CN1723287A - The production method of 2-KGA - Google Patents
The production method of 2-KGA Download PDFInfo
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- CN1723287A CN1723287A CN 03823203 CN03823203A CN1723287A CN 1723287 A CN1723287 A CN 1723287A CN 03823203 CN03823203 CN 03823203 CN 03823203 A CN03823203 A CN 03823203A CN 1723287 A CN1723287 A CN 1723287A
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Abstract
The present invention relates to by method continuous or the semicontinuous fermentation pattern is produced 2-KGA from the L-sorbose, wherein use the mixed culture of Gluconobacter oxydans DSM 4025 and second kind of microbe composition, perhaps, in fermention medium, replenish yeast or yeast product, to replace second kind of microbe composition.
Description
Technical field
The present invention relates to from the L-sorbose 2-keto-L-gulonic acid or its salt be carried out the quantity-produced method, described method comprises: under the situation that has second kind of composition to exist, cultivate the microorganism that belongs to the Gluconobacter genus.
Background technology
2-keto-L-gulonic acid (hereinafter representing with 2-KGA) is the important intermediate that is used to produce the L-xitix.This compound can be converted into the L-xitix according to known Reichstein method.People attempt directly 2-KGA being carried out microorganisms producing with L-sorbose or D-Sorbitol Powder as starting raw material, and example comprises microorganism, particularly Pseudomonas striata and Gluconobacter oxydans is carried out the test of mixed culture.During with this method, the initial concentration of L-sorbose is under the situation of 70g/L, and production concentration is 30g/L; The initial concentration of L-sorbose is under the situation of 100g/L, and production concentration is 37g/L.The production of 2-KGA being carried out from the L-sorbose by Pseudogluconobacter saccharoketogenes under microbe satellite existence and non-existent situation is disclosed among EP 0 221 707 B1.Yet the production concentration of this method is at most 55.3 to 87.6g/L (transforming ratio is 34.2% to 54.1%).
Summary of the invention
An object of the present invention is to provide by continuously fermenting from the method for L-sorbose with high yield production 2-KGA.The salt of 2-KGA is in for example the sodium salt of 2-KGA and calcium salt are also included within.
The present invention relates to by the method for continuously fermenting or semicontinuous fermentation is produced 2-KGA or its salt from the L-sorbose, in the described method, used be selected from by Gluconobacter oxydans DSM 4025 (FERM BP-3812), have Gluconobacter oxydans DSM 4025 (FERM BP-3812) identification mark belong to the microorganism that Gluconobacter belongs to and the microorganism of the group that mutant constituted thereof; Use the substratum that contains yeast or yeast product in the described method, perhaps described method is carried out under the situation that has second kind of microbe composition to exist.
Therefore, an object of the present invention is to provide the method for producing 2-KGA or its salt from the L-sorbose by continuous or semicontinuous fermentation pattern, wherein used be selected from by Gluconobacter oxydansDSM 4025 (FERM BP-3812), have Gluconobacter oxydans DSM 4025 (FERM BP-3812) identification mark belong to the microorganism that Gluconobacter belongs to and the microorganism of the group that mutant constituted thereof; Described method is carried out under the situation that has second kind of microbe composition to exist, and does not additionally replenish yeast or yeast product in the case in fermention medium.
In another object of the present invention, provide by continuously or the semicontinuous fermentation pattern method of producing 2-KGA or its salt from the L-sorbose, wherein used be selected from by Gluconobacter oxydansDSM 4025 (FERM BP-3812), have Gluconobacter oxydans DSM 4025 (FERM BP-3812) identification mark belong to the microorganism that Gluconobacter belongs to and the microorganism of the group that mutant constituted thereof; Wherein, in fermention medium, replenish yeast or yeast product, replace second kind of microbe composition.
Embodiment
More specifically, the invention provides by method continuous or that semicontinuous fermentation is produced 2-KGA from the L-sorbose, described method comprises the steps:
(a) in one or more fermenting containers, cultivation be selected from by Gluconobacter oxydansDSM 4025 (FERM BP-3812), have Gluconobacter oxydans DSM 4025 (FERM BP-3812) identification mark belong to the microorganism that Gluconobacter belongs to and the microorganism of the group that mutant constituted thereof, described cultivation is carried out in the substratum that contains yeast or yeast product, or under the situation that second kind of microbe composition exists, in the substratum that does not have yeast or yeast product, carry out;
(b) in fermenting container continuously supply contain the nutritional medium of L-sorbose, the dilution rate of fermentation is arranged on about 0.01h
-1To about 0.05h
-1, the concentration of described L-sorbose at about 20g/L to approximately between the 250g/L,
(c) from fermenting container, take out fermentation culture continuously; With
(d) from described fermentation culture, reclaim 2-keto-L-gulonic acid or its salt.
The method of above-mentioned steps (a) to (d) is about continuous ferment process, the feature of described continuous ferment process is continuously a supply nutritional medium (above-mentioned steps (b)) in fermenting container, and take out fermentation culture (above-mentioned steps (c)) continuously, make that the working volume in the fermentor tank can keep constant.
In one embodiment, above-mentionedly 2-KGA is carried out the quantity-produced method in a fermenting container, carry out.
Regulation according to " budapest treaty ", G.oxydans DSM 4025 is deposited in the Deutsche Sammlung vonMikroorganismen und Zellkulturen (DSMZ) of G ttingen (Germany) on March 17th, 1987, is numbered DSM No.4025.Depositor is that the eastern science instrument is imported and exported Group Co.,Ltd, and it is that Institute of Microorganism, Academia Sinica for No. 52, People's Republic of China (PRC) Sanlihe Road, Beijing delivers.Effectively depositor is described institute, and its full address is a The People's Republic of China Zhongguangcun, Haidian District, Beijing City Institute of Microorganism, Academia Sinica, 100080.
In addition, the subculture of this bacterial strain also has been deposited in National Institute of Advanced Industrial Science and Technology (AIST) on March 30th, 1992, Tsukuba Central 6,1-1-1 Higashi, Tsukuba, Ibaraki 305-8566, Japan, this also is the regulation according to " budapest treaty ", and deposit number is FERM BP-3812.Depositor is Nippon Roche K.K., 6-1, Shiba 2-chrome, Minato-ku, Tokyo 105-8532Japan.
The second kind of microbe composition that is used for the inventive method can be, for example, and the bacterium that Bacillus and Xanthomonas belong to.The preferred bacterial strains such as B.megaterium DSM 4026 and X.maltophilia IFO 12692 that use.Any above-mentioned bacterial strains can be cultivated 1 to 4 day in 20 ℃ to 40 ℃ quilts in suitable substratum, and the culture that obtains is used as the inoculum of cultivation, cultivates under the situation that described microbe satellite exists.
According to the regulation of " budapest treaty ", on March 17th, 1987 B.megateriumDSM 4026 is delivered the preservation to DSMZ, be numbered DSM 4026.Depositor is that the eastern science instrument is imported and exported Group Co.,Ltd, and it is that Institute of Microorganism, Academia Sinica for No. 52, People's Republic of China (PRC) Sanlihe Road, Beijing delivers.Effectively depositor is described institute, and its full address is a The People's Republic of China Zhongguangcun, Haidian District, Beijing City Institute of Microorganism, Academia Sinica, 100080.According to the regulation of " budapest treaty ", X.maltophilia IFO 12692 in July 31 nineteen sixty-eight by preservation to Institute for Fermentation (Osaka, Japan).
In one embodiment of the invention, be used for producing the method for 2-KGA or its salt from the L-sorbose, used be selected from by Gluconobacter oxydans DSM 4025 (FERM BP-3812), have Gluconobacter oxydans DSM 4025 (FERM BP-3812) identification mark belong to the microorganism that Gluconobacter belongs to and the microorganism of the group that mutant constituted thereof; And used the substratum that contains yeast or yeast product, wherein said yeast or yeast product belong to Ascomycetes subclass.Preferably, described yeast or yeast product belong to Saccharomyces and belong to, for example S.cerevisiae.
Term " yeast product " also comprises fresh yeast, dry yeast and yeast extract.
In one embodiment, the cultivation in the above-mentioned steps (a) is continuously fermented by the multistage and is carried out.Therefore, the present invention relates to continuously ferment and carry out the method that above-mentioned 2-KGA produces with the multistage.It is a kind of embodiment of such multistage fermentation pattern that two stages continuously fermented.Can be continuously fermented system applies the multistage in the 2-KGA fermentation process, obtaining the higher throughput of 2-KGA and higher 2-KGA concentration, and not have L-sorbose residue, this is preferred situation to the separating step in the preparation 2-KGA sodium.
When fermenting process began, the ratio of the quantitative proportion of Bacillus bacterium colony and Gluconobacter bacterium colony and Xanthomonas bacterium colony and Gluconobacter bacterium colony was unimportant.This ratio can be at about 1: 10 to about 1: 300 scope (Bacillus:Gluconobacter and Xanthomonas:Gluconobacter).During the fermentation, this ratio can automatically adjust to self, reaches optimum value.
In culturing micro-organisms in the substratum that is containing L-sorbose and proper nutrition thing, can be easily under aerobic conditions with described microorganism culturing in aqueous culture medium.Yet, can carry out method of the present invention with any traditional fermentation condition.
This fermentation process can be to carry out between about 4.0 to about 9.0 at pH, and pH is preferably between about 6.0 to about 8.0.The temperature range that is preferred for carrying out this fermentation process is between about 13 ℃ to about 36 ℃.More preferably, described fermentation process can be carried out under the interior temperature of about 18 ℃ of extremely about 33 ℃ scopes.Fermentation time can be between about 100 hours to about 1300 hours.In the method, can be as the concentration of the L-sorbose of starting raw material at about 20g/L to approximately between the 250g/L, preferably at about 50g/L to approximately between the 200g/L.
The substratum that is used for this fermentation process contains the nutrition that is useful on microorganism usually, for example can absorbed carbon source, the nitrogenous source that can be digested and inorganics, VITAMIN, trace elements and other promote the factor of growth.As the L-sorbose of starting raw material, can also add other carbon source material, for example glycerine, glucose, N.F,USP MANNITOL, fructose, D-arabitol etc. except that in the method.
Some organic or inorganic materials also can be used as nitrogenous source, for example meat extract, peptone, casein, corn steep liquor, urea, amino acid, nitrate, ammonium salt etc.Inorganics sal epsom, potassiumphosphate, iron protochloride and iron(ic) chloride, lime carbonate etc. also can use.
The system of continuously fermenting can be made of substratum storage tank, one or more fermentor tank and results container.Can be in described fermentor tank in the fermenting process supply contain the nutritional medium of L-sorbose, can be the speed that fermentation culture is discharged from fermentor tank with providing the rate adaptation of nutritional medium, make that the working volume in the fermentor tank can keep constant to described fermentor tank.Preferably, can be carried out in the dilution rate (D) of liquid flow be about 0.01h to the fermentation in the fermentor tank
-1To about 0.05h
-1Situation under, about 0.02h more preferably
-1To about 0.045h
-1There to be the substantive output that increases to produce 2-KGA from the L-sorbose is possible, and promptly production concentration surpasses 112.2g/L (molar yield is 91.0%) when L-sorbose initial concentration is 120g/L, and output was higher when initial concentration was higher.In the steady state of continuous mode, the throughput of 2-KGA is calculated as 3.9g/L/h to 4.8g/L/h.To the calculating of transformation efficiency amount based on 2-KGA that produces and used up L-sorbose.
In the another kind of embodiment of the inventive method, can ferment by semicontinuous (repeated fed-batch), under the situation that has second kind of microbe composition to exist, G.oxydans DSM4025 is cultivated, and described second kind of microbe composition for example is microorganism or the yeast that Bacillus and Xanthomonas belong to.This semicontinuous pattern is to obtain one of method to the high productive capacity of 2-KGA, and wherein, the part of nutrient solution is used as the inoculum of fermentation next time.The semicontinuous pattern of carrying out at the mixed culture of G.oxydansDSM 4025 bacterial strains and second kind of microbe composition obtains by the following method: for example, in fermentor tank, keep 10% (v/v) that cultivated in the whole nutrient solutions that obtain through 48 hours, with its seed, and in described fermentor tank, fill with fresh nutritional medium as second step.According to the same mode described in the top the first step, the part in second step is as the seed of the 3rd step preparation.
, discard biological catalyst biological example material after each batch and caused higher production cost in batches or in the fed-batch mode traditional, the reactor cleaning and (inoculation) the needed time of startup have caused the loss of reactor throughput.Yet semicontinuous pattern can utilize nutrient solution as the seed of cultivating next time.Therefore, this operation can be applied to the fermentation of 2-KGA, and need not to carry out the step of several times from the inoculation of a small amount of seed, and the time that does not need to be used to prepare culture, for example cleaning and start time.In addition, reactor can both keep higher throughput in long fermenting process.
The present invention partly relates to the method for producing 2-KGA from the L-sorbose by the multistage continuous ferment process, wherein, under the situation that has second kind of microbe composition to exist, has used the culture of G.oxydansDSM 4025.The described multistage, the system of continuously fermenting was made up of two or more fermenting container.For the output that makes this method reaches optimum, can carry out fermentation more than one fermenting container by in whole process, using.For example, the fermentation from the L-sorbose to 2-KGA can be carried out two or more fermenting containers, and described fermenting container is placed with consecutive order.
The present invention partly relates to the method for producing 2-KGA from the D-Sorbitol Powder by the rapid fermentation system of multistep, and the rapid fermentation system of described multistep will connect before and after " fermentation process from the D-Sorbitol Powder to the L-sorbose " and " fermentation process from the L-sorbose to 2-KGA ".The bacterium that the nutrient solution that this method is used contains through producing the L-sorbose transforms the L-sorbose that the D-Sorbitol Powder obtains, and described bacterium is not had specific restriction.The known bacterial strain that some Gluconobacter are arranged is G.xylinum and G.suboxydans for example, can both effectively produce the L-sorbose from the D-Sorbitol Powder.
Can be under aseptic condition, with the nutrient solution of the cell of the bacterial strain that contains Gluconobacter of results with mix through the nutritional medium of sterilizing, the concentration of L-sorbose can be adjusted to 13%.In a kind of concrete embodiment of the inventive method, L-sorbose nutrient solution can be used directly as substrate, and need not pass through heat sterilization.Therefore, when setting up the D-Sorbitol Powder to the fermentative production system of 2-KGA, the fermentation system from the D-Sorbitol Powder to the L-sorbose can directly link to each other with the system of continuously fermenting from the L-sorbose to 2-KGA, and does not need L-sorbose fermented liquid is sterilized.Contain the substratum of L-sorbose fermented liquid by use, can utilize mixed culture, in fermentor tank, carry out with the fermentation that pattern carries out of continuously fermenting of two stages to 2-KGA to G.oxydans DSM 4025 and X.maltophilia IFO 12692.In the steady state of described continuous mode, 2-KGA and the acquisition that may produce above 128.9g/L surpass 83.4% molar yield.In addition, the L-sorbose in the mixed-culture medium can all be exhausted.
Microorganism " Gluconobacter oxydans ", " Gluconobacter xylinum ", " Gluconobacter suboxydans ", " Bacillus megaterium " and " Xanthomonasmaltophilia " also comprise: (International Code ofNomenclature of Prokaryotes) is defined by " international prokaryotic organism rules of nomenclature ", the synonym body (synonym) with same physical-chemical attribute or the basinym body (basonym) of these type of species.
Another of the inventive method preferred aspect, fermentation is about 0.1% to carry out preferably about 5% to about 150% air saturation to the situation of about 200% air saturation at dissolved oxygen concentration.
In addition, fermentation preferred in air-flow oxygen concn be between about 0.1% to about 100% and airflow rate is carried out to the situation of about 2.0v/v/min at about 0.01v/v/min.(gas volume in the reactor of the every volume unit of per minute).
Therefore, the present invention relates to above-mentionedly 2-KGA or its salt be carried out the quantity-produced method from the L-sorbose, wherein, to be carried out at dissolved oxygen concentration in the fermenting container be about 5% to the situation of about 100% saturation ratio or to be carried out at oxygen concn in the air-flow be under the situation between about 0.1% to 100% to described continuous production.
To approximately between the 250g/L, and dilution rate is at about 0.01h at about 20g/L for the concentration of L-sorbose in the continuous feeding substratum
-1About 0.05h
-1Between situation under, it also is preferred carrying out fermentation in fermenting container.
Though can under normal pressure, carry out according to fermentation of the present invention, promptly approximately carrying out under the 1bar,, it as a rule is preferred working under about pressure of 1 to about 5bar, is more preferably under about pressure of 1 to about 3bar.
According to the present invention, need not fixing means at microorganism, chemical bond or be used for the physical method that cell keeps for example, for example matrix embedding also need not biological substance control or keeps, and for example the film system just may produce 2-KGA from the L-sorbose.
Can isolate the 2-KGA that obtains according to present method from reaction mixture, for example, separate by forming salt, perhaps by utilizing product to separate with the difference of impurity attribute on every side, described attribute for example is the distribution coefficient between solubleness, absorbability and solvent.Absorption is for example carried out on ion exchange resin, is a kind of method that is used for separated product easily.Available traditional method is carried out further purifying to the product that obtains thus, for example, and by recrystallization or chromatogram.
Perhaps, by esterification and subsequently enolization with lactonize, described reaction mixture can be directly used in the conversion to the L-xitix.
To be set forth the present invention by embodiment below.
Embodiment 1: by G.oxydans DSM 4025 in be supplemented with the zymic substratum from 12%
The L-sorbose carries out the 2-KGA of single phase and continuously ferments
To contain 8.0% L-sorbose (sterilization separately), 0.05% glycerine, 0.25% MgSO
47H
2O, 1.75% corn steep liquor, 5.0% bread yeast (Oriental Yeast IndustryCo.) (pH is 7.0 before the sterilization), 0.5% CaCO
3, 0.5% urea (sterilization separately) and 0.03% defoamer CA-115 (Nissan) seed culture medium be encased in the Erlenmeyer bottle of 500ml (every bottle of 100ml), and sterilized 20 minutes at 121 ℃.The cell inoculation of microorganism G.oxydans DSM 4025 of one inoculation circular rector in this seed culture medium, was cultivated 24 hours at 30 ℃, and the cell of described G.oxydans DSM 4025 is containing 5.0% D-N.F,USP MANNITOL, 0.25% MgSO
47H
2O, 1.75% corn steep liquor, 5.0% bread yeast (pH is 7.0 before the sterilization), 0.5% CaCO
3, 0.5% urea (sterilization separately) and 2.0% agar plate culture medium in cultivated 4 days in 27 ℃ of quilts.The inoculum that obtains is inoculated in 100ml in the 500mlErlenmeyer bottle substratum same as described above, cultivates 24 hours at 30 ℃.
Used during continuously ferment system in single phase the D type the glass fermentor tank (Able, Tokyo, Japan).Described fermentor tank cumulative volume is 3L, and it has that (top drive) system is driven on the top and to the monitor of temperature, pH, dissolved oxygen (DO) and waste gas.Its working volume is 2L, and temperature is controlled in 30 ℃.Stirring velocity and Ventilation Rate are separately positioned on 800rpm and 1.0L/min.With sodium hydroxide solution pH is controlled at 7.0.In addition, prepared the supplemented medium of 10L so that fresh substrate to be provided continuously.During the fermentation beginning, inoculum accounts for 10% (v/v).Control the speed of continuous feeding with peristaltic pump, the nutrient solution that will contain 2-KGA with the another peristaltic pump is continuously collected the results container from fermentor tank, and this moment, working volume remained on 2L.
Produce substratum (2L) and continuous feeding substratum (10L) by 12.0% L-sorbose (sterilization separately), 0.05% glycerine, 0.25% MgSO
47H
2O, 3.0% corn steep liquor, 7.5% bread yeast, 0.15% defoamer CA-115 form.At 121 ℃ to described medium sterilization 20 minutes.With NaOH pH is controlled at 7.0 between yeast phase.The inoculum size of inoculum is 10% (v/v).
After batch fermentation carries out 30 hours, by continuous feeding speed is fine tuning to 81.0ml/h, transfer training mode to continuous mode, dilution rate (D) is set to 0.0405h
-1As a result, continuously ferment and carried out 120 hours, the mean concns of product 2-KGA is 112.2g/L.2-KGA molar yield average out to 91.3%.In the steady state of continuous mode, the throughput of 2-KGA is calculated as 3.9 to 4.8g/L/h.
Embodiment 2: by G.oxydans DSM 4025 in be supplemented with the zymic substratum from 14%
The L-sorbose carries out the 2-KGA of single phase and continuously ferments
With with the identical means described in the embodiment 1, preparation 200ml inoculum, and it is inoculated in the 2L production substratum as described in example 1 above.Batch fermentation changes training mode into continuous mode after carrying out 30 hours.Continuous feeding substratum (10L) is by 14.0% L-sorbose (sterilization separately), 0.05% glycerine, 0.25% MgSO
47H
2O, 3.0% corn steep liquor, 7.5% bread yeast, 0.15% defoamer CA-115 form.With NaOH pH is controlled at 7.0 between yeast phase.Continuously ferment and carried out 100 hours, produced the 2-KGA of 134.3g/L in the steady state of continuous mode.D is calculated as average 0.0356h
-12-KGA molar yield average out to 93.2%.In the steady state of described continuous mode, the throughput of 2-KGA is calculated as 4.78g/L/ hour.
Embodiment 3: by using mixing of G.oxydans DSM 4025 and B.megaterium DSM 4026
Close culture and carry out the 2-KGA fermentation from the L-sorbose with repeated fed-batch (semicontinuous) pattern
On the nutrient agar that contains 0.5% D-glucose, 0.5% beef extract, 0.5% polyprotein peptone (Nippon Seiyaku), 0.30% NaCl (pH is 7.0 before the sterilization) and 2.0% agar, B.megaterium DSM 4026 is carried out one day cultivation in 27 ℃.
Agar culture with the G.oxydans DSM 4025 of the agar culture of the B.megaterium DSM 4026 of a transfering loop and two to three transfering loops, be inoculated into simultaneously in the 100ml substratum in the 500ml Erlenmeyer bottle, and, contain 2% L-sorbose, 0.3% beef extract, 0.3% yeast extract, 0.3% corn steep liquor, 1% polyprotein peptone (Nippon Seiyaku), 0.1% urea, 0.1% KH in the described substratum 30 ℃ of cultivations one day
2PO
4, 0.02% MgSO
47H
2O (is adding CaCO
3And before the sterilization, pH is 6.7), 0.10% CaCO
3With 0.03% defoamer CA-115.Get this culture of 10ml, transfer in the same substratum of 100ml in the 500ml Erlenmeyer bottle, and cultivated 1 day at 30 ℃.With this culture to the culture medium inoculated that is used for ferment tank.
The above-mentioned inoculum of 200ml is inoculated into 2L and contains 8% L-sorbose (sterilization separately), 2.5% corn steep liquor, 0.0086% MgSO
47H
2O, 0.086% KH
2PO
4, 0.067% defoamer CA-115 the production substratum in.Batch fermentation continues to gather in the crops out the nutrient solution of 1.8L after 48 hours.0.2L nutrient solution is left in the fermentor tank, as the seed that ferments next time.In fermentor tank, fill with and contain 10% L-sorbose (sterilization separately), 2.5% corn steep liquor, 0.0086% MgSO
47H
2O, 0.086% KH
2PO
4, 0.067% defoamer CA-115 fresh culture.With NaOH pH is controlled at 7.0 between yeast phase.As a result, the 2-KGA molar yield of the first step and second step is respectively 84.7% and 90.1%.Maximum value to the throughput of 2-KGA in second step is calculated as 2.55g/L/h.
Embodiment 4: by using G.oxydans DSM 4025 and B.megaterium DSM 4026
Mixed culture carries out the 2-KGA of single phase from the L-sorbose and continuously ferments
Produce substratum (2L) and continuous feeding substratum (10L) by 10% L-sorbose (sterilizing separately), 2.5% corn steep liquor, 0.0086% MgSO
47H
2O, 0.086% KH
2PO
4Form with 0.067% defoamer CA-115.121 ℃ of sterilizations of above-mentioned substratum being carried out 20 minutes.With NaOH pH is controlled at 7.0 between yeast phase.With with the identical means described in the embodiment 3, preparation 200ml inoculum, and it is inoculated in the production substratum of 2L.
After batch fermentation carries out 30 hours, by continuous feeding speed is fine tuning to 61.0ml/h, change training mode into continuous mode, D is at 0.0305h
-1To 0.0407h
-1Scope in the change.The mean concns of 2-KGA is 41.2g/L, has also obtained 92.7% 2-KGA molar average transformation efficiency.In the steady state of described continuous mode, the throughput of 2-KGA is calculated as 1.51g/L/h.
Embodiment 5: pass through to use G.oxydans DSM 4025 and X.maltophilia in the 3L fermentor tank
The mixed culture of IFO 12692 carries out two stage 2-KGA from the L-sorbose and continuously ferments
In the two stage system of continuously fermenting, equipped same facility as described in example 2 above for the glass fermentor tank.This fermentation system is made of two fermentor tanks.Usually, during the fermentation beginning, inoculum accounts for 10% (v/v).The working volume of two fermentor tanks all is 2L, by with the single phase pattern in identical way, with peristaltic pump continuous feeding speed is controlled.Transfer in the subordinate phase fermentor tank with the nutrient solution that second pump will take out from first fermentor tank.Finally, with the 3rd peristaltic pump nutrient solution is got in the results storage tank.Temperature is controlled in 30 ℃, and stirring velocity and Ventilation Rate are set to 800rpm and 1.0L/min respectively.With sodium hydroxide solution pH is controlled at 7.0.
Containing 1% polyprotein peptone, 0.2% yeast extract, 0.1% MgSO
47H
2On the nutrient agar of O (sterilization before pH be 7.0) and 2% agar, in 27 ℃ with X.maltophilia IFO 12692 cultivations 2 days.
Agar culture with the G.oxydans DSM 4025 of the agar culture of the X.maltophilia IFO 12692 of a transfering loop and two to three transfering loops, be inoculated into simultaneously in the 100ml substratum in the 500ml Erlenmeyer bottle, and cultivated one day at 30 ℃, described substratum is identical with the substratum that is used for G.oxydans DSM 4025 and B.megaterium DSM 4026 are carried out mixed culture.Get the 10ml culture, transfer in the identical substratum of 100ml in the 500ml Erlenmeyer bottle, and cultivated 1 day at 30 ℃.With this culture to the culture medium inoculated that is used for ferment tank.
Produce substratum (2L) by 12% L-sorbose (sterilization separately), 3% corn steep liquor, 0.4% yeast extract, 0.25% MgSO
47H
2O, 0.05% glycerine and 0.067% defoamer CA-115 form.121 ℃ of sterilizations of above-mentioned substratum being carried out 20 minutes.With NaOH pH is controlled at 7.0 between yeast phase.The above-mentioned inoculum of 200ml is inoculated in the production substratum of 2L.
Two stages, the system of continuously fermenting was made up of fs and subordinate phase fermentor tank.The fermentation culture that will obtain from the fs fermentor tank is transferred in the subordinate phase fermentor tank and is gone, so that the L-sorbose is converted into 2-KGA fully.For first and second fermentor tanks, D is being respectively 0.0277 to 0.070h appropriate opportunity
-1Scope in and 0.0314 to 0.0549h
-1Scope in change, to work out the D value that the L-sorbose is converted into 2-KGA fully and in final results jar, does not have the residual necessity of L-sorbose.Be terminated secondary fermentation in 1,331.5 hour.Summarized the fermentation activity of the mixture of G.oxydansDSM 4025 and X.maltophilia IFO 12692 in the table 1 by two stage continuous modes.In this two stages continuous mode, apparent D is 0.0380h
-1Situation under, reality has obtained to contain the nutrient solution of 113.1g/L 2-KGA, has shown total dilution rate in second hurdle, it is calculated as 0.019h
-1Total 2-KGA molar yield is 90.1%.Under the steady state of this continuous mode, in first fermentor tank throughput of 2-KGA and total throughput to 2-KGA are calculated as 3.34g/L/h and 2.15g/L/h respectively.
Table 1: the fermentation activity that uses two stage continuous modes of 3L fermentor tank
| State | Gross activity | Fs | Subordinate phase |
| Dilution rate is (average: h -1) | 0.0190 | 0.0382 | 0.0380 |
| 2-KGA concentration is (average: g/L) | 113.1 | 87.5 | 113.1 |
| Throughput (g/L/h) to 2-KGA | 2.14 | 3.34 | - |
| 2-KGA molar yield (%) | 90.1 | 90.4 | 90.1 |
Embodiment 6: use G.oxydans DSM 4025 and X.maltophilia in the 30L fermentor tank
The mixed culture of IFO 12692 carries out two stage 2-KGA from the L-sorbose and continuously ferments
With the fermentor tank of 30L, carry out and the identical pattern of cultured continuously pattern that is applied to the 3L fermentor tank.Described continuously fermenting carried out surpassing 180 hours, when D is set to 0.041h
-1The time, observe stable 2-KGA production in subordinate phase, and do not had L-sorbose residue.As a result, in the fermentor tank of 30L scale, reproduced the throughput that obtains in the 3L fermentor tank to 2-KGA.Table 2 pair fermentation activity is summarized.In the steady state of continuous mode, produced the 2-KGA of 118.5g/L.In the steady state of described continuous mode, the throughput to 2-KGA in first fermentor tank is calculated as 4.72g/L/h.In addition, total throughput is calculated as 2.55g/L/ hour, and total 2-KGA molar yield is 89.6%.
Table 2: the fermentation activity that uses two stage continuous modes of 30L fermentor tank
| State | Gross activity | Fs | Subordinate phase |
| Dilution rate is (average: h -1) | 0.0215 | 0.0430 | 0.0430 |
| 2-KGA concentration is (average: g/L) | 118.5 | 109.9 | 118.5 |
| Throughput (g/L/h) to 2-KGA | 2.55 | 4.72 | - |
| 2-KGA molar yield (%) | 89.6 | 89.0 | 89.6 |
Embodiment 7: pass through to use G.oxydans DSM 4025 and X.maltophilia in the 3L fermentor tank
The mixed culture of IFO 12692 carries out from the L-sorbose nutrient solution that is converted by the D-Sorbitol Powder
Two stage 2-KGA continuously ferments
Carried out continuously fermenting from the D-Sorbitol Powder in the present embodiment to 2-KGA, wherein set up the multistep fermentation system that couples together before and after " fermentation process " and " fermentation process ", and the fermentation activity from the D-Sorbitol Powder to 2-KGA has been studied from the L-sorbose to 2-KGA from the D-Sorbitol Powder to the L-sorbose.The bacterium that contains by producing the L-sorbose is used as L-sorbose source from the nutrient solution that the D-Sorbitol Powder transforms the L-sorbose that gets, and the bacterium of described product L-sorbose for example is the bacterial strain of Gluconobacter.Under aseptic condition, with the nutrient solution that contains the Gluconobacter cell gathered in the crops with contain 3% corn steep liquor, 0.4% yeast extract, 0.25% MgSO through sterilization
47H
2O, 0.05% glycerine and the substratum of 0.067% defoamer CA-115 mix, and the concentration of L-sorbose is adjusted to 13%.G.oxydans DSM4025 and X.maltophilia IFO 12692 are carried out mixed culture, in the fermentor tank of 3L, carry out the 2-KGA fermentation of two stage cultured continuously patterns.
Fermentation has been carried out 290 hours.Kept metastable fermentation activity in this period, the average D in first fermentor tank and second fermentor tank is respectively 0.0410h
-1And 0.0404h
-1Table 3 is couple result summarize.In the steady state of continuous mode, produced the 2-KGA of 128.9g/L.In the steady state of described continuous mode, the throughput to 2-KGA in first fermentor tank is calculated as 4.31g/L/h.Total throughput is calculated as 2.34g/L/h, is 83.4% from the molar yield of L-sorbose.
Table 3: the fermentation activity that uses two stage continuous modes of L-sorbose nutrient solution
| State | Gross activity | Fs | Subordinate phase |
| Dilution rate is (average: h -1) | 0.0202 | 0.0410 | 0.0404 |
| 2-KGA concentration is (average: g/L) | 115.9 | 105.2 | 115.9 |
| Throughput (g/L/h) to 2-KGA | 2.34 | 4.31 | - |
| 2-KGA molar yield (%) | 83.4 | 83.7 | 83.4 |
Claims (10)
1. one kind by the method for continuously fermenting or semicontinuous fermentation is produced 2-keto-L-gulonic acid or its salt from the L-sorbose, in the described method, used be selected from by Gluconobacter oxydans DSM4025 (FERM BP-3812), have Gluconobacter oxydans DSM 4025 (FERMBP-3812) identification mark belong to the microorganism that Gluconobacter belongs to and the microorganism of the group that mutant constituted thereof; Use the substratum that contains yeast or yeast product in the described method, perhaps described method is carried out under the situation that has second kind of microbe composition to exist.
2. the method for claim 1 comprises the steps:
(a) in one or more fermenting containers, preferably in a fermenting container, cultivate and be selected from Gluconobacter oxydans DSM 4025 (FERM BP-3812), have Gluconobacteroxydans DSM 4025 (FERM BP-3812) identification mark belong to the microorganism that Gluconobacter belongs to and the microorganism of mutant thereof; Described cultivation is carried out in the substratum that contains yeast or yeast product, or carries out in the substratum that is not having yeast or yeast product under the situation that second kind of microbe composition exists;
(b) in fermenting container continuously supply contain the nutritional medium of L-sorbose, the dilution rate of fermentation is arranged on about 0.01h
-1To about 0.05h
-1, the concentration of described L-sorbose at about 20g/L to approximately between the 250g/L,
(c) from fermenting container, take out fermentation culture continuously; With
(d) from described fermentation culture, reclaim 2-keto-L-gulonic acid or its salt.
3. the method for claim 1, wherein second kind of microbe composition is selected from the group that is made of Bacillus and Xanthomonas, preferably the group that is made of Bacillus megaterium DSM 4026 and Xanthomonas maltophilia IFO 12692.
4. the method for claim 1, wherein said yeast or yeast product belong to Ascomycetes subclass, preferably belong to Saccharomyces and belong to.
5. method as claimed in claim 2 wherein uses the multistage to continuously ferment.
6. the method for claim 1, wherein said fermentation are carried out in pH under about 4.0 situations to about 9.0 the scope.
7. the method for claim 1, wherein said fermentation are carried out in temperature under about 13 ℃ of situations to about 36 ℃ scope.
8. the method for claim 1, wherein said fermentation be carried out in the fermenting container dissolved oxygen concentration about 5% to oxygen concn between about 100% saturation ratio or in the air-flow under the situation between about 0.1% to about 100%.
9. the method for claim 1 is wherein used semicontinuous (repeated fed-batch) fermentation, and wherein, the part of fermentation culture is used as the inoculum of next time cultivating, and this process is repeated continuously.
10. the method for claim 1, wherein, the nutrient solution that contains the L-sorbose that is converted from the D-Sorbitol Powder is used as the substrate of fermentation, and described L-sorbose is by producing the bacterium of L-sorbose, for example Gluconobacter or Acetobacter are converted from the D-Sorbitol Powder.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| EP02021606.5 | 2002-09-27 | ||
| EP02021606 | 2002-09-27 |
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| CNB038232030A Expired - Lifetime CN100560728C (en) | 2002-09-27 | 2003-09-22 | The production method of 2-KGA |
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| CN (1) | CN100560728C (en) |
| AU (1) | AU2003283253A1 (en) |
| WO (1) | WO2004029265A2 (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102757928A (en) * | 2012-08-09 | 2012-10-31 | 山东天力药业有限公司 | 2-keto-L-gulonic acid high tolerance type gluconobacteroxydans and application thereof in vitamin C fermentation production |
| CN103290071A (en) * | 2013-06-09 | 2013-09-11 | 山东天力药业有限公司 | Method for preparing 2-keto-L-gulonic acid |
| CN103789387A (en) * | 2012-10-30 | 2014-05-14 | 山东天力药业有限公司 | Method for increasing production efficiency of vitamin C intermediate 2-keto-L-gulonic acid |
Family Cites Families (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DK171869B1 (en) * | 1985-10-22 | 1997-07-21 | Takeda Chemical Industries Ltd | Process for preparing 2-keto-L-gulonic acid and biologically pure microorganism culture for use in the process |
| US4935359A (en) * | 1987-02-07 | 1990-06-19 | Institute Of Microbiology | Fermentation process |
| DK173507B1 (en) * | 1988-09-30 | 2001-01-15 | Hoffmann La Roche | Process for the preparation of 2-keto-L-gulonic acid |
| RU2102481C1 (en) * | 1991-06-13 | 1998-01-20 | Ф.Хоффманн-Ля Рош, Аг | Method of 2-keto-l-gulonic acid or its salt preparing |
| EP0972843A1 (en) * | 1998-07-17 | 2000-01-19 | F. Hoffmann-La Roche Ag | Continuous fermentation process |
-
2003
- 2003-09-22 WO PCT/EP2003/010488 patent/WO2004029265A2/en not_active Application Discontinuation
- 2003-09-22 AU AU2003283253A patent/AU2003283253A1/en not_active Abandoned
- 2003-09-22 CN CNB038232030A patent/CN100560728C/en not_active Expired - Lifetime
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102757928A (en) * | 2012-08-09 | 2012-10-31 | 山东天力药业有限公司 | 2-keto-L-gulonic acid high tolerance type gluconobacteroxydans and application thereof in vitamin C fermentation production |
| CN103789387A (en) * | 2012-10-30 | 2014-05-14 | 山东天力药业有限公司 | Method for increasing production efficiency of vitamin C intermediate 2-keto-L-gulonic acid |
| CN103290071A (en) * | 2013-06-09 | 2013-09-11 | 山东天力药业有限公司 | Method for preparing 2-keto-L-gulonic acid |
| CN103290071B (en) * | 2013-06-09 | 2015-02-18 | 山东天力药业有限公司 | Method for preparing 2-keto-L-gulonic acid |
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| AU2003283253A8 (en) | 2004-04-19 |
| WO2004029265A2 (en) | 2004-04-08 |
| AU2003283253A1 (en) | 2004-04-19 |
| WO2004029265A3 (en) | 2004-05-27 |
| CN100560728C (en) | 2009-11-18 |
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