure vorlagen. Die Gärung wurde daher abgestellt und die Gärlösungen wurden kombiniert und zentrifugiert. Der Ueberstand wurde mit 38 ml 50%iger Schwefelsäure auf pH 1,5 angesäuert und dreimal mit 1 Liter Diäthyläther extrahiert. Die vereinigten Aetherextrakte wurden dreimal mit 10 ml gesättigter Nairiumchloridlösung gewaschen und unter vermindertem Druck eingedampft. Dem Rückstand wurden 100 ml Toluol zugesetzt und das Gemisch wurde unter vermindertem Druck zwecks Entfernung von Wasser eingeengt. Der gelblich-braune, ölige Rückstand wurde in 400 ml Petroläther gelöst und die Lösung über Nacht im Kühlschrank gehalten. Das Kristallisat wurde durch Filtrieren gesammelt, mit 50 ml kaltem Petroläther gewaschen und im Vakuum getrocknet. Man erhielt 28,1 g D-2-Hydroxy-4-methyl-valeriansäure in Form von farblosen Kristallen, Schmelzpunkt 79,9°C,

Figure imgb0018
(c=2, 1 N NaOH).Ten baffled 500 ml Erlenmeyer flasks, each containing 100 ml of medium B, were inoculated with Coryneform strain NRS-130KH20B (FERM-P No. 3657, NRRL B-1 1088) from the slant culture and 24 hours under the same Conditions incubated as in Example 1. 8.19 g of sodium DL-2-hydroxy-4-methylvalerianate was added as a substrate to each flask, and fermentation was continued for 120 hours under the same conditions. According to the method described above, it was found that 120 mg after the addition of the substrate 35 mg / ml of 4-methyl-2-oxo-valeric acid and 37 mg / ml of pure D-hydroxy acid were present. The fermentation was therefore stopped and the fermentation solutions were combined and centrifuged. The supernatant was acidified to pH 1.5 with 38 ml of 50% sulfuric acid and extracted three times with 1 liter of diethyl ether. The combined ether extracts were washed three times with 10 ml of saturated sodium chloride solution and evaporated under reduced pressure. 100 ml of toluene was added to the residue, and the mixture was concentrated under reduced pressure to remove water. The yellowish brown, oily residue was dissolved in 400 ml of petroleum ether and the solution was kept in the refrigerator overnight. The crystals were collected by filtration, washed with 50 ml of cold petroleum ether and dried in vacuo. 28.1 g of D-2-hydroxy-4-methylvaleric acid were obtained in the form of colorless crystals, melting point 79.9 ° C.
Figure imgb0018
 (c = 2.1 N NaOH).

Beispiel 4Example 4

Die 4-Methyl-2-oxovaleriansäure und eine kleine Menge D-2-Hydroxy-4-methylvaleriansäure enthaltende Mutterlauge und Waschwässer aus Beispiel 3 wurden vereinigt und in einen Scheidetrichter verbracht. Dann wurde unter Schütteln 1 N NaOH zugesetzt bis das pH der wässrigen Schichten 7,0 erreichte (Zugabe von 299 ml 1 N NaOH). Die wässrige Schicht wurde abgetrennt und unter vermindertem Druck auf etwa 150 ml eingeengt. Dem Rückstand wurden 100 ml destilliertes Wasser, enthaltend Raney-Nickel T4 (hergestellt aus 36 g einer 42% Nickel enthaltenden Nickel/Aluminiumlegierung nach der Methode von Nishimura, Bull. Chem. Soc. Japan, 32, 61-64, 1959), zugesetzt. Das Gemisch wurde bei Raumtemperatur unter normalem Druck bis zum Ende der Wasserstoffaufnahme hydriert. Der Katalysator wurde abfiltriert. Das Filtrat wurde mit 25 ml 50%iger Schwefelsäure angesäuert und dann veirmal mit 500 ml Aethyläther extrahiert. Die Aetherextrakte wurden vereinigt, mit 10 ml gesättigter Natriumchloridlösung gewaschen und unter vermindertem Druck eingeengt. Dem Rückstand wurden 100 ml Toluol zugesetzt und das Gemisch wurde nochmals unter vermindertem Druck zu einem leicht gelben Oel eingeengt. Zwecks Kristallisation wurden 160 ml Petroläther zugesetzt. Das Gemisch wurde über Nacht im Kühlschrank behalten, dann filtriert und die Kristalle wurden mit kaltem Petroläther gewaschen, wobei man 32,3 g 2-Hydroxy-4-methylvaleriansäure in Form von fablosen Kristallen erhielt Schmelzpunkt 73,9°C,

Figure imgb0019
(c=2, 1 N NaOH).The mother liquor and washings from Example 3 containing 4-methyl-2-oxovaleric acid and a small amount of D-2-hydroxy-4-methylvaleric acid were combined and transferred to a separatory funnel. Then 1N NaOH was added with shaking until the pH of the aqueous layers reached 7.0 (addition of 299 ml of 1N NaOH). The aqueous layer was separated and concentrated to about 150 ml under reduced pressure. 100 ml of distilled water containing Raney nickel T4 (prepared from 36 g of a nickel / aluminum alloy containing 42% nickel by the method of Nishimura, Bull. Chem. Soc. Japan, 32, 61-64, 1959) were added to the residue . The mixture was hydrogenated at room temperature under normal pressure until hydrogen uptake ceased. The catalyst was filtered off. The filtrate was acidified with 25 ml of 50% sulfuric acid and then extracted veirmal with 500 ml of ethyl ether. The ether extracts were combined, washed with 10 ml of saturated sodium chloride solution and concentrated under reduced pressure. 100 ml of toluene were added to the residue and the mixture was again concentrated under reduced pressure to a light yellow oil. 160 ml of petroleum ether was added for the purpose of crystallization. The mixture was kept in the refrigerator overnight, then filtered and the crystals washed with cold petroleum ether to give 32.3 g of 2-hydroxy-4-methylvaleric acid in the form of fabulous crystals, melting point 73.9 ° C.,
Figure imgb0019
 (c = 2.1 N NaOH).

Beispiel 5Example 5

DL-2-Hydroxy-4-methylvaleriansäure wurde bis zu einer Konzentration von 3,0 mg/ml im Medium A gelöst und das pH durch Zugabe von 5N NaOH auf 7,0 gestellt. Drei 500 ml Erlenmeyer-Kolben, enthaltend je 100 ml des so hergestellten Mediums wurden im Autoklaven sterilisiert und dann mit einem der folgenden Stämme beimpft: Bacillus megaterium NRS-85KH20B (FERM-P No. 3164, NRRL B-11084), Pseudomonas fluorescens NRS-127CzH5B (FERM-P No. 3658, NRRL 11089) und Streptomyces sp. NRS79KH1A (FERM-P No. 3160, NRRL 11083). Die Kultivierung wurde unter Schütteln (180 Bewegungen/Minute) bei 27°C durchgeführt. Nach 72 und 144 Stunden wurde das Substrat, wie oben beschrieben, gaschromatographisch analysiert. Es wurden folgende Resultate ermittelt:

Figure imgb0020
DL-2-hydroxy-4-methylvaleric acid was dissolved in medium A to a concentration of 3.0 mg / ml and the pH was adjusted to 7.0 by adding 5N NaOH. Three 500 ml Erlenmeyer flasks, each containing 100 ml of the medium thus prepared, were sterilized in an autoclave and then inoculated with one of the following strains: Bacillus megaterium NRS-85KH20B (FERM-P No. 3164, NRRL B-11084), Pseudomonas fluorescens NRS -127CzH5B (FERM-P No. 3658, NRRL 11089) and Streptomyces sp. NRS79KH1A (FERM-P No. 3160, NRRL 11083). The cultivation was carried out with shaking (180 movements / minute) at 27 ° C. After 72 and 144 hours, the substrate was analyzed by gas chromatography as described above. The following results were found:
Figure imgb0020

Beispiel 6Example 6

Nach der Methode von Beispiel 5 wurden folgende Stämme im Medium A, das 3,0 mg/mi DL-2-Hydroxy-4-methylvaleriansäure enthielt, kultiviert: Bacillus freudenreichii NRS-137KH20B (FERM-P No. 3169, ATCC 31301), Pseudomonas putida NRS-146KH6B (FERM-P No. 3172, ATCC 31303), Pseudomonas sp. NRS-139KH6B (FERM-P No. 3170, ATCC 31302), Coryne-Form NRS-135KH9B (FERM-P No. 3168, NRRL B-11087) und Streptomyces sp. NRS-125KH27A (FERM-P No. 3660, NRRL 11091). Die Gärlösung wurde unter Verwendung der oben angegebenen Methode nach 72 und 144 Stunden analysiert. Die Resultate sind'in der folgenden Tabelle angegeben:

Figure imgb0021
The following strains were cultured according to the method of Example 5 in medium A which contained 3.0 mg / ml DL-2-hydroxy-4-methylvaleric acid: Bacillus freudenreichii NRS-137KH20B (FERM-P No. 3169, ATCC 31301), Pseudomonas putida NRS-146KH6B (FERM-P No. 3172, ATCC 31303), Pseudomonas sp. NRS-139KH6B (FERM-P No. 3170, ATCC 31302), Coryne form NRS-135KH9B (FERM-P No. 3168, NRRL B-11087) and Streptomyces sp. NRS-125KH27A (FERM-P No. 3660, NRRL 11091). The fermentation solution was analyzed after 72 and 144 hours using the method given above. The results are shown in the following table:
Figure imgb0021

Beispiel 7Example 7

Ein mit Schikane versehener, 100 ml Medium A enthaltender Erlenmeyer-Kolben wurde mit einem der Stämme FERM-P No. 3166, (NRRL B-11086), FERM-P No. 3657 (NRRL B-11088), FERM-P No. 3658 (NRRL 11089), FERM-P No. 3167 (ATCC 31300), FERM-P No. 3659 (NRRL 11090) und FERM-P No. 3170 (ATCC 31302) ab einer 2-10 Tage alten Agar-Schrägkultur beimpft und 24-72 Stunden unter den gleichen Bedingungen wie in Beispiel 2 kultiviert. Die Kulturbrühe wurde 10 Minuten unter Kühlung zentrifugiert und die ausgeschiedenen Zellen wurden zweimal mit 100 ml 0,85% NaCI enthaltendem Phosphatpuffer (pH 7,0) gewaschen und dann in 100 ml dieses Puffers suspendiert. Die Zellsuspension wurde in 5-ml-Portionen mit DL-a-Hydroxycarbonsäure versetzt, sodass die Konzentration 20 mg/ml betrug. Das Reaktionsgemisch wurde 20 Minuten bei 27°C inkubiert. Die Aktivität jedes Organismus für die verschiedenen Substrate wurde durch die oben beschriebene spektrophotometrische Analyse der entsprechenden entstandenen 2-Oxocarbonsäure ermittelt. Die Resultate sind in der nachstehenden Tabelle angegeben, worin die Reaktivität als relative Reaktivität gegenüber dem Substrat DL-2-Hydroxy-4-methylvaleriansäure (100) ausgedrückt ist. Die spezifische Aktivität jedes Stamms mit 2-Hydroxy-4-methylvaleriansäure ist: 5,8; 6,0; 0,95; 5,5; 3,5 und 0,54 nMol/min/mg Protein mit den Stämmen FERM-No. 3166, 3657, 3658, 3167, 3170 bzw. 3659.A baffled Erlenmeyer flask containing 100 ml Medium A was filled with one of the strains FERM-P No. 3166, (NRRL B-11086), FERM-P No. 3657 (NRRL B-11088), FERM-P No. 3658 (NRRL 11089), FERM-P No. 3167 (ATCC 31300), FERM-P No. 3659 (NRRL 11090) and FERM-P No. 3170 (ATCC 31302) from a 2-10 day old agar slant culture and inoculated for 24-72 hours under the same conditions as in Example 2. The culture broth was centrifuged for 10 minutes with cooling and the separated cells were washed twice with 100 ml of phosphate buffer (pH 7.0) containing 0.85% NaCl and then suspended in 100 ml of this buffer. The cell suspension was mixed in 5 ml portions with DL-a-hydroxycarboxylic acid so that the concentration was 20 mg / ml. The reaction mixture was incubated at 27 ° C for 20 minutes. The activity of each organism for the different substrates was determined by the above-described spectrophotometric analysis of the corresponding 2-oxocarboxylic acid formed. The results are shown in the table below, in which reactivity is expressed as relative reactivity to the substrate DL-2-hydroxy-4-methylvaleric acid (100). The specific activity of each strain with 2-hydroxy-4-methylvaleric acid is: 5.8; 6.0; 0.95; 5.5; 3.5 and 0.54 nmol / min / mg protein with the strains FERM-No. 3166, 3657, 3658, 3167, 3170 and 3659, respectively.

Die Zellen wurden durch Beschallung (70 W, 15 Minuten) aufgebrochen und 10 Minuten unter Kühlung zentrifugiert. Der erhaltene Ueberstand (Rohextrakt), wurde wie oben beschrieben, mit verschiedenen Substraten in einem 0,02 M Phosphatpuffer (pH 7,0) oder einem 0,02 M Trishydrochloridpuffer (pH 8,0) inkubiert. Es wurden ähnliche relative Aktivitäten gefunden, obwohl die spezifische Aktivität kleiner war, z.B. 0,50 nMol/Min/mg Protein mit dem Stamm No. 3657.

Figure imgb0022
Substrat:
Figure imgb0023
 The cells were disrupted by sonication (70 W, 15 minutes) and centrifuged for 10 minutes with cooling. The supernatant obtained (crude extract) was incubated as described above with various substrates in a 0.02 M phosphate buffer (pH 7.0) or a 0.02 M trishydrochloride buffer (pH 8.0). Similar relative activities were found, although the specific activity was lower, for example 0.50 nmol / min / mg protein with strain No. 3657.
Figure imgb0022
 Substrate:
Figure imgb0023

Beispiel 8Example 8

Ein mit Schikane versehener, 100 ml Medium A enthaltender Erlenmeyer-Kolben wurde mit Brevibacterium albidum NRS-130KH20B (FERM-P No. 3657, NRRL B-11088) von der Schrägkultur beimpft und unter den gleichen Bedingungen wie in Beispiel 2 24 Stunden kultiviert. Die Kulturbrühe (90 ml) wurde zu 22 ml sterilisiertem 50%igem Glycerin gegeben, suspendiert und bei -196°C in flüssigem Stickstoff aufbewahrt.A baffled Erlenmeyer flask containing 100 ml of Medium A was inoculated with Brevibacterium albidum NRS-130KH20B (FERM-P No. 3657, NRRL B-11088) from the slant culture and cultured under the same conditions as in Example 2 for 24 hours. The culture broth (90 ml) was added to 22 ml of sterilized 50% glycerol, suspended and stored at -196 ° C in liquid nitrogen.

Ein mit Schikane versehener, 100 ml des Mediums B enthaltender Erlenmeyer-Kolben wurde durch Zugabe von 2 ml der obigen, aufgetauten Zellsuspension beimpft und 24 Stunden unter identischen Bedingungen kultiviert. Dieser Kultur wurden 8,8 g Natrium-DL-2-hydroxy-4-methyl- valerianat als Substrat zugesetzt und die Fermentation wurde fortgesetzt. 72 und 120 Stunden nach Zusatz des Substrats wurden die zurückbleibenden Mengen des Substrats in der Brühe, die optische Reinheit (ausgedrückt in % des D-Enantiomeren) und die Menge der erthaltenen 4-Methyl-2-oxo- valeriansäure durch die oben beschriebenen Methoden ermittelt. Die Resultate sind in der folgenden Tabelle angegeben:

Figure imgb0024
A baffled Erlenmeyer flask containing 100 ml of Medium B was inoculated by adding 2 ml of the above thawed cell suspension and cultured under identical conditions for 24 hours. 8.8 g of sodium DL-2-hydroxy-4-methyl valerianat was added as substrate and the fermentation was continued. 72 and 120 hours after addition of the substrate, the remaining amounts of the substrate in the broth, the optical purity (expressed in% of the D-enantiomer) and the amount of 4-methyl-2-oxo-valeric acid obtained were determined by the methods described above . The results are shown in the following table:
Figure imgb0024

Beispiel 9Example 9

Ein 1 Liter-Glasfermentor mit 300 ml des Mediums B wurde sterilisiert, dann mit 6 ml der wie in Beispiel 8 hergestellten Zellsuspension No. 3657 beimpft und 16 Stunden bei 27°C unter aeroben Bedingungen (Luftströmung 0,5 1/Min, 300 Umdrehungen/Minute) kultiviert. Als Substrat wurden 24,6 g Natrium-2-hydroxy-4-methylvalerianat zugesetzt und das pH der Kultur durch Zusatz von 2N HCI auf 8,5 eingestellt. Die Fermentation wurde bei pH 8,5 unter den gleichen Bedingungen fortgeführt (Luftströmung 0,3 1/Min, 300 Umdrehungen/Minute). Die Analyse des Substrats, 72 und 120 Stunden nach Substratzugabe, ist in der folgenden Tabelle angegeben:

Figure imgb0025
A 1 liter glass fermentor with 300 ml of medium B was sterilized, then with 6 ml of the cell suspension no. 3657 inoculated and cultivated for 16 hours at 27 ° C under aerobic conditions (air flow 0.5 1 / min, 300 revolutions / minute). 24.6 g of sodium 2-hydroxy-4-methylvalerianate were added as the substrate and the pH of the culture was adjusted to 8.5 by adding 2N HCl. The fermentation was continued at pH 8.5 under the same conditions (air flow 0.3 l / min, 300 revolutions / minute). The analysis of the substrate, 72 and 120 hours after substrate addition, is given in the following table:
Figure imgb0025

Beispiel 10Example 10

Ein mit Schikane versehener 500 ml Erlenmeyer-Kolben mit 100 ml des Mediums B wurde mit 1 ml der gemäss Beispiel 8 hergestellten Zellsuspension von B. albidum NRS-130KH20B (FERM-P No. 3657, NRRL B-11088) beimpft und 24 Stunden bei 27°C unter Rühren und den in Beispiel 2 beschriebenen Bedingungen kultiviert. Dann wurden 1,48 g des Natriumsalzes der DL-a-Hydroxy-3-pyridylessigsäure zugesetzt und die Fermentation wurde 120 Stunden fortgeführt. Die Kulturbrühe (pH 9,2) wurde zentrifugiert und durch eine Säule mit 71 ml Dowex-1x4 (Acetatform, 200-400 mesh) geführt. Der Anionenaustauscher wurde dann mit Wasser gewaschen und mit einem Gemisch von 2N Essigsäure/Methanol (1:1 v/v) eluiert. Das Eluat wurde durch Dünnschichtchromatographie (Platte: Silicagel, Lösungsmittel: Aethylacetat-Essigsäure-Wasser-Methanol, 10:2:1:2, v/v; Nachweis: UV-Strahlen) kontrolliert. Die UV-Absorptionsflecken (Rf 0,12) aufweisenden Fraktionen wurden vereinigt und unter vermindertem Druck eingeengt. Der Rückstand wurde in 20 ml Wasser gelöst und zu 793 mg Pulver lyophilisiert. Nach Kristallisation aus Methanol-Aethylacetat erhielt man 625 mg optisch aktive a-Hydroxy-3-pyridylessigsäure in Form von farblosen Kristallen, Schmelzpunkt 140°C,

Figure imgb0026
(c=1, H20), Mol. Formel: C7H7NO3.A baffled 500 ml Erlenmeyer flask with 100 ml of medium B was inoculated with 1 ml of the cell suspension of B. albidum NRS-130KH20B (FERM-P No. 3657, NRRL B-11088) prepared according to Example 8 and added for 24 hours Cultivated at 27 ° C with stirring and the conditions described in Example 2. Then 1.48 g of the sodium salt of DL-a-hydroxy-3-pyridylacetic acid was added and the fermentation was continued for 120 hours. The culture broth (pH 9.2) was centrifuged and passed through a column with 71 ml of Dowex-1x4 (acetate form, 200-400 mesh). The anion exchanger was then washed with water and eluted with a mixture of 2N acetic acid / methanol (1: 1 v / v). The eluate was checked by thin layer chromatography (plate: silica gel, solvent: ethyl acetate-acetic acid-water-methanol, 10: 2: 1: 2, v / v; detection: UV rays). The fractions containing UV absorption spots (Rf 0.12) were combined and concentrated under reduced pressure. The residue was dissolved in 20 ml of water and lyophilized to 793 mg powder. After crystallization from methanol-ethyl acetate, 625 mg of optically active a-hydroxy-3-pyridylacetic acid were obtained in the form of colorless crystals, melting point 140 ° C.
Figure imgb0026
 (c = 1, H 2 0), mol. Formula: C 7 H 7 NO 3 .

Die Säule wurde dann mit einem Gemsich von 1N HCI und Aceton (1:1, v/v) eluiert. Das Eluat wurde nach dem gleichen Dünnschichtchromatographiesystem kontrolliert. Die UV-Absorptionsflecken (Rf 0,37) aufweisenden Fraktionen wurden vereinigt, mit Wasser verdünnt und dann durch eine Säule, enthaltend 30 ml Dowex-50 (H+-Form), gegeben. Die Säule wurde mit Wasser gewaschen und mit 1%igem wässrigem Ammoniak eluiert. Das Eluat wurde unter vermindertem Druck eingeengt, lyophilisiert und das entstandene Pulver aus Methanol kristallisiert. Man erhielt 326 mg cx-Oxo-3-pyridylessig- säure in Form von farblosen Kristallen, Schmelzpunkt 177-179°C (Zers.), Mol. Formel: C7H5NO3.The column was then eluted with a mixture of 1N HCl and acetone (1: 1, v / v). The eluate was checked using the same thin-layer chromatography system. The fractions having UV absorption spots (Rf 0.37) were combined, diluted with water and then passed through a column containing 30 ml of Dowex-50 (H + form). The column was washed with water and eluted with 1% aqueous ammonia. The eluate was concentrated under reduced pressure, lyophilized and the resulting powder crystallized from methanol. 326 mg of cx-oxo-3-pyridylacetic acid were obtained in the form of colorless crystals, melting point 177-179 ° C. (dec.), Mol. Formula: C 7 H 5 NO 3 .

Beispiel 11 1Example 11 1

Ein mit Schikane versehener 500 ml Erlenmeyer-Kolben, enthaltend 100 ml des Mediums B, wurde mit 2 ml der nach Beispiel 8 hergestellten Zellsuspension von B. albidum NRS-130KH20B (FERM-No. 3657, NRRL B-11088) beimpft und 24 Stunden bei 27°C unter Schütteln (180 Bewegungen/Minute) kultiviert. Die Kulturbrühe wurde 10 Minuten zentrifugiert, die ausgeschiedenen Zellen wurden dreimal mit 100 ml 0,85% Natriumchlorid enthaltendem 0,02M Phosphatpuffer (pH 7,5) gewaschen und dann in 100 ml des gleichen Puffers suspendiert. 50 ml dieser Suspension wurden in einen 500 ml Erlenmeyer-Kolben umgeschüttet, mit 1,14 g Natrium DL-2-Hydroxyoctanoat versetzt und der Kolben 48 Stunden bei 27°C geschüttelt. Die Zellen wurden durch Zentrifugieren aus dem Reaktionsgemisch entfernt, der Ueberstand durch Zusatz von 50%iger Schwefelsäure auf pH 1,5 angesäuert und dreimal mit 50 ml Diäthyläther extrahiert. Die vereinigten Aetherextrakte wurden mit Wasser gewaschen und unter vermindertem Druck eingedampft. Der Rückstand wurde mit 26 ml 2,4-Dinitrophenylhydrazin-Reagens, hergestellt nach der Methode von C.D. Johnson, J. Am. Chem. Soc. 73, 5888, (1951), versetzt und das Gemisch über Nacht bei Raumtemperatur stehen gelassen. Die entstandenen gelben Kristalle wurden gesammelt und aus Benzol umkristallisiert. Man erhielt 761 mg 2,4-Dinitrophenylhydrazon der 2-Oxooctansäure, Schmelzpunkt 137°C, Mol. Formel: C14H18N4O6.A baffled 500 ml Erlenmeyer flask containing 100 ml of medium B was inoculated with 2 ml of the cell suspension of B. albidum NRS-130KH20B (FERM-No. 3657, NRRL B-11088) prepared according to Example 8 and 24 hours cultivated at 27 ° C with shaking (180 movements / minute). The culture broth was centrifuged for 10 minutes, the secreted cells were washed three times with 100 ml of 0.82% sodium chloride containing 0.02M phosphate buffer (pH 7.5) and then suspended in 100 ml of the same buffer. 50 ml of this suspension were poured into a 500 ml Erlenmeyer flask, 1.14 g of sodium DL-2-hydroxyoctanoate were added and the flask was shaken at 27 ° C. for 48 hours. The cells were removed from the reaction mixture by centrifugation, the supernatant was acidified to pH 1.5 by adding 50% strength sulfuric acid and extracted three times with 50 ml of diethyl ether. The combined ether extracts were washed with water and evaporated under reduced pressure. The residue was treated with 26 ml of 2,4-dinitrophenylhydrazine reagent, prepared by the method of CD Johnson, J. Am. Chem. Soc. 73, 5888, (1951), and the mixture was left overnight at room temperature. The resulting yellow crystals were collected and recrystallized from benzene. This gave 761 mg of 2,4-dinitrophenylhydrazone of 2-oxooctanoic acid, melting point 137 ° C., mol. Formula: C 14 H 18 N 4 O 6 .

Die Hydrazon-Mutterlauge wurde zweimal mit 50 m! Diäthyläther extrahiert. Die vereinigten Aetherextrakte wurden mit einer kleinen Menge Wasser gewaschen und das pH de wässrigen Schicht unter Schütteln und Zugabe von 1 N NaOH auf 7,0 eingestellt. Die wässrige Schicht wurde zweimal mit 20 ml Diäthyläther gewaschen und unter vermindertem Druck eingeengt. Das Konzentrat wurde zu 237 mg Natrium D-2-Hydroxyoctanoat,

Figure imgb0027
(c=2, H20) lyophilisiert.The hydrazone mother liquor was washed twice with 50 m! Extracted diethyl ether. The combined ether extracts were washed with a small amount of water and the pH of the aqueous layer was adjusted to 7.0 with shaking and addition of 1N NaOH. The aqueous layer was washed twice with 20 ml of diethyl ether and concentrated under reduced pressure. The concentrate became 237 mg sodium D-2-hydroxyoctanoate,
Figure imgb0027
 (c = 2, H 2 0) lyophilized.

Beispiel 12Example 12

Die Spaltung von verschiedenen DL-a-Hydroxycarbonsäuren wird wie in Beispiel 11 mit den restlichen Zellen von B. albidum NRS-130KH20B (FERM-P No. 3657, NRRL B-11088) durchgeführt. Die Resultate sind in der folgenden Tabelle angegeben:

Figure imgb0028
The cleavage of various DL-a-hydroxycarboxylic acids is carried out as in Example 11 with the remaining cells from B. albidum NRS-130KH20B (FERM-P No. 3657, NRRL B-11088). The results are shown in the following table:
Figure imgb0028

Beispiel 13Example 13

Das gewaschene Zellpräparat, hergestellt aus 100 mi der nach Beispiel 11 hergestellten Kultur von B. albidum NRS-130KH20B (FERM-P No. 3657, NRRL B-1 1088) wurde nochmals mit destilliertem Wasser gewaschen und lyophilisiert, wobei man 478 mg Lyophilisat (trockenes Zellpulver) erhielt. Das trockene Zellpulver wurde in 100 ml 0,02 M Phosphatpuffer (pH 7,5) suspendiert. Das Gemisch wurde mit 2,34 g Natrium-DL-2-hydroxy-4-methylvalerianat versetzt und nach Einstellen des pH auf 7,5 bei 27°C geschüttelt (180 Bewegungen/Minute). Nach 48 und 72 Stunden Schütteln wurde das im Puffer zurückbleibende Substrat, die optische Reinheit und die Menge der erhaltenen 4-Methyl-2-oxovaleriansäure durch die oben beschriebene Methode ermittelt. Die Resultate sind in der folgenden Tabelle angegeben:The washed cell preparation, prepared from 100 ml of the culture of B. albidum NRS-130KH20B (FERM-P No. 3657, NRRL B-1 1088) prepared according to Example 11, was washed again with distilled water and lyophilized, whereby 478 mg of lyophilisate ( dry cell powder). The dry cell powder was suspended in 100 ml of 0.02 M phosphate buffer (pH 7.5). The mixture was mixed with 2.34 g of sodium DL-2-hydroxy-4-methylvalerianate and, after adjusting the pH to 7.5, shaken at 27 ° C. (180 movements / minute). After shaking for 48 and 72 hours, the substrate remaining in the buffer, the optical purity and the amount of 4-methyl-2-oxovaleric acid obtained were determined by the method described above. The results are shown in the following table:

Figure imgb0029
Figure imgb0029

Claims (4)

1. Process for the manufacture of optically active a-hydroxycarboxylic acids, characterized by asymmetrically dehydrogenating a D,L-a-hydroxycarboxylic acid of the general formula
Figure imgb0032
wherein R represents a branched or straight-chain alkyl residue with 1-13 C-atoms or a pyridyl residue,
or a salt thereof to the a-ketocarboxylic acid or to a salt thereof by means of microorganisms belonging to the genera Streptomyces, Pseudomonas, Bacillus or to the Coryneform group and having enantiospecific dehydrogenase activity, liberating the acid from a salt obtained and finally isolating the non-dehydrogenated enantiomer of the a-hydroxycarboxylic acid and, if desired, isolating the α-ketocarboxylic acid from the reaction medium.
2. Process according to claim 1, characterized in that the a-ketocarboxylic acid obtained is hydrogenated in a manner known per se to the α-hydroxycarboxylic acid and this is led back into the process as the'starting material.
3. Process according to claim 1 or 2, characterized in that 2-hydroxypropionic acid, 2-hydroxybutyric acid, 2-hydroxyvaleric acid, 2-hydroxy-3-methylbutyric acid, 2-hydroxycaproic acid, 2-hydroxy-4-methylvaleric acid, 2-hydroxy-3,3-dimethylbutyric acid, 2-hydroxyheptylic acid, 2-hydroxy-4-methyl- caproic acid, 2-hydroxycaprylic acid, 2-hydroxypelargonic acid, 2-hydroxydecanoic acid, 2-hydroxy- undecanoic acid, 2-hydroxydodecanoic acid, 2-hydroxytridecanoic acid, 2-hydroxytetradecanoic acid, 2-hydroxypentadecanoic acid or α-hydroxy-3-pyridylacetic acid is used as the starting acid of formula I.
4. Process according to claim 1, 2 or 3, characterized in that the strain Bacillus megaterium NRRL B-1 1084; Bacillus sp. NRRL 11085; Bacillus freudenreichii ATCC 31301; one of the strains NRRL B-1 1086; ATCC 31300; NRRL B-1 1088; or NRRL B-1 1087 belonging to the Coryneform group; Pseudomonas fluorescens NRRL 11089; Pseudomonas putida ATCC 31303 or NRRL 11090; Pseudomonas sp. ATCC 31302, Streptomyces sp. NRRL 11083 or NRRL 11091 is used as the microorganism.
EP78100464A 1977-07-26 1978-07-21 Process for the preparation of optically active alpha-hydroxycarboxylic acids Expired EP0000560B1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CH921877A CH628009A5 (en) 1977-07-26 1977-07-26 METHOD FOR PRODUCING OPTICALLY ACTIVE ALPHA HYDROXYCARBONIC ACIDS.
CH9218/77 1977-07-26

Publications (2)

Publication Number Publication Date
EP0000560A1 EP0000560A1 (en) 1979-02-07
EP0000560B1 true EP0000560B1 (en) 1982-02-03

Family

ID=4349625

Family Applications (1)

Application Number Title Priority Date Filing Date
EP78100464A Expired EP0000560B1 (en) 1977-07-26 1978-07-21 Process for the preparation of optically active alpha-hydroxycarboxylic acids

Country Status (10)

Country Link
US (1) US4204044A (en)
EP (1) EP0000560B1 (en)
JP (1) JPS5426397A (en)
AT (1) AT365641B (en)
CH (1) CH628009A5 (en)
DE (2) DE2861599D1 (en)
FR (1) FR2398713A1 (en)
GB (1) GB2001646A (en)
IT (1) IT1097447B (en)
NL (1) NL7807332A (en)

Families Citing this family (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IT1109506B (en) * 1978-05-23 1985-12-16 Snam Progetti ENZYMATIC-MICROBIOLOGICAL PROCESS FOR THE PRODUCTION OF OPTICALLY ACTIVE AMINO ACIDS FROM IDANTOINE AND / OR CARBAMY-DERIVATIVES RACEMI
US4560656A (en) * 1981-09-28 1985-12-24 Fritzsche Dodge & Olcott Inc. Production of γ-decalactone
EP0103210B1 (en) * 1982-09-14 1987-11-25 Degussa Aktiengesellschaft L-2-hydroxy-4-methylpentanoic acid dehydrogenase, process for its production and its use
DE3320495A1 (en) * 1983-06-07 1984-12-13 Degussa Ag, 6000 Frankfurt MICROBIOLOGICALLY PRODUCED D-2-HYDROXY-4-METHYLPENTANIC ACID DEHYDROGENASE, METHOD FOR THEIR DETERMINATION AND THEIR USE
CA1215924A (en) * 1983-07-27 1986-12-30 David W. Bewick Process for producing optically active aryloxypropionic acids and derivatives thereof useful as herbicides
JPS626697A (en) * 1985-07-03 1987-01-13 Osaka Soda Co Ltd Production of optically active epichlorohydrin
DE3536662A1 (en) * 1985-10-15 1987-05-27 Degussa MICROBIOLOGICALLY PRODUCED D (-) - MANDELATE DEHYDROGENASE, METHOD FOR THEIR PRODUCTION AND THEIR USE
JPH0817718B2 (en) * 1987-10-16 1996-02-28 東レ株式会社 Method for producing D-(-)-tartaric acid
ATE98697T1 (en) * 1988-06-06 1994-01-15 Ciba Geigy Ag PROCESS FOR THE PREPARATION OF HYDROXYACIDS.
IT1223758B (en) * 1988-08-04 1990-09-29 S G A Di G Sacerdote E C Sas PROCEDURE FOR THE MICROBIOLOGICAL PRODUCTION OF R RANANOLIDE RANGE AND / OR R OCTANOLIDE RANGE
US5155043A (en) * 1989-04-06 1992-10-13 Idemitsu Kosan Company Limited Method for preparing optically active substances
GB8910958D0 (en) * 1989-05-12 1989-06-28 Bruce Neil C Assay
US4912042A (en) * 1989-08-17 1990-03-27 Eastman Kodak Company Preparation of D-malic acid or derivative
US4921798A (en) * 1989-09-25 1990-05-01 Eastman Kodak Company Synthesis of (aryl or arylalkyl)-3-hydroxy propionic acids and aryl alkanediols having high optical purity
US5240835A (en) * 1989-10-10 1993-08-31 Genencor International, Inc. Methods for enzymatically preparing polymerizable monomers
US5843765A (en) * 1997-01-31 1998-12-01 National Research Council Of Canada Streptomyces microorganism useful for the preparation of (R)-baclofen from the racemic mixture
US6274766B1 (en) * 1999-05-19 2001-08-14 Bayer Corporation Process for purifying α-keto acids
JP4845372B2 (en) * 2004-11-30 2011-12-28 広栄化学工業株式会社 Process for producing optically active 2-hydroxyester compound

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3823069A (en) * 1972-04-05 1974-07-09 Bristol Myers Co Preparation of l(-)-ypsilon-amino-alpha-hydroxybutyric acid

Also Published As

Publication number Publication date
CH628009A5 (en) 1982-02-15
DE2861599D1 (en) 1982-03-11
IT7826132A0 (en) 1978-07-26
IT1097447B (en) 1985-08-31
EP0000560A1 (en) 1979-02-07
GB2001646A (en) 1979-02-07
FR2398713B3 (en) 1980-10-24
US4204044A (en) 1980-05-20
AT365641B (en) 1982-02-10
ATA540478A (en) 1981-06-15
NL7807332A (en) 1979-01-30
FR2398713A1 (en) 1979-02-23
DE2832230A1 (en) 1979-02-08
JPS5426397A (en) 1979-02-27

Similar Documents

Publication Publication Date Title
EP0000560B1 (en) Process for the preparation of optically active alpha-hydroxycarboxylic acids
DE69123041T2 (en) MANUFACTURING PROCESS FOR OPTICALLY ACTIVE 3-PHENYL-1,3-PROPANDIOL
US4460765A (en) Enzyme inhibitor produced by cultivation of streptomyces microorganisms
DE69219530T2 (en) Process for the preparation of R (-) - mandelic acid and its derivative
DE3586935T2 (en) INKEPHALINASE-B INHIBITORS THEIR PRODUCTION AND THEIR PHARMACEUTICAL COMPOSITIONS.
DE3587230T2 (en) ENZYMATIC PROCEDURE.
DE2825245C2 (en)
EP0599159B1 (en) Heterogeneous protein mixture with alpha-L-rhamnosidase activity, process for preparation and use.
EP0164573B1 (en) Process for the production of cyclopropane carboxylic acids
DE60007338T2 (en) METHOD FOR PRODUCING MYCOPHENOLIC ACID AND DERIVATIVES THEREOF
DE2757980C2 (en)
DE3234203C2 (en)
EP0233570A2 (en) Process for the production of coniferyl aldehyde, and micro-organism therefor
EP0502525A1 (en) Biotechnological process for the production of S-(+)-2,2-dimethylcyclopropanecarboxamide and R-(-)-2,2-dimethylcyclopropanecarboxylic acid
DE3850589T2 (en) Cytochrome P-450 enzymes.
US4249008A (en) 3,4-Dihydro-4-hydroxy-5-(3-hydroxy-2-pyridinyl)-4-methyl-2H-pyrrole-2-carboxamide
DE69839248T2 (en) PROCESS FOR THE PREPARATION OF INHIBITORS OF HMG-CoA REDUCTASE.
DE3039132C2 (en) Process for the stereospecific preparation of carbamyl derivatives of α-hydroxy acids
US4339535A (en) Process for preparing antibiotic EM 4940
DE2746209A1 (en) NEW ANTIBIOTIC AND METHOD FOR MANUFACTURING IT
CH637137A5 (en) Antibiotic and process for its preparation
DE2510868C3 (en) Nanaomycin A and process for the preparation of the antibiorica nanaomycin A and nanaomycin B.
DE2921052C2 (en)
FR2489691A1 (en) AM-3603 ANTIBIOTIC AND PROCESS FOR PRODUCING THE SAME
CA1039214A (en) Antibiotic s15-1 production by streptomyces hw-64 and hw-70

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

AK Designated contracting states

Designated state(s): BE CH DE FR GB NL

17P Request for examination filed
GRAA (expected) grant

Free format text: ORIGINAL CODE: 0009210

AK Designated contracting states

Designated state(s): BE CH DE FR GB NL

REF Corresponds to:

Ref document number: 2861599

Country of ref document: DE

Date of ref document: 19820311

PGFP Annual fee paid to national office [announced via postgrant information from national office to epo]

Ref country code: FR

Payment date: 19820423

Year of fee payment: 5

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: CH

Effective date: 19820731

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: BE

Effective date: 19830121

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: NL

Effective date: 19830201

NLV4 Nl: lapsed or anulled due to non-payment of the annual fee
REG Reference to a national code

Ref country code: CH

Ref legal event code: PL

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: DE

Effective date: 19830401

GBPC Gb: european patent ceased through non-payment of renewal fee
PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: FR

Free format text: LAPSE BECAUSE OF NON-PAYMENT OF DUE FEES

Effective date: 19840330

REG Reference to a national code

Ref country code: FR

Ref legal event code: ST

PG25 Lapsed in a contracting state [announced via postgrant information from national office to epo]

Ref country code: GB

Effective date: 19881117

PLBE No opposition filed within time limit

Free format text: ORIGINAL CODE: 0009261

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: NO OPPOSITION FILED WITHIN TIME LIMIT