CN100999745A - Process of preparing gamma-poly glutaminic acid - Google Patents
Process of preparing gamma-poly glutaminic acid Download PDFInfo
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Abstract
The present invention discloses poly-gamma-glutamic acid preparing process. The process includes the steps of culturing different kinds of poly-gamma-glutamic acid producing bacterial strains in culture medium containing carbon source, nitrogen source, NaCl and intermediate to obtain poly-gamma-glutamic acid fermenting liquor, precipitating in organic solvent, dialysis and drying to obtain poly-gamma-glutamic acid product. The process has the intermediate as the precursor for preparing poly-gamma-glutamic acid, and is simple, low in cost and high in yield.
Description
Technical field
The present invention relates to a kind of method for preparing gamma-polyglutamic acid-.
Background technology
(Poly γ-glumatic acid, γ-PGA) a kind ofly can pass through the equal amino-acid compound of microorganism synthetic to gamma-polyglutamic acid-.As a kind of bioabsorbable polymer material, γ-PGA have biodegradability good, edible, to the advantage of human body and environment toxicological harmless.Therefore, γ-PGA and derivative thereof have purposes widely at aspects such as food, makeup, medicine and water treatments.
The method of at present synthetic γ-PGA mainly contains chemical synthesis, extraction method and microorganism synthesis method etc.But the chemical synthesis synthetic route is long, by product is many, yield is low, and the molecular weight of product little, be difficult to satisfied requirement as the new drug carrier material, do not have industrial application value.And extraction method is lower owing to γ-PGA concentration, and with the condition difference, content is big.Therefore, extraction process is very complicated, and production cost is very high, is difficult to carry out large-scale industrial production equally.
The main at present Production by Microorganism Fermentation γ-PGA that adopts.γ-PGA is that nineteen thirty-seven Ivanovics at first finds (Ivanovics G, Bruckner V.Immunit atsforch.1937,90,304~318) in the pod membrane of Bacillus anthracis.The Korea S researchist adopts stream to add the high-density cultured method to Bacillus licheniformisATCC9945a in 2.5 liters of fermentor tanks, the ultimate capacity (Yoon SH, DO JH, the Lee SY.Biotechnol.Lett that ferment and to reach 35g/L after 35 hours, 2000,22:585~588).Ogawa studies show that to what the scale operation of Bacillus subtilis MR 141 was carried out optimizing culture condition in the fermentor tank of 30L can make γ-PGA maximum production reach 35g/L (Ogawa Y.Yamaguchi F, Yuasa K.BiosciBiotec Biochem, 1997,61:1684~1687).Kubota separates a strain Bacillus subtilis F201 who obtains in Osaka City University soil, Bacillus subtilis F201 bacterial strain can reach the production peak of 50g/L under best fermentative production condition, this is the high yield output (Kubota H.Biosci Biotec Biochem, 1993:1212~1213) of bibliographical information.This bacterial strain successfully is used for large-scale industrialization by Meiji Seika Kaisha company and produces γ-PGA (Tanaka T, Yaguchi T, Hiruta O, et al.BiosciBiotechnol Biochem, 1993,57,1809~1810; Tanaka T, Hiruta O, Futamura T, et al.Biosci Biotechnol Biochem, 1993,57,2148~2153).
Present most γ-PGA produce bacterial strain with L-glutamic acid as precursor, and utilize the intermediate product of relative low price to replace L-glutamic acid will help reducing the cost of γ-PGA.Intermediate product in glutamic acid fermentation and the separation and Extraction process mainly contains three kinds: 1, glutami acid fermentation liquor: obtain after the fermented liquid behind the glutamic acid fermentation process is removed thalline, its L-glutamic acid content is generally 10~15% (m/v); 2, glutamic acid crystallization mother liquor: with glutami acid fermentation liquor experience freeze isoelectric precipitation, again with obtaining in the NaOH solution and after dissolving, its L-glutamic acid content is generally 40~45% (m/v); 3, bran acid: with the monosodium glutamate crude product that the further concentrate drying of glutamic acid crystallization mother liquor obtains, its L-glutamic acid content is generally 75~80% (m/v).
Can predict, the intermediate product that adopts gourmet powder fermenting production is as the biosynthetic glutamate precursor of γ-PGA, because its price is more cheap than the L-glutamic acid finished product, can reduce the production cost of γ-PGA under identical production capacity.
Summary of the invention
The object of the present invention is to provide a kind of efficient, low cost to utilize intermediate product that glutamic acid fermentation produces method as precursor fermentative preparation gamma-polyglutamic acid-.
Purpose of the present invention can reach by following measure:
The microorganism of using
Be used to produce microbial bacteria bacillus subtilis (Bacillussubtills) zju-7 of gamma-polyglutamic acid-of the present invention, this bacterial strain is inventor's isolation and selection and getting from the soy sauce production waste material, be deposited in China Microbial Culture Preservation Commission common micro-organisms center on November 15th, 2004, it abbreviates CGMCC as, and deposit number is: CGMCC No.1250;
The microorganism strains bacillus natto (Bacillus natto) that is used to produce gamma-polyglutamic acid-of the present invention is available from Beijing institute of microbiology of the Chinese Academy of Sciences;
The microbial bacteria bacillus licheniformis (Bacilluslicheniformis) that is used to produce gamma-polyglutamic acid-of the present invention is that University Of Science and Technology Of Tianjin is so kind as to give.
The glutamic acid fermentation intermediate product
Being used for producing the employed glutamic acid fermentation intermediate product of gamma-polyglutamic acid-of the present invention is various fermented liquid, crystalline mother solution or the bran acid that contain high density L-glutamic acid of glutamic acid fermentation production process.
The method for preparing gamma-polyglutamic acid-of the present invention is characterized in that concrete steps are as follows:
1) with the fermentative production bacterial strain of gamma-polyglutamic acid-, 37 ℃ of slant medium activation 8 hours, substratum was counted by the quality percent by volume: glucose 1~10%, peptone 1~8%, L-glutamic acid 2~6%, MgSO
40.05~0.5%, NaCl1~8%, K
2HPO
40.01~0.05%, supply volume with water;
2) the fermentative production inoculation of the gamma-polyglutamic acid-after will activating is in liquid nutrient medium, liquid nutrient medium contains that to count 1~8% carbon source, 1~8% nitrogenous source, 1~5%NaCl and L-glutamic acid content by the quality percent by volume be 1~30% glutamic acid fermentation intermediate product, the consumption of glutamic acid fermentation intermediate product is 1~15% for making the whole content of L-glutamic acid, supply volume with water, cultivated 36 hours for 32~40 ℃;
3) with the thalline in the centrifugation method removal fermented liquid, with hydrochloric acid the pH value of supernatant liquor is transferred to 3~5, add organic solvent then, add-on is 2~5 times of supernatant liquor volume, precipitation is dissolved in throw out in 100 times of volume water again, removes by filter insolubles, small-molecule substance is removed in dialysis then, again solution lyophilize or vacuum-drying is obtained gamma-polyglutamic acid-.
Among the present invention, the fermentative production bacterial strain of said gamma-polyglutamic acid-can be subtilis (Bacillussubtills), bacillus natto (Bacillus natto) or Bacillus licheniformis (Bacillus licheniformis);
Glutamic acid fermentation intermediate product in the said liquid nutrient medium is glutami acid fermentation liquor, glutamic acid crystallization mother liquor or bran acid; Carbon source can be glucose, sucrose, fructose, maltose, lactose, Zulkovsky starch or starch, and is comparatively suitable with dextrose plus saccharose; Nitrogenous source can be an organic nitrogen source, as peptone, yeast extract paste, corn steep liquor, soybean cake powder or Semen Maydis powder etc., or inorganic nitrogen-sourced NH
4NO
3Or (NH
4)
2SO
4, above-mentioned nitrogenous source can use separately, also can mix use.
Contain 1~15% L-glutamic acid in the liquid nutrient medium of the present invention, be preferably in 6~12%, in the process for preparation of liquid medium within, make the content of L-glutamic acid in the liquid nutrient medium between 1~15% according to its requirement the glutamic acid fermentation intermediate product.When the L-glutamic acid that adds very little, the gamma-polyglutamic acid-turnout reduces, even does not produce fully; Add-on is excessive, the thalli growth variation, and the L-glutamic acid residue in the fermented liquid too much causes waste.
The gamma-polyglutamic acid-that the present invention obtains has following physico-chemical property:
1) this product is water-soluble, is insoluble to methyl alcohol, ethanol, acetone and other organic solvent;
2) this product ninhydrin reaction is negative, and is positive with ninhydrin reaction after the 6MHCl hydrolysis;
3) detect with HPLC and thin layer chromatography after the 6MHCl hydrolysis, find to generate in the hydrolyzate single amino acid-L-glutamic acid.Prove that this product is the high molecular polymer of L-glutamic acid;
4) prove that by methods such as SDS-PAGE and gel filtration chromatographies the molecular weight of this product is 200~1000KDa;
Have a large amount of free carboxies on the gamma-polyglutamic acid-molecule of the present invention's preparation, thereby have good hygroscopic property and performance of keeping humidity, can be used as the wetting Agent for Printing Inks of insoluble drug carrier, food thickener, starch protective agent and makeup.
The present invention compared with prior art has the following advantages:
1) the present invention uses the glutamate precursor of the intermediate product of glutamic acid fermentation production as the gamma-polyglutamic acid-fermentative production, compares with glutamate precursor, has reduced the separation and purification process of L-glutamic acid, has reduced the production cost of gamma-polyglutamic acid-;
2) intermediate product of glutamic acid fermentation production not only contains the glutamate precursor of high density, also contains nutritive substance residual in the Corynebacterium glutamicum fermenting process, can further utilize in the gamma-polyglutamic acid-fermentation production process, helps reducing producing consuming;
3) by to the optimization of strain culturing condition, particularly by adjusting the concentration of L-glutamic acid and salt thereof in substratum, the content that makes the gamma-polyglutamic acid-in the fermented liquid is up to 45~55g/L, thereby provides a kind of efficient cheapness to prepare the method for gamma-polyglutamic acid-.
Embodiment
The following examples elaborate to the present invention, but to the present invention without limits.
Embodiment 1
1) with subtilis (Bacillus subtills) zju-7 through 37 ℃ of slant activation, slant medium: glucose 1.5%, peptone 1%, L-glutamic acid 3%, MgSO
40.1%, NaCl 1%, K
2HPO
40.01%, supply volume with water; Activate 8 hours.
2) liquid nutrient medium: glucose 2%, peptone 1.5%, MgSO
40.1%, NaCl 1%, K
2HPO
40.01%, be 3% with the Corynebacterium glutamicum fermented liquid in latter stage of centrifugal removal thalline according to the concentration of the concentration dilution of its L-glutamic acid L-glutamic acid to the shake-flask culture base simultaneously, supply volume with water, pH7, liquid amount 50ml/500ml shakes bottle, sterilizes 20 minutes for 115 ℃.
After sterilization finished, above-mentioned subtilis (Bacillus subtills) zju-7 was inoculated in cooling, and inoculum size is 3%, 37 ℃ of shake-flask culture, and rotating speed 200rpm carries out the shake flask fermentation experiment under these conditions, cultivates 24 hours.
3) after the fermentation ends, thalline in the centrifugal removal fermented liquid, with hydrochloric acid the pH value of supernatant liquor is transferred to 4, add 4 times of volumes methanol then, precipitation is dissolved in throw out in 100 times of volume water again, remove by filter insolubles, small-molecule substance is removed in dialysis then, again the solution lyophilize is obtained gamma-polyglutamic acid-, and output can reach 15g/L.
Embodiment 2
With embodiment 1, gamma-polyglutamic acid-is produced bacterial strain be replaced by bacillus natto (Bacillus natto), through 37 ℃ of slant activation, slant medium: glucose 1.5%, peptone 1%, L-glutamic acid 3%, MgSO
40.1%, NaCl 1%, K
2HPO
40.01%, supply volume with water, activate 8 hours.
Liquid nutrient medium: glucose 2%, peptone 2%, L-glutamic acid 5%, MgSO
40.2%, NaCl 2%, K
2HPO
40.05%, pH7, liquid amount 30/250ml shakes bottle, sterilizes 20 minutes for 115 ℃.The sterilization postcooling, inoculation, inoculum size is 3%, supplies volume with water, cultivates 24 hours for 37 ℃, shaking bottle rotating speed is 200rpm.
After the fermentation ends, with embodiment 1 separation and purification gamma-polyglutamic acid-, output can reach 10.3g/L.
Embodiment 3
With embodiment 1, adopt the surrogate of crystalline mother solution as glutamate precursor, 37 ℃ of shake-flask culture, rotating speed 200rpm cultivated 24 hours.
After the fermentation ends, thalline in the centrifugal removal fermented liquid, with hydrochloric acid the pH value of supernatant liquor is transferred to 4, add 4 times of volumes methanol then, precipitation is dissolved in throw out in 100 times of volume water again, remove by filter insolubles, small-molecule substance is removed in dialysis then, again the solution lyophilize is obtained gamma-polyglutamic acid-, and output can reach 12g/L.
Embodiment 4
With glucose 2%, peptone 2%, MgSO
40.1%, NaCl 2%, K
2HPO
40.05%, as the glutamate precursor surrogate, make that the aminoglutaric acid concentration in the shake-flask culture base is 8% with the solution behind the centrifugal removal thalline of Corynebacterium glutamicum fermented liquid in latter stage, supply volume with water, regulate pH7, liquid amount 30ml/200ml shakes bottle, sterilized 20 minutes for 115 ℃, after sterilization finishes, cooling, inoculation subtilis (Bacillus subtills) zju-7, inoculum size is 3%, 37 ℃ of shake-flask culture, 200rpm, cultivated 24 hours
After the fermentation ends, according to the method separation and purification gamma-polyglutamic acid-of embodiment 1, its output can reach 32g/L.
Embodiment 4
With embodiment 3, the glucose in the shake-flask culture base is replaced with sucrose, the gamma-polyglutamic acid-concentration that the result obtains in the fermented liquid is 38g/L.
Embodiment 5
With glucose 10%, peptone 8%, MgSO
40.2%, NaCl 5%, K
2HPO
40.05%, the fermented liquid that the adding Corynebacterium glutamicum is fermented latter stage makes that substratum two-story valley histidine content is 15%, supplies volume with water, pH7, and liquid amount 50/500ml shakes bottle, sterilizes 20 minutes for 115 ℃.The sterilization postcooling inserts Bacillus licheniformis (Bacillus licheniformis 9945A), cultivates 24 hours for 37 ℃, and shaking bottle rotating speed is 200rpm.
After the fermentation ends, the thalline in the centrifugal removal fermented liquid, the method separation and purification of being narrated according to embodiment 1 obtains-polyglutamic acid, and output can reach 24g/L.
Embodiment 6
With glucose 1%, peptone 1%, MgSO
40.05%, NaCl 1%, K
2HPO
40.01%, add bran acid and make that aminoglutaric acid concentration is 2% in the substratum, supply volume with water, pH7, liquid amount 50ml/500ml shakes bottle, sterilizes 20 minutes for 121 ℃, after sterilization finishes, cooling inserts bacillus natto (Bacillus natto), and inoculum size is 1%, 37 ℃ of shake-flask culture, rotating speed 100~400rpm carries out the shake flask fermentation experiment under these conditions, cultivates 24 hours.
After the fermentation ends, according to the method separation and purification gamma-polyglutamic acid-of embodiment 1, its output can reach 11g/L.
Embodiment 7
Through 37 ℃ of slant activation, slant medium is: glucose 1%, peptone 2%, L-glutamic acid 2%, MgSO with subtilis (Bacillus subtills) zju-7
40.05%, NaCl 1%, K
2HPO
40.01%, supply volume with water, activate 12 hours.
Picking list bacterium colony is to the first order seed substratum on the inclined-plane, and the first order seed substratum is: glucose 2%, peptone 2%, L-glutamic acid 3%, MgSO
40.05%, NaCl 1%, K
2HPO
40.1%, supply volume with water, pH.7, liquid amount 10ml/100ml shakes bottle, 115 sterilizations 15~30 minutes, after sterilization finishes, cooling, inoculation subtilis (Bacillus subtills) zju-7, inoculum size is that 3%, 37 ℃ of rotating speed 200rpm cultivates 12h.
With sucrose 1%, peptone 1%, L-glutamic acid 4%, MgSO
40.1%, NaCl.0.5%, K
2HPO
40.05% is secondary seed medium, supply volume with water, pH7, liquid amount 100ml/1000ml shakes bottle, sterilized 20 minutes for 115 ℃, after sterilization finished, cooling inserted subtilis (Bacillus subtills) zju-7, inoculum size is 3%, 37 ℃ of shake-flask culture, 200rpm cultivated 12 hours.
With sucrose 4%, peptone 4%, MgSO
40.1%, NaCl 3%, K
2HPO
40.05%, add Corynebacterium glutamicum fermented liquid in latter stage and make that the aminoglutaric acid concentration in the fermention medium is 8%, supply volume with water, pH7, liquid amount 3L/5L fermentor tank, sterilized 20 minutes for 115 ℃, insert the secondary seed of above-mentioned subtilis (Bacillussubtills) zju-7, inoculum size is 1%, control fermented liquid pH value is 6.5~7.0, air flow is 3.0vvm, and temperature is 37 ℃, cultivates 36h.
After the fermentation ends, according to the method separation and purification gamma-polyglutamic acid-of embodiment 1, experimental result shows that gamma-polyglutamic acid-output reaches 51.3g/L, and productivity reaches 1.225gh
-1L
-1
Embodiment 8
With embodiment 7, glutamate precursor in the fermentation cylinder for fermentation substratum is substituted with crystalline mother solution, insert the secondary seed of above-mentioned subtilis (Bacillus subtills) zju-7, inoculum size is 1%, control fermented liquid pH value is 6.5~7.0, air flow is 3.0vvm, and temperature is 37 ℃, cultivates 36h.
After the fermentation ends, according to the method separation and purification gamma-polyglutamic acid-of embodiment 1, experimental result shows that gamma-polyglutamic acid-output reaches 42.7g/L, and productivity reaches 0.957gh
-1L
-1
Embodiment 9
With embodiment 7, glutamate precursor in the fermentation cylinder for fermentation substratum is substituted with bran acid, insert the secondary seed of above-mentioned subtilis (Bacillus subtills) zju-7, inoculum size is 1%, control fermented liquid pH value is 6.5~7.0, air flow is 3.0vvm, and temperature is 37 ℃, cultivates 36h.
After the fermentation ends, according to the method separation and purification gamma-polyglutamic acid-of embodiment 1, experimental result shows that gamma-polyglutamic acid-output reaches 46.2g/L,
Embodiment 10
Through 37 ℃ of slant activation, slant medium is with bacillus natto (Bacillus natto): glucose 1%, peptone 1% activates 12 hours.
Picking list bacterium colony is to the first order seed substratum on the inclined-plane, and the first order seed substratum is: glucose 2%, peptone 2%, L-glutamic acid 3%, MgSO
40.05%, NaCl 1%, K
2HPO
40.1%, supply volume with water, pH.7, liquid amount 10ml/100ml shakes bottle, 115 sterilizations 15~30 minutes, after sterilization finishes, cooling, inoculation bacillus natto (Bacillus natto), inoculum size is that 3%, 37 ℃ of rotating speed 200rpm cultivates 12h.
With glucose 1%, peptone 1%, L-glutamic acid 4%, MgSO
40.1%, NaCl.0.5%, K
2HPO
40.05% is secondary seed medium, supplies volume with water, pH7, and liquid amount 100ml/1000ml shakes bottle, sterilized 20 minutes for 115 ℃, after sterilization finished, cooling inserted bacillus natto (Bacillus natto), inoculum size is 3%, 37 ℃ of shake-flask culture, and 200rpm cultivated 12 hours.
With glucose 4%, peptone 4%, MgSO
40.1%, NaCl 3%, K
2HPO
40.05%, add Corynebacterium glutamicum fermented liquid in latter stage and make that the aminoglutaric acid concentration in the fermention medium is 8%, supply volume with water, pH7, liquid amount 3L/5L fermentor tank, sterilized 20 minutes for 115 ℃, insert the secondary seed of above-mentioned bacillus natto (Bacillusnatto), inoculum size is 1%, control fermented liquid pH value is 6.5~7.0, air flow is 3.0vvm, and temperature is 37 ℃, cultivates 36h.
After the fermentation ends, according to the method separation and purification gamma-polyglutamic acid-of embodiment 1, experimental result shows that gamma-polyglutamic acid-output reaches 31g/L.
Embodiment 11
With embodiment 10, the glutamate precursor in the fermentation cylinder for fermentation substratum is substituted with crystalline mother solution, insert the secondary seed of above-mentioned bacillus natto (Bacillus natto), inoculum size is 1%, and control fermented liquid pH value is 6.5~7.0, and air flow is 3.0vvm, temperature is 37 ℃, cultivates 36h.
After the fermentation ends, according to the method separation and purification gamma-polyglutamic acid-of embodiment 1, experimental result shows that gamma-polyglutamic acid-output reaches 26.5g/L.
Claims (5)
1. method for preparing gamma-polyglutamic acid-is characterized in that concrete steps are as follows:
1) with the fermentative production bacterial strain of gamma-polyglutamic acid-, 37 ℃ of slant medium activation 8 hours, substratum was counted by the quality percent by volume: glucose 1~10%, peptone 1~8%, L-glutamic acid 2~6%, MgSO
40.05~0.5%, NaCl 1~8%, K
2HPO
40.01~0.05%, supply volume with water;
2) the fermentative production inoculation of the gamma-polyglutamic acid-after will activating is in liquid nutrient medium, liquid nutrient medium contains that to count 1~8% carbon source, 1~8% nitrogenous source, 1~5%NaCl and L-glutamic acid content by the quality percent by volume be 1~30% glutamic acid fermentation intermediate product, the consumption of glutamic acid fermentation intermediate product is 1~15% for making the whole content of L-glutamic acid, supply volume with water, cultivated 36 hours for 32~40 ℃;
3) with the thalline in the centrifugation method removal fermented liquid, with hydrochloric acid the pH value of supernatant liquor is transferred to 3~5, add organic solvent then, add-on is 2~5 times of supernatant liquor volume, precipitation is dissolved in throw out in 100 times of volume water again, removes by filter insolubles, small-molecule substance is removed in dialysis then, again solution lyophilize or vacuum-drying is obtained gamma-polyglutamic acid-.
2. the method for preparing gamma-polyglutamic acid-according to claim 1, the fermentative production bacterial strain that it is characterized in that said gamma-polyglutamic acid-are subtilis (Bacillus subtills), bacillus natto (Bacillus natto) or Bacillus licheniformis (Bacillus licheniformis)
3.. the method for preparing gamma-polyglutamic acid-according to claim 1 is characterized in that said glutamic acid fermentation intermediate product is glutami acid fermentation liquor, glutamic acid crystallization mother liquor or bran acid.
4. the method for preparing gamma-polyglutamic acid-according to claim 1, the carbon source that it is characterized in that substratum are glucose, sucrose, fructose, maltose, lactose, Zulkovsky starch or starch.
5. the method for preparing gamma-polyglutamic acid-according to claim 1, the nitrogenous source that it is characterized in that substratum is an organic nitrogen source, as peptone, yeast extract paste, corn steep liquor, soybean cake powder or Semen Maydis powder etc., or inorganic nitrogen-sourced NH
4NO
3Or (NH
4)
2SO
4
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