Classifications

• • 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
  • C12P33/00—Preparation of steroids
• • 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
  • Y10S435/863—Mycobacterium
  • Y10S435/866—Mycobacterium smegmatis

Definitions

• the present invention relates to a microbiological method for producing 2Obeta-hydr0xy- 9-ox0- 1,2,3,4,10,l9-hexanor-5,9-seco-pregnan-5-oic acid, an
• the compound of formula I is utilized as an intermediate in the preparation of known steroids by procedures described in Belgian Pat. No. 741,826.
• Mycobacterium phlei ATCC 19249 it is intended to cover all microorganisms of the genus Mycobacterium phlei which produce the compound of formula land which cannot be definitely differentiated from the strain deposited with the American Type Culture Collection, Rockville, Maryland under ATCC No. 19249 and its subcultures includingmutants and variants.
• the hexanor-S,9-seco-pregnan-5-oic acid of formula I is manufactured by fermenting 3beta,20betadihydrOXypregn-S-ene or 2Obeta-hydroxypregn-4-en- 3-one with Mycobacterium phlei ATCC 19249 adapted to aliphatic hydrocarbons.
• the adaption of Mycobacterium phlei ATCC 19249 to aliphatic hydrocarbons can be carried out in a manas the carbon source, the hexanor-S,9-seco-pregnan-5- oic acid of formula I. Those cultures which show no growth on such media are used for the present fermentation.
• the present process can be carried out by any conventional procedure of aerobic fermentation.
• suitable culture substrates are liquid media which contain a source of assimilable nitrogen and a source of assimilable carbon as well as inorganic salts.
• Any conventional source of assimilable nitrogen and assimilablecarbon can be utilized.
• As the source of assimilable nitrogen there can be used animal, vegetable, microbial and inorganic nitrogen compounds such as meat extracts, peptone, cornsteep, yeast extract, glycine and sodium nitrate.
• As the source of assimilable carbon there can be used sugars such as glucose and, especially, the steroid starting material to be fermented.
• the nutrient medium may, if desired, also contain trace elements such as iron, sulfur and phosphorus as well as growth factors (e.g., vitamins such as biotin or pyridoxine) or auxins (e.g., indolylacetic acid).
• growth factors e.g., vitamins such as biotin or pyridoxine
• auxins e.g., indolylacetic acid.
• the medium can be sterilized and/or can be provided with substances such as benzoates, antibiotics, etc., which inhibit the growth of foreign organisms.
• the fermentation is preferably carried out in a neutral or weakly acidic pI-I-range (e.g.,
• the fermentation can be carried out at a temperature of between 18C. and 40C. It is preferably carried out at about 28C.
• the steroid used as the starting material is preferably added to the fermentation culture in solution.
• Any conventional inert organic solvent can be used as the solvent medium in accordance with this invention.
• the preferred solvents are included dimethyl sulfoxide, dimethylformamide, ethanol or acetone. It has proved to be advantageous to add the steroid gradually to the fermentation solution and to wait for the complete decomposition of the previously added steroid before each new addition.
• the hexanor-5,9- seco-pregnan-S-oic acid aforesaid can be isolated from the culture solution by conventional procedures.
• ner known per se by inoculating the microorganism from culture substrates which contain glucose on to those substrates in which the glucose is replaced in increasing quantity by aliphatic hydrocarbons, especially C -C alkanes.
• mutants as used herein can be spontaneous' mutants or mutants produced in a physical or chemical manner. Mutants can be produced by irradiation (e.g., with ultraviolet light or gamma rays) or by treatment with mutating agents (e.g., N-methyl-nitronitrosoguanidine or bromouracil). Spontaneous mutants are preferably used in the present process. The selection of the mutants can be carried out by spreading single colonies on agar plates which contain, as the carbon source, the steroid starting material to be fermented. Single colonies obtained from such agar substrates are finally spread on agar plates which contain,
• Mycobacterium phlei ATCC 19249 adapted to C C n-aIkane, was incoluated from sloping agar cultures on to a culture medium 'of the following composition:
• the cultures were incubated for days at 28C. with aeration and stirring. From the thus-obtained cultures, there were taken plate-cultures on agar plates which contained only 3B,20B-dihydroxypregn-S-ene as the carbon source. The plate-cultures were repeated several times, by which means the steroid content in the agar plates was increased. Finally, further platecultures were used in which the agar contained only B-hydroxy-9-oxo-l ,2,3,4, 1 0,l9-hexanor-5,9-secopregnan-S-oic acid'as the carbon source. Those cultures which showed no growth on these plates were used for the fermentation. In order to store the cultures, a suspension in sterile 10 percent skim milk was prepared from the biomass and was filled into 2 ml. ampoules and stored under liquid nitrogen.
• Example 2 in tap water. pH 7.0 (adjusted with sodium hydroxide), was inoculated with the contents of 2 ampoules prepared according to Example 1. The culture was incubated for 48 hours at 28C. with shaking. This preculture .was used to inoculate 8 liters of nutrient medium in a small fermenter following which incubation was carried out for 2 days at 28C. with stirring and aeration (640 r.p.m., 2 liters air/minute), the pH value being adjusted to pH 7.0 by the addition of 28 percent sodium hydroxide. The contents of this fermenter were used as the inoculant for 180 liter fermenter containing the same nutrient solution.
• pH 7.0 adjusted with sodium hydroxide
• the fermentation mass was centrifuged, rinsed with 20 liters of water and adjusted to pH 2.0 with concentrated aqueous hydrochloric acid.
• the solution was extracted twice with the same volume and once with half the volume of methylene chloride.
• the extracts were concentrated in a rotary evaporator and worked up by countercurrent extractionv
• the yield of 20,8-hydroxy-9- oxo-1,2,3,4, l0,l9-hexanor-5,9-seco-pregnan-5-oic acid amounted to 82 percent. 10 percent of9,20-dioxo- 1,2,3,4,l0,l9-hexanor-5-seco-pregnan-5-oic acid was also isolated.
• a solution of 13.4 g of 20beta-hydroxy-9-oxo- 1,2,3,4,1O,19-hexan0r-5,9-seco-pregnan-5oic acid and 4 g of sodium acetate in 250 ml of acetic anhydride is boiled at reflux under nitrogen for 4 hours.
• the acetic anhydride is removed in vacuum, the residue twice evaporated with toluene and finally taken up in 750 ml of methylene chloride.
• the methylene chloride solution is washed five times with water, dried over Na SO and evaporated in vacuum.

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• Organic Chemistry (AREA)
• Chemical & Material Sciences (AREA)
• Engineering & Computer Science (AREA)
• Zoology (AREA)
• Life Sciences & Earth Sciences (AREA)
• Wood Science & Technology (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Microbiology (AREA)
• General Chemical & Material Sciences (AREA)
• Biotechnology (AREA)
• Health & Medical Sciences (AREA)
• Biochemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• General Engineering & Computer Science (AREA)
• General Health & Medical Sciences (AREA)
• Genetics & Genomics (AREA)
• Preparation Of Compounds By Using Micro-Organisms (AREA)
• Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)
• Steroid Compounds (AREA)

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Cited By (2)

Citations (2)

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• 1972
  • 1972-08-31 CH CH1283872A patent/CH585171A5/xx not_active IP Right Cessation
• 1973
  • 1973-07-10 IL IL42711A patent/IL42711A/en unknown
  • 1973-07-10 ZA ZA734650A patent/ZA734650B/xx unknown
  • 1973-07-24 AU AU58431/73A patent/AU468583B2/en not_active Expired
  • 1973-08-08 DE DE19732340162 patent/DE2340162A1/de active Pending
  • 1973-08-16 HU HUHO1605A patent/HU168234B/hu unknown
  • 1973-08-16 US US00389041A patent/US3853707A/en not_active Expired - Lifetime
  • 1973-08-20 CA CA179,199A patent/CA992480A/en not_active Expired
  • 1973-08-24 PH PH14956*UA patent/PH9700A/en unknown
  • 1973-08-28 FR FR7331045A patent/FR2197868B1/fr not_active Expired
  • 1973-08-29 SE SE7311760A patent/SE410731B/xx unknown
  • 1973-08-30 JP JP48096788A patent/JPS4962692A/ja active Pending
  • 1973-08-30 GB GB4093173A patent/GB1397163A/en not_active Expired
  • 1973-08-30 BE BE135087A patent/BE804196A/xx unknown
  • 1973-08-30 AT AT752773A patent/AT332001B/de not_active IP Right Cessation
  • 1973-08-31 NL NL7312061A patent/NL7312061A/xx not_active Application Discontinuation

Patent Citations (2)

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