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
  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/62—Carboxylic acid esters
  • C12P7/625—Polyesters of hydroxy carboxylic acids
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08G—MACROMOLECULAR COMPOUNDS OBTAINED OTHERWISE THAN BY REACTIONS ONLY INVOLVING UNSATURATED CARBON-TO-CARBON BONDS
  • C08G63/00—Macromolecular compounds obtained by reactions forming a carboxylic ester link in the main chain of the macromolecule
  • C08G63/02—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds
  • C08G63/06—Polyesters derived from hydroxycarboxylic acids or from polycarboxylic acids and polyhydroxy compounds derived from hydroxycarboxylic acids

Definitions

• the invention relates to the field of organic synthesis.
• it relates to a new process for obtaining poly- ⁇ -hydroxy-octanoic acid, a polymer constituted by repeating units of formula + CH (C 5 H 11 ) - CH 2 - C (0) - 0 ⁇ n
• the method of the invention is based on a method of extracting poly- ⁇ -hydroxy-octanoic acid from a culture of bacteria containing it using an organic solvent.
• Poly-hydroxy-alkanoic acids are storage compounds found in certain families of bacteria. It is in the cytoplasmic solution that these acids are deposited in the form of an aggregate when the cells are cultivated in a medium rich in carbon and in which one of the nutritive agents is growth-limiting.
• the present invention provides a new solution to this problem.
• the process of the invention consists in fact in cultivating appropriate bacteria in an adequate nutritive medium.
• the bacteria are then separated after fermentation by usual techniques, for example by centrifugation, and the wet biomass thus obtained is dispersed in acetone or isopropanol or other dehydrating solvent known in the prior art.
• the still wet biomass is stirred in the organic dispersion solvent chosen until a homogeneous suspension is obtained.
• the separation of the solid cell mass from the suspension is carried out according to the usual methods, for example by decantation, filtration or centrifugation.
• a simple filtration using a Buchner filter allows the separation of the cells from the bacteria in the form of a now dry powder. Further drying can optionally be carried out by direct exposure to air of the separated solid.
• poly- ⁇ -hydroxy-octanoic acid is completely soluble in anhydrous acetone, the presence of water, even in very small quantities, makes the polymer perfectly insoluble.
• the extraction of poly- ⁇ -hydroxy-octanoic acid takes place in non-chlorinated organic solvents defined by the following group: acetone, methyl isobutyl ketone, diisopropyl ketone, diethyl ketone, ethyl acetate, propyl acetate, butyl acetate, diethyl ether, diisopropyl ether and tetrahydrofuran.
• acetone or tetrahydrofuran is used.
• Pseudomonas in a nutritive medium in which one of the agents is growth-limiting, gives rise to the formation of poly- ⁇ -hydroxy-octanoic acid.
• Such bacteria cultures can be obtained from depositaries such as the ATCC (American Type Culture Collection, Rockville, Maryland, USA) or the DSM (Deutsche Sammlung von Mikroorganismen, Gôttingen, Germany). Specifically, in the process of the invention, bacteria of one of the following strains were used: Pseudomonas oleovorans ATCC 29347, Pseudomonas sp. DSM 1650, and Pseudomonas citronellonis DSM 50332.
• the invention therefore also relates to a process for obtaining poly- ⁇ -hydroxy-octanoic acid by aerobic fermentation of bacteria capable of accumulating said acid when cultivated in a nutrient solution rich in carbon and in which one nutritive agents is growth limiting, characterized in that the nutritive solution is added to the culture so as to maintain a dilution rate of between approximately 0.050 and 0.100 h -1 , which is then separated from the biomass of the culture medium, that the separated biomass is suspended in acetone or isopropanol or other known dehydrating solvent, that the suspension obtained is filtered and the dried solid biomass is brought into contact with an inert organic solvent chosen from the group that here: acetone, methyl isobutyl ketone, diisopropyl ketone, diethyl ketone, ethyl acetate, propyl acetate, butyl acetate, diethyl ether, diisopropyl ether and tetrahydrofura Finally, separate the
• This nutrient solution was added to the fermentation medium so as to maintain the dilution rate at a value of 0.085 h -1 .
• the cell mass After reaching a steady state, i.e. a constant concentration of biomass and a complete consumption of carbon and nitrogen, the cell mass is stored in a tank before centrifugation (30 min), washed with demineralized water and centrifuged again.
• the wet mass (51.5 g) harvested from 21 of culture was suspended in 250 ml of isopropanol and stirred at room temperature until a homogeneous suspension is obtained, then it is filtered using a B ⁇ chner type filter and 12.4 g of dry cell mass was obtained after air drying.
• This dry mass was suspended in 155 ml of acetone and stirred for 1 h at room temperature, then it was filtered.
• the clear filtrate was finally evaporated in air to provide 2.43 g of a translucent polymer film.
• the polymer content of the cell mass was determined by gas chromatography of the methyl ester of ⁇ -hydroxy-octanoic acid obtained by methanolysis of the polymer [see for example Appl. About. Microbiol. 54 (1988), 1977].
• the proportion of polymer was 20% by weight.
• a dried biomass was obtained according to the method described in the previous example.
• MW 2.11. 10 2 . [ ⁇ ] W * according to Makromolekulare Chemie 176 (1975), 2655, in which MW represents the molecular weight and [ ⁇ ] defines the intrinsic viscosity as measured at 30 ° by a digital Brookfiêld viscometer.
• the polymer was solubilized at different concentrations in cyclohexanone.
• Other tests have been carried out by suspending the dried biomass in solvents other than acetone, for example methyl isobutyl ketone, diisopropyl ketone, diethyl ketone, ethyl acetate, propyl acetate, acetate butyl, diethyl ether or even diisopropyl ether.
• a dried biomass was obtained according to the method described in Example 1.
• the nutrient solution had the following composition (proportions expressed relative to 1 l of distilled water).
• octanoic acid 15.12 g
• Example 2 The operation was carried out as indicated in Example 1, adding the nutritive solution to the fermentation medium so as to maintain the dilution rate at the value indicated in the following table. Poly- ⁇ -hydroxy-octanoic acid was thus obtained in the indicated proportions expressed as the content of the polymer in the cell mass.

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• Preparation Of Compounds By Using Micro-Organisms (AREA)

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• 1992
  • 1992-11-13 EP EP92923487A patent/EP0569569A1/de not_active Ceased
  • 1992-11-13 US US08/094,198 patent/US5422257A/en not_active Expired - Fee Related
  • 1992-11-13 JP JP5509757A patent/JPH06505403A/ja active Pending
  • 1992-11-13 WO PCT/EP1992/002608 patent/WO1993011656A1/fr not_active Application Discontinuation

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