CN1644677A - Bacillus and its use of preparation of gama-polycysteine - Google Patents
Bacillus and its use of preparation of gama-polycysteine Download PDFInfo
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- CN1644677A CN1644677A CN 200410010509 CN200410010509A CN1644677A CN 1644677 A CN1644677 A CN 1644677A CN 200410010509 CN200410010509 CN 200410010509 CN 200410010509 A CN200410010509 A CN 200410010509A CN 1644677 A CN1644677 A CN 1644677A
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- bacillus
- zju
- polyglutamic acid
- gamma
- subtilis
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- 241000193830 Bacillus <bacterium> Species 0.000 title claims abstract description 35
- 238000002360 preparation method Methods 0.000 title description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims abstract description 40
- 239000007788 liquid Substances 0.000 claims abstract description 25
- 238000000034 method Methods 0.000 claims abstract description 22
- 229920002643 polyglutamic acid Polymers 0.000 claims abstract description 21
- 239000011780 sodium chloride Substances 0.000 claims abstract description 20
- 238000000855 fermentation Methods 0.000 claims abstract description 16
- 230000004151 fermentation Effects 0.000 claims abstract description 16
- 244000063299 Bacillus subtilis Species 0.000 claims abstract description 7
- 235000014469 Bacillus subtilis Nutrition 0.000 claims abstract description 7
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims abstract description 6
- 229910052799 carbon Inorganic materials 0.000 claims abstract description 6
- 239000003960 organic solvent Substances 0.000 claims abstract description 4
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims abstract 8
- 229910052757 nitrogen Inorganic materials 0.000 claims abstract 4
- 239000001963 growth medium Substances 0.000 claims abstract 3
- 239000002244 precipitate Substances 0.000 claims abstract 3
- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 claims abstract 2
- 235000013922 glutamic acid Nutrition 0.000 claims abstract 2
- 239000004220 glutamic acid Substances 0.000 claims abstract 2
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 claims description 50
- 229960002989 glutamic acid Drugs 0.000 claims description 25
- 239000001888 Peptone Substances 0.000 claims description 19
- 108010080698 Peptones Proteins 0.000 claims description 19
- 235000019319 peptone Nutrition 0.000 claims description 19
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 18
- 239000008103 glucose Substances 0.000 claims description 17
- 239000002609 medium Substances 0.000 claims description 14
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 claims description 12
- 239000002054 inoculum Substances 0.000 claims description 12
- 238000012807 shake-flask culturing Methods 0.000 claims description 12
- 230000001954 sterilising effect Effects 0.000 claims description 12
- 238000004659 sterilization and disinfection Methods 0.000 claims description 12
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 9
- 241000894006 Bacteria Species 0.000 claims description 8
- 238000001816 cooling Methods 0.000 claims description 8
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- 239000005720 sucrose Substances 0.000 claims description 7
- 229920002472 Starch Polymers 0.000 claims description 6
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 claims description 6
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- 239000002253 acid Substances 0.000 claims description 4
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- 240000008042 Zea mays Species 0.000 claims description 3
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 3
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 3
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- 239000008101 lactose Substances 0.000 claims description 3
- OWEGMIWEEQEYGQ-UHFFFAOYSA-N 100676-05-9 Natural products OC1C(O)C(O)C(CO)OC1OCC1C(O)C(O)C(O)C(OC2C(OC(O)C(O)C2O)CO)O1 OWEGMIWEEQEYGQ-UHFFFAOYSA-N 0.000 claims description 2
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 claims description 2
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 2
- GUBGYTABKSRVRQ-PICCSMPSSA-N Maltose Natural products O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-PICCSMPSSA-N 0.000 claims description 2
- GUBGYTABKSRVRQ-QUYVBRFLSA-N beta-maltose Chemical compound OC[C@H]1O[C@H](O[C@H]2[C@H](O)[C@@H](O)[C@H](O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@@H]1O GUBGYTABKSRVRQ-QUYVBRFLSA-N 0.000 claims description 2
- 229940041514 candida albicans extract Drugs 0.000 claims description 2
- 238000009629 microbiological culture Methods 0.000 claims description 2
- 125000001477 organic nitrogen group Chemical group 0.000 claims description 2
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- 235000019764 Soybean Meal Nutrition 0.000 claims 1
- 235000013312 flour Nutrition 0.000 claims 1
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 claims 1
- 239000004455 soybean meal Substances 0.000 claims 1
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- 239000000047 product Substances 0.000 abstract description 7
- 238000012216 screening Methods 0.000 abstract 1
- 108700022290 poly(gamma-glutamic acid) Proteins 0.000 description 13
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- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 description 7
- 230000004913 activation Effects 0.000 description 7
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- 241000726221 Gemma Species 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 4
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 description 4
- WFJIVOKAWHGMBH-UHFFFAOYSA-N 4-hexylbenzene-1,3-diol Chemical group CCCCCCC1=CC=C(O)C=C1O WFJIVOKAWHGMBH-UHFFFAOYSA-N 0.000 description 3
- 238000000605 extraction Methods 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 229920001817 Agar Polymers 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 229940024606 amino acid Drugs 0.000 description 2
- -1 amino-acid compound Chemical class 0.000 description 2
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- 230000001788 irregular Effects 0.000 description 2
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 2
- 235000015097 nutrients Nutrition 0.000 description 2
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- 238000003786 synthesis reaction Methods 0.000 description 2
- 239000002699 waste material Substances 0.000 description 2
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- 241000193738 Bacillus anthracis Species 0.000 description 1
- 241000311115 Bacillus paralicheniformis ATCC 9945a Species 0.000 description 1
- KRKNYBCHXYNGOX-UHFFFAOYSA-K Citrate Chemical compound [O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O KRKNYBCHXYNGOX-UHFFFAOYSA-K 0.000 description 1
- 108010010803 Gelatin Proteins 0.000 description 1
- 125000003338 L-glutaminyl group Chemical group O=C([*])[C@](N([H])[H])([H])C([H])([H])C([H])([H])C(=O)N([H])[H] 0.000 description 1
- 241001052560 Thallis Species 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 229940065181 bacillus anthracis Drugs 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
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- CEQFOVLGLXCDCX-WUKNDPDISA-N methyl red Chemical compound C1=CC(N(C)C)=CC=C1\N=N\C1=CC=CC=C1C(O)=O CEQFOVLGLXCDCX-WUKNDPDISA-N 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
本发明提供了一种采用发酵制备γ－聚谷氨酸的方法，该方法将经过筛选得到的枯草芽孢杆菌(Bacillus subtills)zju－7菌株，即CGMCC No.1250，在含有碳源、氮源、谷氨酸、MgSO4、NaCl和K2HPO4的培养基中培养，得到发酵液，再将发酵液通过有机溶剂沉淀、透析，得到γ－聚谷氨酸产品。本发明方法工艺简单，高效价廉，产量高。The invention provides a method for preparing γ-polyglutamic acid by fermentation. In the method, Bacillus subtilis (Bacillus subtills) zju-7 strain obtained through screening, that is, CGMCC No.1250, is mixed with carbon source and nitrogen source , glutamic acid, MgSO 4 , NaCl and K 2 HPO 4 culture medium to obtain a fermented liquid, and then precipitate and dialyze the fermented liquid with an organic solvent to obtain a γ-polyglutamic acid product. The method of the invention has the advantages of simple process, high efficiency, low price and high output.
Description
Technical field
The present invention relates to a kind of high yield gamma-polyglutamic acid-(microorganism strains of γ-PGA), and use it for the 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.Immunitatsforch.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 Bacilluslicheniformis ATCC9945a 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 subtilisMR 141 was carried out optimizing culture condition in the fermentor tank of 30L can make γ-PGA maximum production reach 35g/L (Ogawa Y, YamaguchiF, Yuasa K.Biosci Biotec Biochem, 1997,61:1684～1687).Kubota separates a strain Bacillus subtilis F201 who obtains in Osaka City University soil, this 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.Biosci Biotechnol Biochem, 1993,57,1809～1810; Tanaka T, Hiruta O, Futamura T, et al.Biosci Biotechnol Biochem, 1993,57,2148～2153).
Though the fermentative production of γ-PGA has obtained bigger progress, still exist the consumption raw material more, the production cycle is longer, and production efficiency is low, the production cost problem of higher, therefore, efficient, low cost preparing gamma-PGA still awaits further research.
Summary of the invention
The object of the present invention is to provide a kind of high yield gamma-polyglutamic acid-(microorganism strains of γ-PGA), and the method for preparing gamma-polyglutamic acid-with this microorganism strains.
Purpose of the present invention reaches by following measure:
The microorganism of using
The microorganism strains that is used to produce gamma-polyglutamic acid-of the present invention is subtilis (Bacillussubtills) zju-7, this bacterial strain system 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.
Subtilis (Bacillus subtills) zju-7 has following character:
1. morphological specificity
Vegetative cell is the bacillus of 0.6～0.9 * 1.5～3.0 μ m sizes on the peptone nutrient agar, cultivates for 40 ℃ and forms gemma in 7 days, and the gemma size is 0.6～0.8 * 1.0～2.0 μ m, is Long Circle or cylindrical.
2. the feature on various substratum:
(1) the peptone agar glucose is dull and stereotyped cultivates: but 24 hours raised growths of 30～40 ℃ of cultivations.Bacterium colony is white in color, and there is mycoderm on the bacterium colony surface, and gauffer is arranged, and is opaque, do not glisten, and the edge is irregular.
(2) peptone agar glucose slant culture: but 24 hours raised growths of 30～40 ℃ of cultivations.Bacterium colony is white in color, and there is mycoderm on the bacterium colony surface, and gauffer is arranged, and is opaque, do not glisten, and the edge is irregular.
(3) the peptone liquid of glucose is cultivated: form mycoderm on the nutrient solution surface.
(4) peptone glucose stab culture: thalline is in surface growth, does not grow in the bottom.
3. the Physiology and biochemistry character of subtilis (Bacillus subtills) zju-7 sees Table 1
4. by bacterium 16s RNA comparison, prove that this microorganism is subtilis (Bacillussubtills)
In sum, what the present invention used has been the gas genus bacillus, is specially subtilis (Bacillussubtills) zju-7.
Table 1 subtilis (Bacillus subtills) zju-7 Physiology and biochemistry character
| Test subject is the test subject result as a result |
| Gram-positive is produced acid from carbohydrate |
| The shaft-like glucose of thalline shape+ |
| Cell dia＞1 micron-sucrose+formation gemma+fructose+ |
| Gemma expands-lactose+ |
| The growth of gemma circle-utilize Citrate trianion+ |
| Parasporal crystal-50 ℃ growth+ |
| Anaerobic growth-pH＜growth+ |
| Methyl red test+7%NaCl growth+ |
| PH＜6.0+hydrolyzed starch+ |
| PH＞7.0-liquefy gelatin+ |
Utilize subtilis to prepare the method for gamma-polyglutamic acid-, may further comprise the steps:
1) with subtilis (Bacillus subtills) zju-7 CGMCC No.1250, activates at slant medium;
Substratum is: 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%, each components contents is percent weight in volume, promptly g/100ml is together following.Activate after 8 hours visible colony growth.
2) preparation substratum: carbon source 1～10%, nitrogenous source 1～8%, L-glutamic acid 2～15%, MgSO
40.05～0.5%, NaCl 1～8%, K
2HPO
40.01～0.05%, each components contents is percent weight in volume, pH6～7, and liquid amount 20～200ml/500ml shakes bottle, after the sterilization, cooling inserts subtilis (Bacillus subtills) zju-7 after activating, and inoculum size is 1～10%, 20～40 ℃ of shake-flask culture, rotating speed 100～400rpm carries out shake flask fermentation under these conditions, cultivates 24～72 hours; Or adorn liquid in fermentor tank, and sterilization inserts subtilis (Bacillus subtills) zju-7 after activating, and inoculum size is 1～10%, and air flow is 1.0～3.0vvm, cultivates 24～72 hours under 35～40 ℃ of temperature;
3) thalline in the centrifugal removal fermented liquid, with acid the pH value of supernatant liquor is transferred to 3～5, add methyl alcohol, ethanol or acetone organic solvent then, add-on is 2～5 times of supernatant liquor volume, and precipitation is dissolved in the water throw out, remove by filter insolubles, small-molecule substance is removed in dialysis, and drying obtains gamma-polyglutamic acid-.
The carbon source of using among the present invention can be glucose, sucrose, fructose, maltose, lactose, Zulkovsky starch or starch, and is comparatively suitable with dextrose plus saccharose; Nitrogenous source can be organic nitrogen source peptone, yeast extract paste or corn steep liquor; Also can be inorganic nitrogen-sourced NH
4NO
3Or (NH
4)
2SO
4, above-mentioned nitrogenous source can use separately, also can mix use.
In the above-mentioned substratum, preferred L-glutamic acid content is 7～12%, because 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 L-glutamic acid that uses among the present invention also can be the form of its salt.Preferred NaCl content is 3～5%, because the NaCl content in the fermented liquid is low excessively, produces a large amount of foams during the fermentation; The NaCl too high levels can suppress the growth of thalline.
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) subtilis (Bacillus subtills) the zju-7 microorganism strains of the present invention's use can high efficient fermentation be produced gamma-polyglutamic acid-:
(2). subtilis (Bacillus subtills) zju-7 strain culturing condition is extensive, can use multiple different carbon source and nitrogenous source;
(3) the substratum color and luster is more shallow, helps the product separation and purification in later stage;
(4) by optimization to subtilis (Bacillus subtills) zju-7 strain culturing condition, particularly by in substratum, adding L-glutamic acid and salt thereof, the content that makes the gamma-polyglutamic acid-in the fermented liquid is up to 50～60g/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
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%, activate 8 hours.
Shake-flask culture base: glucose 2%, peptone 1.5%, L-glutamic acid 3%, MgSO
40.1%, NaCl 1%, K
2HPO
40.01%, 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.
After the fermentation ends, the thalline in the centrifugal removal fermented liquid is removed the thalline in the fermented liquid, with hydrochloric acid the pH value of supernatant liquor is transferred to 4, adds 4 times of volumes methanol then, precipitation.Throw out is dissolved in 100 times of volume water again, removes by filter insolubles, small-molecule substance is removed in dialysis then, at last the solution lyophilize is obtained the white powder material, and this white mass is a gamma-polyglutamic acid-, and output can reach 15g/L.
Embodiment 2
With subtilis (Bacillus subtills) zju-7 CGMCC No.1250, in the slant medium activation, with embodiment 1;
Shake-flask culture base: 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%, and bacterial classification is subtilis (Bacillus subtills) zju-7, cultivates 24 hours for 37 ℃, and shaking bottle rotating speed is 200rpm.
After the fermentation ends, with embodiment 1 separation and purification gamma-polyglutamic acid-, output can reach 18g/L.
Embodiment 3
With subtilis (Bacillus subtills) zju-7 CGMCC No.1250, in the slant medium activation, with embodiment 1;
With glucose 2%, peptone 2%, L-glutamic acid 8%, MgSO
40.1%, NaCl 2%, K
2HPO
40.05% is the shake-flask culture base, pH7, and liquid amount 30ml/200ml shakes bottle, sterilizes 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 23g/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 28g/L.
Embodiment 5
With subtilis (Bacillus subtills) zju-7 CGMCC No.1250, in the slant medium activation, with embodiment 1;
With glucose 10%, peptone 8%, L-glutamic acid 15%, MgSO
40.2%, NaCl 5%, K
2HPO
40.05% is substratum, pH7, and liquid amount 50/500ml shakes bottle, sterilizes 20 minutes for 115 ℃.The sterilization postcooling inserts subtilis (Bacillus subtills) zju-7, 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 narrating according to EXAMPLE l obtains-polyglutamic acid, and output can reach 54g/L.
Embodiment 6
With subtilis (Bacillus subtills) zju-7 CGMCC No.1250, in the slant medium activation, with embodiment 1;
With glucose 1%, peptone 1%, L-glutamic acid 2%, MgSO
40.05%, NaCl 1%, K
2HPO
40.01% is substratum, pH7, liquid amount 50ml/500ml shakes bottle, sterilizes 20 minutes for 121 ℃, after sterilization finishes, cooling inserts subtilis (Bacillus subtills) zju-7, 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 15g/L.
Embodiment 7
With subtilis (Bacillus subtills) zju-7 CGMCC No.1250, in the slant medium activation, with embodiment 1;
With sucrose 5%, peptone 5%, L-glutamic acid 5%, MgSO
40.1%, NaCl 2%, K
2HPO
40.05% is the shake-flask culture base, pH7, and liquid amount 50ml/200ml shakes bottle, sterilizes 20 minutes for 115 ℃, after sterilization finished, cooling inserted subtilis (Bacillus subtills) zju-7, and inoculum size is 3%, 37 ℃ of shake-flask culture, 200rpm cultivated 48 hours.
After the fermentation ends, the method separation and purification gamma-polyglutamic acid-according to embodiment 1 the results are shown in Table 2.
Experimental result shows: do not add in the control bottle of L-glutamic acid and do not generate gamma-polyglutamic acid-, and increase along with L-glutamic acid and sodium salt add-on thereof, the content of gamma-polyglutamic acid-increases thereupon in the fermented liquid, the content of L-glutamic acid surpasses 10% in fermention medium, the final gamma-polyglutamic acid-that generates 50g/L in the nutrient solution.
Table 2 L-glutamic acid content is to the influence of gamma-polyglutamic acid-output
| Aminoglutaric acid concentration (%) | Gamma-polyglutamic acid-growing amount (g/L) |
| Do not add | ????0 |
| ????2 | ????14 |
| ????4 | ????21 |
| ????6 | ????25 |
| ????8 | ????28 |
| ????10 | ????50 |
Embodiment 8
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%, 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%, 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, pH7, and liquid amount 100ml/1000ml shakes bottle, sterilizes 20 minutes for 115 ℃, after sterilization finished, cooling inserted subtilis (Bacillus subtills) zju-7, and inoculum size is 3%, 37 ℃ of shake-flask culture, 200rpm cultivated 12 hours.
With sucrose 5%, peptone 5%, L-glutamic acid 10%, MgSO
40.1%, NaCl 3%, K
2HPO
40.05% is substratum, pH7, liquid amount 3L/5L fermentor tank, sterilized 20 minutes for 115 ℃, 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 51.3g/L, and productivity reaches 1.425gh
-1L
-1
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