WO2000049133A1 - Procede de preparation de l-sorbose - Google Patents

Procede de preparation de l-sorbose Download PDF

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WO2000049133A1
WO2000049133A1 PCT/EP2000/001370 EP0001370W WO0049133A1 WO 2000049133 A1 WO2000049133 A1 WO 2000049133A1 EP 0001370 W EP0001370 W EP 0001370W WO 0049133 A1 WO0049133 A1 WO 0049133A1
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sorbitol
sorbose
medium
fermentation
culture
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PCT/EP2000/001370
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German (de)
English (en)
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Anne-Mette Jakobsen
Kirsten Hogh Nielsen
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Basf Aktiengesellschaft
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Priority to AU29124/00A priority Critical patent/AU2912400A/en
Priority to KR1020017010525A priority patent/KR20010102256A/ko
Priority to CA002362926A priority patent/CA2362926A1/fr
Priority to JP2000599859A priority patent/JP2003521232A/ja
Priority to EP00907584A priority patent/EP1153120A1/fr
Priority to IL14465100A priority patent/IL144651A0/xx
Publication of WO2000049133A1 publication Critical patent/WO2000049133A1/fr
Priority to NO20013995A priority patent/NO20013995L/no

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    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
    • 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
    • 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
    • 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
    • C12P19/00—Preparation of compounds containing saccharide radicals
    • C12P19/02—Monosaccharides
    • 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
    • C12P7/58—Aldonic, ketoaldonic or saccharic acids
    • C12P7/60—2-Ketogulonic acid
    • 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

Definitions

  • the present invention relates to an improved process for the production of L-sorbose, which in particular facilitates the industrial production of this substance, and to special non-recombinant microorganisms which can be used particularly advantageously in the context of this production process.
  • L-sorbose is the starting product for the production of 2-keto-L-gulonic acid from which L-ascorbic acid is synthesized on an industrial scale.
  • the production of ascorbic acid or the precursors thereof, e.g. L-sorbose, microbiologically by fermentation offers the advantage of producing the desired product in enantiomerically pure form.
  • EP-A-0 758 679 describes an L-sorbose-producing G. oxydans strain with the designation G624 (deposit number FERM-BP 4415), which can be cultivated in 20% sorbitol medium. After 4 days of cultivation, this strain converted D-sorbitol into L-sorbose in high yield. This strain is also transformed with an expression construct that carries the coding DNA for the enzymes L-sorbose dehydrogenase and L-sorbosone dehydrogenase. The transformant thus obtained is used to produce 2-keto-L-gulonic acid from D-sorbitol. However, the culture medium used for this fermentation only has a D-sorbitol concentration of 5%. A 5-day cultivation is required to complete the implementation.
  • the object of the present invention was therefore to create the conditions for a more efficient fermentative production of L-sorbose.
  • This object was achieved in particular by providing a semi-continuous, fermentative process for the production of L-sorbose from D-sorbitol using microorganisms of the Gluconobacter oxydans species. This task was also solved by providing optimized bacteria of the Gluconobacter oxydans species.
  • a first object of the present invention thus relates to highly tolerant bacteria of the Gluconobacter oxydans species, which are obtainable by conditioning a low-tolerant G. oxydans strain by cultivating it step by step with increasing D-sorbitol Concentration adapted to an increased D-sorbitol concentration in the culture medium.
  • a "highly tolerant" G. oxydans strain which is adapted to high D-sorbitol concentrations according to the invention is present when it can be cultivated in media which have an initial D-sorbitol concentration in the range from about 20 to 40% by weight. %, such as, for example, about 25 to 38% by weight or 28 to 35% by weight, cultivation at a D-sorbitol concentration of less than 20% by weight is natural. lent also possible. When cultivated, such a strain should preferably also ferment the D-sorbitol contained in the medium in high, for example 70 to 100%, yield to L-sorbose.
  • the highly tolerant strains according to the invention are obtainable by conditioning the D-sorbitol concentration in the course of conditioning from initially approximately 5 to 10% by weight to a final concentration in the range from approximately 20 to 40% by weight, e.g. about 25 to 38% by weight or 28 to 35% by weight, gradually increased.
  • Conditioning can be carried out as follows, for example:
  • a sterile, known per se liquid medium suitable for the cultivation of G. oxydans is prepared, the initial D-sorbitol content of which is adjusted such that after inoculation it has a D-sorbitol content of, for example, about 5 to 15% by weight, such as about 10% by weight.
  • Suitable formulations for culture media are described, for example, in "Industrial Microbiology, Hans-Jürgen Rehm, Springer Verlag Berlin, Heidelberg, New York, 2nd Edition, p. 500.
  • a 10% sorbitol medium pH 5
  • yeast extract the 0 , 5% by weight of yeast extract and optionally 0.1% by weight of glucose were added This medium is inoculated with a freshly prepared inoculum of an unconditioned G.
  • the oxydans strain and the inoculated medium is cultured at about 30 to 37 ° C. aerobic conditions and, for example, gentle shaking or stirring. The fermentation is continued until no significant L-sorbose formation can be observed. An aliquot is removed from the fermentation broth and inoculated therewith another sterile culture medium of the second cultivation stage, the D Sorbitol content (after inoculation) is higher than the content in the medium of the previous cultivation stage
  • the ratio of inoculum to culture to be inoculated should be in the range from 1: 5 to 1:20, such as 1: 5 to 1:10.
  • the above procedure is repeated with a gradual increase in the sorbitol concentration until a bacterial culture is obtained which grows in the medium with the desired high initial D-sorbitol concentration.
  • the gradual increase in the D-sorbitol concentration can be selected depending on the response of the parent strain to the increase in concentration.
  • the increase can take place in the same or different sizes.
  • a gradual increase in the D-sorbitol concentration can take place in the same 0.5% to 3% steps, such as 1% or 2% steps.
  • bacteria of the type G. oxydans are provided which, in addition to an increased D-sorbitol tolerance, also have an essentially constant, ie stable, high L-sorbose synthesis performance.
  • Bacteria with such a property profile can be obtained by repeating a sorbitol-highly tolerant bacterium as described above at an increased sorbitol concentration, for example in an approximately 25 to 35% by weight D-sorbitol medium, until termination of L-sorbose formation.
  • a satisfactorily "high" L-sorbose synthesis performance is achieved when about 90 to 100 mol%, such as, for example, about 93 to 98 mol%, of the sorbitol used after not more than about 48 hours, in particular after no more than about 36 hours, such as after about 15 to 25 hours or 18 to 22 hours. Since the synthesis performance can be influenced by the culture conditions, the above criteria apply in particular under the following standard conditions: D-sorbitol starting content: 28-30% by weight; pH of the medium: 4.5-5.0; Ventilation 0.5-1.5 1 air / 1 medium / h; Volume ratio of inoculum to culture medium presented about 1: 5 to 1: 6; Standard medium: containing per kg medium
  • a satisfactorily "constant” or “stable” L-sorbose synthesis performance is given according to the invention if the cultivation (preferably under the above standard conditions) is achieved by serially inoculating an aliquot of the culture broth onto freshly prepared culture medium, preferably at a volume ratio of inoculum to culture medium provided about 1: 5 to 1: 6, can be repeated several times, and without a significant decrease in the L-sorbose synthesis performance of the production strain used.
  • a fluctuation of the synthesis performance within the ranges defined above for turnover and duration of the implementation is possible.
  • a constant synthesis performance should be repeated after 1 to 5 times or more, e.g. Vaccination 10 to 50 times or more may be observed.
  • the conditioning methods according to the invention can in principle be carried out with all unconditioned G. oxydans strains. However, the conditioning is preferably carried out using Gluconobacter oxydans NRRL-B72. This strain is in the deposit position (Northern Regional Research Laboratory, USA) is freely available.
  • the present invention relates in particular in the above manner conditioned bacteria of the species G. oxydans which higt befä- ⁇ are to convert, under aerobic conditions D-sorbitol to L-sorbose and wherein the bacterium is also not in the form of individual, paired or short chains mobile, gram-negative rods are present, good growth in semi-solid sorbitol / glucose / yeast extract agar medium and in liquid sorbitol / corn steep liquor medium at a pH of about 3.5 - 6, a temperature of about 25 - 40 ° C. and a D-sorbitol concentration of up to about 40% by weight, such as, for example, over 20 to 40% by weight, in particular about 25 to 35% by weight.
  • a particularly preferred G. oxydans strain obtained on the basis of the above-described conditioning process, starting from NRRL-B72 and obtained under the internal name “2B 3 ", has the following properties:
  • T 25 - 40 ° C, optimum at 32 ° C - pH range: growth at pH 3.5 - 6, optimum at pH 4.5 - 5.0
  • the strain produced thus corresponds to the essential features of G. oxydans according to the information in Bergey's Manual of Systematic Bacteriology, (9th edition), p. 275 ff. With regard to further non-limiting characteristics, reference can be made to the information in this standard set of provisions.
  • the invention also relates to mutants and variants thereof which have essentially the same properties and are suitable for carrying out the process according to the invention for producing L-sorbose, which is described in more detail in the following sections will be described.
  • suitable methods for producing mutants and variants of the specifically described microorganisms according to the invention include, but are not limited to: mutagenesis by irradiation with ultraviolet light or X-rays; treatment with a chemical mutagen such as nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine), methyl methanesulfonate, nitrogen mustard and the like; Gene integration techniques, and transduction using bacteriophages.
  • mutagenesis by irradiation with ultraviolet light or X-rays treatment with a chemical mutagen such as nitrosoguanidine (N-methyl-N'-nitro-N-nitrosoguanidine), methyl methanesulfonate, nitrogen mustard and the like
  • Gene integration techniques, and transduction using bacteriophages are known from the prior art and are described, for example, in: J.H. Miller, Experiments in Molecular Genetics, Cold Spring Harbor Laboratory Press, Cold spring Harbor, New York (1972); J.H. Miller,
  • the present invention also relates to a process for the semi-continuous, fermentative production of L-sorbose, wherein
  • Culture medium containing sorbitol in a weight ratio of 1: 4 to 1: 8 is inoculated and cultivated until the D-sorbitol reaction is essentially complete; and d) after the reaction has ended, the L-sorbose formed is isolated from the culture broth;
  • the inoculum has, for example, a cell density corresponding to 1 to 10 g of biomass (dry substance) per liter of inoculum.
  • the fermentation cycle comprising steps a) to c) is repeated as often as required until a sufficient amount of L-sorbose has been formed.
  • An "arbitrary" repetition of fermentation cycles in the context of the present invention means e.g. the 1 to 5 times or more, e.g. repeating the fermentation cycle 10 to 50 times or 50 to 200 times.
  • the method according to the invention offers the particular advantage over the prior art that it does not require a new inoculum for inoculating the production culture medium. Rather, the required inoculum can be branched off directly from the culture broth of the previous fermentation cycle, preferably after the end of the D-sorbitol reaction. Surprisingly, it was also found according to the invention that numerous fermentation cycles can be run through, although the initial D-sorbitol concentration is unusually high, for example in the range 25-35% by weight. It is also surprising that each of the fermentation cycles provides L-sorbose in almost quantitative yield, ie in the range of approximately 95-98 mol%, within a reaction time of approximately 15-25 hours, in particular approximately 20 hours, per cycle. The ratio of inoculum to culture medium chosen according to the invention ensures that the production approach is practically no Phase goes through, but rather contains a bakery culture of high vitality.
  • the fermentation temperature is preferably approximately 25-40 ° C., in particular approximately 32-36 ° C.
  • a fermentation temperature of about 35 ° C. which lies between the optimum growth of the bacterium (32 ° C.) and the optimum temperature of the enzymatically catalyzed partial oxidation of D-sorbitol to L-sorbose (36 ° C.), is particularly preferred.
  • temperatures below 25 ° C and above 40 ° C no significant increase in the number of bacteria can be determined.
  • the fermentation it is also preferred to carry out the fermentation with the supply of about 0.5-1.5 liters of atmospheric air per liter of medium and per minute.
  • the bacterial growth and the fermentative conversion of D-sorbitol can be further improved in a known manner by enriching the air with oxygen, for example by up to about 20% by volume.
  • the oxygen transition can also be regulated in a manner known per se by varying the pressure.
  • the oxygen transition is influenced in particular by the stirring performance of the stirring device used. Stirring powers of approximately 0.5 to 4 kW / m 3 of medium are preferred.
  • the blade stirrer is an example of a type of stirrer commonly used.
  • the bacteria preferred according to the invention have an optimum growth at pH 4.5-5.0. With a pH of over 6 to about 7.5, growth and productivity are reversibly prevented, while the pH in the course of the fermentation also briefly values of 3.5 or less, e.g. can take up to about 3.0 without negatively affecting sorbitol oxidation in the current batch as well as growth and productivity in the next fermentation cycle.
  • the sorbitol substrate does not have too high a proportion of D-glucose impurities, since this causes an extraordinary drop in pH during the fermentation due to the oxidation of D-glucose to gluconic acid.
  • the generation time of the microorganisms preferably used according to the invention for fermentation also increases with increasing sorbitol concentration in the medium.
  • the generation time is 3 times longer in a 27% medium than in one 10% medium with otherwise identical fermentation parameters.
  • the generation time increases sharply above a concentration range of about 30-32% by weight of sorbitol.
  • the growth stops at a concentration of about 40% by weight.
  • the bacteria reach optimal bacterial density after inoculation with an inoculum in a ratio of 1: 5.6 (inoculum to culture medium) within about 12 to 18 hours.
  • the oxidation of D-sorbitol to L-sorbose only starts after the culture has reached a certain cell density, such as about 30 to 50% of the final density.
  • the substrate D-sorbitol used should preferably have no contamination of D-mannitol if L-sorbose is to be isolated in crystalline form.
  • D-mannitol is namely converted enzymatically to D-fructose. The latter inhibits the crystallization process in small amounts.
  • Another advantage according to the invention can be seen in the fact that the production method according to the invention can be carried out using a comparatively simple, inexpensive nutrient or culture medium. This preferably contains per kg of liquid medium:
  • the culture medium is sterilized in a manner known per se before inoculation.
  • the pH of the medium is set to 5.5, for example with approximately 70-80% acetic acid, before sterilization.
  • the pH is only controlled by the calcium carbonate added.
  • An additional control such as by adding sodium hydroxide solution to the ongoing fermentation process, is not necessary.
  • the pH of the culture is approximately 5.5-6 and during the log phase (logarithmic multiplication of the bacteria) of growth, the pH falls to a range of approximately 4.0-4.5 .
  • further components can be added to the nutrient medium used according to the invention if necessary.
  • one or more other carbon sources may be included, such as Starch hydrolysates, cellulose hydrolysates or molasses; and alcohols such as glycerin.
  • One or more nitrogen sources may also be included, e.g. Ammonia, ammonium salts of inorganic or organic acids, such as ammonium chloride, ammonium nitrate, ammonium phosphate, ammonium sulfate and ammonium acetate; Urea; Nitrate and nitrite salts and other nitrogenous materials such as amino acids, meat extract, peptone, fish meal, fish hydrolysates, casein hydrolyzates, soybean hydrolyzates, yeast extract, dried yeast, ethanol yeast distillate, soybean meal, cottonseed meal and the like.
  • Ammonia ammonium salts of inorganic or organic acids, such as ammonium chloride, ammonium nitrate, ammonium phosphate, ammonium sulfate and ammonium acetate
  • Urea Nitrate and nitrite salts and other nitrogenous materials such as amino acids, meat extract, peptone, fish meal, fish hydrolysates, casein hydrolyzates, soybean hydrolyzates
  • inorganic salts such as e.g. Salts of potassium, calcium, sodium, magnesium, manganese, iron, cobalt, zinc, copper and other trace elements as well as phosphoric acid.
  • Suitable trace elements and growth factors can also be added individually or in combination, e.g. Coenzyme A, pantothenic acid, biotin, thiamine, riboflavin, flavin mononucleotide, flavin adenine dinucleotide and other vitamins, amino acids such as cysteine, sodium thiosulfate, p-aminobenzoic acid, niazinamide and the like. These can be used in pure form or in the form of natural materials which contain these substances.
  • the preferred fermentation technique used in each case depends primarily on the size of the batch. While an aerobic shake culture is suitable in principle for smaller batches, fermentation under submerged aerobic conditions is preferred for carrying out the process according to the invention on an industrial scale.
  • the conversion of D-sorbitol is monitored in a conventional manner, for example by HPLC analysis of a sample taken.
  • a suitable HPLC column of the type OA KC 7.8 x 300 mm, from Merck, Mobile Phase 0.05 N sulfuric acid, 0.4 ml / min flow rate is suitable for determining the L-sorbose content.
  • the L-sorbose formed is isolated continuously or batchwise using conventional methods.
  • the resulting culture broth is first concentrated in a conventional manner, if appropriate after separating the cell mass, for example by filtration or centrifugation. Concentration takes place, for example, by evaporation at elevated temperature, such as 50 to 80 ° C, and reduced pressure, such as 0.01 to 0.1 bar.
  • the crystals which precipitate are filtered and, if necessary, recrystallized. Further purification steps, such as solvent extraction, chromatography, precipitation or salting out, can be used individually or in combination if necessary.
  • Corn steep liquor 50% dry matter 240 g (about 200 ml) ammonium sulfate 22.2 g
  • the laboratory cultures of Gluconobacter oxydans are stored in slant agar glasses with this solid medium.
  • the inoculation cultures for the initial inoculation of the fermenter are produced on this solid medium in Roux bottles immediately before inoculation.
  • Sorbitol syrup 70% by weight dry matter 75.0 g (about 5% sorbitol) corn steep liquor / salt mixture 7.5 g
  • Agar agar powder 25.0 g Demineralized water 1 000 ml
  • Sorbitol syrup, corn steep liquor / salt mixture and water are mixed together, heated to 60 ° C. and filtered.
  • the agar powder is then slowly added to the boiling filtrate.
  • the warm medium is then distributed to the culture vessels (9 ml each 30 ml inclined agar glass and 100 ml each 650 ml Roux bottle).
  • the tubes are then closed and autoclaved at 121 ° C for 20 minutes.
  • This medium is used to check the purity of the culture during fermentation.
  • the medium is suitable for the growth of G. oxydans and is unsuitable for most of the commonly occurring contaminating microorganisms.
  • G. oxydans forms characteristic curved, round, whitish-yellow, shiny cultures after 1 to 2 days of incubation at 32 ° C.
  • Sorbitol syrup 70% by weight dry substance 75.0 g (about 5% sorbitol)
  • Sorbitol syrup, yeast extract and glucose are mixed together and then mixed with water.
  • the pH is adjusted to 5.3-5.4 using 12% by weight acetic acid.
  • the agar powder is slowly added to the boiling mixture.
  • the still warm medium is distributed on glass bottles with screw caps and autoclaved for 20 minutes at 121 ° C. After cooling to about 50 ° C, the medium is distributed to sterile plastic petri dishes.
  • This medium is used to check the purity of the culture during fermentation. It is a nutrient medium for most commonly occurring contaminating microorganisms. It is not suitable for the growth of G. oxydans. After 2 days of incubation at 32 ° C, G. oxydans forms faintly visible fuzzy colonies on this medium. No growth is usually observed. Meat extract 3.0 g
  • the medium was produced in analogy to medium c) above. Only the pH is set to 7.
  • Sorbitol syrup (70% dry matter; corresponding to 400 kg sorbitol) is diluted with tap water and mixed with the corn steep liquor. Then add the mineral salts, pre-dissolved or suspended in tap water. Then the pH is adjusted to 5.5 with 70-80% acetic acid. Finally, add the anti-foaming agent. The medium is sterilized in a manner known per se at 141 ° C. for 2 minutes.
  • the pre-cultures of G. oxydans are stored in the form of agar cultures (medium b) at 5 ° C.
  • medium b medium b
  • five new agar cultures are prepared from an agar culture by incubation at 32 ° C. for 2 days.
  • the bacteria from eight such agar cultures are used to inoculate the same medium in Roux bottles.
  • bacteria of an agar culture are suspended in 2 x 5 ml of sterile 0.9% saline. The suspension is transferred to a Roux bottle.
  • a total of eight inoculated bottles are incubated at 32 ° C for two days and stored at 5 ° C if necessary.
  • the 50 m 3 fermenter is filled with 15,000 kg of production medium (compare example 1 medium e)), but the sorbitol concentration is only 15% by weight and the content of corn steep liquor and mineral salts has been doubled).
  • the temperature of the medium is set at 35 ° C.
  • a 200 ml inoculum prepared according to Example 2 is transferred sterile to the fermenter.
  • the fermenter is aerated with 1600 - 1800 m 3 of sterile air per hour.
  • the pressure in the fermenter is about 1.3 atm.
  • the initial pH is about 5.5 - 6.0.
  • the fermentation time in this first step is about 30 to 40 hours.
  • the resulting culture broth is either worked up to isolate the L-sorbose formed or used as an inoculum for the second fermentation stage.
  • a second 50 m 3 fermenter is filled with 33,000 kg of sterile production medium (28% by weight sorbitol) and inoculated with 6,400 liters of inoculum (corresponding to 7,000 kg of culture from the first fermentation step).
  • the ratio of inoculum to production medium is 1: 4.7. It is then fermented over a period of about 18 to 22 hours until the sorbitol concentration has dropped to less than 0.1% by weight.
  • the L-sorbose yield is about 8.3 kg / m 3 fermenter volume per hour.
  • Another inoculum of 6,400 liters is branched off from the fermentation broth obtained for the next fermentation cycle. The remaining amount of fermentation broth is processed to isolate L-sorbose.
  • a fermentation broth obtained by a process according to Example 3 comprises about 25 to 26% by weight of L-sorbose, less than about 0.1% by weight of D-sorbitol, residues from the growth medium (dissolved or suspended) and 1 to 2 g Bacterial mass per liter.
  • the fermentation broth is kept at about 50 ° C to avoid further growth of the bacteria and loss of sorbose.
  • the fermentation broth is concentrated in a first concentration step to a sorbose content of approximately 42% by weight at a temperature in the range from 60 to 80 ° C. and a pressure of 0.02 bar in a conventional falling film evaporator.
  • a second step further concentration takes place in a conventional long tube evaporator at 57 ° C and a pressure of 0.02 bar.
  • This second evaporation step leads to oversaturation of the concentrated L-sorbose solution.
  • the crystal content is in the range from about 30 to 50% by volume.
  • the crude crystal suspension obtained in this way is then transferred to a conventional sedimentation container. After a while, a very dense crystal suspension forms in its bottom area, which is essentially free of bacterial mass. This is separated from the supernatant, centrifuged, washed with water and dried at about 90 ° C. to a residual moisture content of less than 0.1% by weight.
  • the mother liquor obtained, the washing liquid and the supernatant from the sedimentation container can then, as described above, be worked up further, if appropriate, after combining with the fermentation broth from another batch.
  • the total yield, based on the D-sorbitol used, is usually in the range from about 89 to 92 mol% after complete working up.
  • the product produced in this way serves as a starting product for the production of L-ascorbic acid.
  • a sterile, agar-free liquid medium (850 ml total volume in a 2 l culture vessel) is produced analogously to medium c) above (cf. Example 1) and has a D-sorbitol content of approximately 10% by weight.
  • This medium is inoculated with 150 ml of a freshly prepared inoculum of an unconditioned G. oxydans strain.
  • the inoculated medium (pH 5) is incubated at 32 ° C under aerobic conditions (atmospheric air) and vigorous shaking (80 rpm). The fermentation is continued until no significant L-sorbose formation can be observed. A volume of 150 ml is withdrawn from the fermentation broth and inoculated therewith another sterile culture medium, the D-sorbitol content of which is 11% by weight. The above procedure is repeated with a gradual (1% increment) increase in sorbitol concentration until a bacterial culture is obtained which grows in about 30% by weight D-sorbitol medium.
  • the G. oxydans culture obtained in this way can then be inoculated serially in 30% by weight D-sorbitol medium (150 ml inoculum to 850 ml medium) in order to further optimize the L-sorbose synthesis performance.
  • Optimal synthesis performance is achieved when about 90 to 98 mol% of the sorbitol used has been converted after about 24 hours of cultivation.

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Abstract

Bactéries à tolérance élevée au D-sorbitol de l'espèce Gluconobacter oxydans, procédé de création desdites bactéries et procédé de production en semi-continu et par fermentation de L-sorbose à partir de D-sorbitol.
PCT/EP2000/001370 1999-02-19 2000-02-18 Procede de preparation de l-sorbose WO2000049133A1 (fr)

Priority Applications (7)

Application Number Priority Date Filing Date Title
AU29124/00A AU2912400A (en) 1999-02-19 2000-02-18 Method for producing l-sorbose
KR1020017010525A KR20010102256A (ko) 1999-02-19 2000-02-18 Ｌ-소르보스의 제조 방법
CA002362926A CA2362926A1 (fr) 1999-02-19 2000-02-18 Procede de preparation de l-sorbose
JP2000599859A JP2003521232A (ja) 1999-02-19 2000-02-18 Ｌ−ソルボースの製法
EP00907584A EP1153120A1 (fr) 1999-02-19 2000-02-18 Procede de preparation de l-sorbose
IL14465100A IL144651A0 (en) 1999-02-19 2000-02-18 Method for producing l-sorbose
NO20013995A NO20013995L (no) 1999-02-19 2001-08-16 Fremgangsmåte for fremstilling av L-sorbose

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
DE19907115A DE19907115A1 (de) 1999-02-19 1999-02-19 Verfahren zur Herstellung von L-Sorbose
DE19907115.2 1999-02-19

Publications (1)

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WO2000049133A1 true WO2000049133A1 (fr) 2000-08-24

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PCT/EP2000/001370 WO2000049133A1 (fr) 1999-02-19 2000-02-18 Procede de preparation de l-sorbose

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Country Link
EP (1) EP1153120A1 (fr)
JP (1) JP2003521232A (fr)
KR (1) KR20010102256A (fr)
CN (1) CN1346401A (fr)
AU (1) AU2912400A (fr)
CA (1) CA2362926A1 (fr)
DE (1) DE19907115A1 (fr)
IL (1) IL144651A0 (fr)
NO (1) NO20013995L (fr)
WO (1) WO2000049133A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004029262A2 (fr) * 2002-09-27 2004-04-08 Dsm Ip Assets B.V. Production de 2-kga
WO2006084646A2 (fr) * 2005-02-11 2006-08-17 Dsm Ip Assets B.V. Gene sms 22

Families Citing this family (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2876693B1 (fr) * 2004-10-15 2007-01-26 Roquette Freres Procede de preparation de l-iditol
CN102286567B (zh) * 2011-07-21 2013-07-10 安徽丰原发酵技术工程研究有限公司 一种生产山梨糖的方法
CN112625955B (zh) * 2020-12-24 2022-07-08 浙江新和成股份有限公司 一种氧化葡糖杆菌及其在生产古龙酸中的应用

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Publication number Priority date Publication date Assignee Title
JPS6244194A (ja) * 1985-04-22 1987-02-26 Takeda Chem Ind Ltd Ｌ−ソルボ−スの製造法
JPS62275692A (ja) * 1986-02-11 1987-11-30 Takeda Chem Ind Ltd Ｌ―ソルボースの製造法
EP0518136A2 (fr) * 1991-06-13 1992-12-16 F. Hoffmann-La Roche Ag Procédé de fermentation pour la production de l'acide-2-céto-L-gulonique
EP0728840A2 (fr) * 1995-02-27 1996-08-28 F. Hoffmann-La Roche AG Déshydrogénase de D-Sorbitol
EP0758679A1 (fr) * 1994-02-25 1997-02-19 Fujisawa Pharmaceutical Co., Ltd. Procede de production d'acide 2-ceto-l-gulonique
EP0972843A1 (fr) * 1998-07-17 2000-01-19 F. Hoffmann-La Roche Ag Procédé de fermentation en continu

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPS6244194A (ja) * 1985-04-22 1987-02-26 Takeda Chem Ind Ltd Ｌ−ソルボ−スの製造法
JPS62275692A (ja) * 1986-02-11 1987-11-30 Takeda Chem Ind Ltd Ｌ―ソルボースの製造法
EP0518136A2 (fr) * 1991-06-13 1992-12-16 F. Hoffmann-La Roche Ag Procédé de fermentation pour la production de l'acide-2-céto-L-gulonique
EP0758679A1 (fr) * 1994-02-25 1997-02-19 Fujisawa Pharmaceutical Co., Ltd. Procede de production d'acide 2-ceto-l-gulonique
EP0728840A2 (fr) * 1995-02-27 1996-08-28 F. Hoffmann-La Roche AG Déshydrogénase de D-Sorbitol
EP0972843A1 (fr) * 1998-07-17 2000-01-19 F. Hoffmann-La Roche Ag Procédé de fermentation en continu

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Title
PATENT ABSTRACTS OF JAPAN vol. 011, no. 232 (C - 437) 29 July 1987 (1987-07-29) *
PATENT ABSTRACTS OF JAPAN vol. 012, no. 165 (C - 496) 18 May 1988 (1988-05-18) *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2004029262A2 (fr) * 2002-09-27 2004-04-08 Dsm Ip Assets B.V. Production de 2-kga
WO2004029262A3 (fr) * 2002-09-27 2004-07-01 Dsm Ip Assets Bv Production de 2-kga
WO2006084646A2 (fr) * 2005-02-11 2006-08-17 Dsm Ip Assets B.V. Gene sms 22
WO2006084646A3 (fr) * 2005-02-11 2006-09-28 Dsm Ip Assets Bv Gene sms 22

Also Published As

Publication number Publication date
NO20013995L (no) 2001-10-17
CA2362926A1 (fr) 2000-08-24
JP2003521232A (ja) 2003-07-15
NO20013995D0 (no) 2001-08-16
IL144651A0 (en) 2002-05-23
AU2912400A (en) 2000-09-04
KR20010102256A (ko) 2001-11-15
DE19907115A1 (de) 2000-08-24
CN1346401A (zh) 2002-04-24
EP1153120A1 (fr) 2001-11-14

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