CN101486977A - Bacillus subtilis and method for preparing gamma-polyglutamic acid by using same - Google Patents
Bacillus subtilis and method for preparing gamma-polyglutamic acid by using same Download PDFInfo
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- 244000063299 Bacillus subtilis Species 0.000 title claims abstract description 42
- 235000014469 Bacillus subtilis Nutrition 0.000 title claims abstract description 18
- 238000000034 method Methods 0.000 title claims description 23
- 229920002643 polyglutamic acid Polymers 0.000 title abstract description 6
- 239000007788 liquid Substances 0.000 claims abstract description 42
- 238000000855 fermentation Methods 0.000 claims abstract description 18
- 230000004151 fermentation Effects 0.000 claims abstract description 18
- 238000002360 preparation method Methods 0.000 claims abstract description 4
- 238000004321 preservation Methods 0.000 claims abstract description 4
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 claims description 32
- 239000001888 Peptone Substances 0.000 claims description 22
- 108010080698 Peptones Proteins 0.000 claims description 22
- 235000019319 peptone Nutrition 0.000 claims description 22
- 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 16
- 230000001580 bacterial effect Effects 0.000 claims description 16
- 239000008103 glucose Substances 0.000 claims description 16
- 239000011780 sodium chloride Substances 0.000 claims description 16
- 229960002989 glutamic acid Drugs 0.000 claims description 15
- QJGQUHMNIGDVPM-UHFFFAOYSA-N nitrogen group Chemical group [N] QJGQUHMNIGDVPM-UHFFFAOYSA-N 0.000 claims description 15
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 13
- 229940041514 candida albicans extract Drugs 0.000 claims description 13
- 229910052799 carbon Inorganic materials 0.000 claims description 13
- 239000012138 yeast extract Substances 0.000 claims description 13
- PXEDJBXQKAGXNJ-QTNFYWBSSA-L disodium L-glutamate Chemical compound [Na+].[Na+].[O-]C(=O)[C@@H](N)CCC([O-])=O PXEDJBXQKAGXNJ-QTNFYWBSSA-L 0.000 claims description 10
- 239000002054 inoculum Substances 0.000 claims description 10
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- 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 6
- 229930006000 Sucrose Natural products 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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- 241000894006 Bacteria Species 0.000 claims description 5
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- 239000000284 extract Substances 0.000 claims description 5
- 229920001817 Agar Polymers 0.000 claims description 4
- 239000008272 agar Substances 0.000 claims description 4
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- 239000006228 supernatant Substances 0.000 claims description 4
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 3
- 235000015278 beef Nutrition 0.000 claims description 3
- 230000014759 maintenance of location Effects 0.000 claims description 3
- 238000003860 storage Methods 0.000 claims description 3
- 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
- 229930091371 Fructose Natural products 0.000 claims description 2
- 239000005715 Fructose Substances 0.000 claims description 2
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-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
- 229910002651 NO3 Inorganic materials 0.000 claims description 2
- NHNBFGGVMKEFGY-UHFFFAOYSA-N Nitrate Chemical compound [O-][N+]([O-])=O NHNBFGGVMKEFGY-UHFFFAOYSA-N 0.000 claims description 2
- 240000008042 Zea mays Species 0.000 claims description 2
- 235000005824 Zea mays ssp. parviglumis Nutrition 0.000 claims description 2
- 235000002017 Zea mays subsp mays Nutrition 0.000 claims description 2
- 235000005822 corn Nutrition 0.000 claims description 2
- 229960000935 dehydrated alcohol Drugs 0.000 claims description 2
- 238000000502 dialysis Methods 0.000 claims description 2
- 239000008101 lactose Substances 0.000 claims description 2
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- 150000003384 small molecules Chemical class 0.000 claims description 2
- 230000001954 sterilising effect Effects 0.000 claims description 2
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- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 claims 1
- 238000000605 extraction Methods 0.000 abstract description 2
- 239000002609 medium Substances 0.000 description 23
- WHUUTDBJXJRKMK-VKHMYHEASA-N L-glutamic acid Chemical compound OC(=O)[C@@H](N)CCC(O)=O WHUUTDBJXJRKMK-VKHMYHEASA-N 0.000 description 14
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 10
- 239000001963 growth medium Substances 0.000 description 9
- 238000004816 paper chromatography Methods 0.000 description 9
- 238000011218 seed culture Methods 0.000 description 9
- 238000004519 manufacturing process Methods 0.000 description 8
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- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 6
- 241000193830 Bacillus <bacterium> Species 0.000 description 5
- 244000005700 microbiome Species 0.000 description 5
- 229910052757 nitrogen Inorganic materials 0.000 description 5
- 241000194108 Bacillus licheniformis Species 0.000 description 3
- 238000001514 detection method Methods 0.000 description 3
- 238000003786 synthesis reaction Methods 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Natural products CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- 241000194103 Bacillus pumilus Species 0.000 description 2
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 2
- 230000008901 benefit Effects 0.000 description 2
- 239000003795 chemical substances by application Substances 0.000 description 2
- 229960004756 ethanol Drugs 0.000 description 2
- 235000013305 food Nutrition 0.000 description 2
- 239000000463 material Substances 0.000 description 2
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 2
- 108090000623 proteins and genes Proteins 0.000 description 2
- 238000001308 synthesis method Methods 0.000 description 2
- 108020004465 16S ribosomal RNA Proteins 0.000 description 1
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 1
- 241000193738 Bacillus anthracis Species 0.000 description 1
- 241001037822 Bacillus bacterium Species 0.000 description 1
- 241000196324 Embryophyta Species 0.000 description 1
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 1
- 244000046052 Phaseolus vulgaris Species 0.000 description 1
- 235000010627 Phaseolus vulgaris Nutrition 0.000 description 1
- 108010020346 Polyglutamic Acid Proteins 0.000 description 1
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- 238000001994 activation Methods 0.000 description 1
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- QVGXLLKOCUKJST-UHFFFAOYSA-N atomic oxygen Chemical compound [O] QVGXLLKOCUKJST-UHFFFAOYSA-N 0.000 description 1
- 229940065181 bacillus anthracis Drugs 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
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- 238000009833 condensation Methods 0.000 description 1
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- 229910000365 copper sulfate Inorganic materials 0.000 description 1
- ARUVKPQLZAKDPS-UHFFFAOYSA-L copper(II) sulfate Chemical compound [Cu+2].[O-][S+2]([O-])([O-])[O-] ARUVKPQLZAKDPS-UHFFFAOYSA-L 0.000 description 1
- 230000008878 coupling Effects 0.000 description 1
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- HHCCNQLNWSZWDH-UHFFFAOYSA-N n-hydroxymethanimine oxide Chemical compound O[N+]([O-])=C HHCCNQLNWSZWDH-UHFFFAOYSA-N 0.000 description 1
- FEMOMIGRRWSMCU-UHFFFAOYSA-N ninhydrin Chemical compound C1=CC=C2C(=O)C(O)(O)C(=O)C2=C1 FEMOMIGRRWSMCU-UHFFFAOYSA-N 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The Bacillus subtilis strain is Bacillus subtilis XN01 and is preserved in China Center for Type Culture Collection (CCTCC) at 3.18.2008, and the serial number is as follows: CCTCC NO: m208044; the strain is used for preparing the gamma-polyglutamic acid through the steps of strain culture preservation, seed liquid preparation, shake flask liquid fermentation and gamma-polyglutamic acid extraction.
Description
Technical field
The present invention relates to a kind of bacillus and prepare the method for gamma-polyglutamic acid-, be specifically related to a kind of Bacillus subtillis and prepare the method for gamma-polyglutamic acid-with this bacterium with this bacterium.
Background technology
Gamma-polyglutamic acid-(γ-Poly glutamic acid, γ-PGA) is with γ-carboxyl and alpha-amino group a kind of polyamino acid of condensation mutually by D-type or L-type L-glutamic acid monomer, it is the macromolecular material with falling property of biology, following (the Shih I of its structure, Van Y.Bioresour Technol, 2001,79:207~225):
The gamma-polyglutamic acid-relative molecular weight is generally 100,000 to 1,000,000, there are a large amount of peptide bonds and free carboxy on its main chain, therefore it has good biodegradability, water-soluble and edibility, can be used as thickening material, wetting Agent for Printing Inks, frostproofer, pharmaceutical carrier, biodegradable fiber, high-performance water-retaining agent, flocculation agent, fodder additives etc., (Shih I is with a wide range of applications in fields such as food, makeup, medicine and water treatments, VanY.Bioresour Technol, 2001,79:207~225).
The preparation method of gamma-polyglutamic acid-mainly contains chemical synthesis and microorganism synthesis method.Chemical synthesis adopts traditional polypeptide synthesis, L-glutamic acid is connected one by one or adopts fragment combination to connect into peptide, and this process generally comprises steps such as radical protection, activation, coupling and deprotection, and this method synthetic route is grown, by product is many, yield is low.Gamma-polyglutamic acid-(Iv á novics G, Bruckner V.Z at first in the pod membrane of Bacillus anthracis, have been found as far back as nineteen thirty-seven by Iv á novics etc.
, 1937,90:304~318), nineteen forty-two Bovarnick etc. discovers that some Bacillus bacterium can accumulate gamma-polyglutamic acid-(Bovarnick M.J Biol Chem, 1942,145:415~424) in fermention medium.
Have that production process is easy to control, fermentation yield stable, extraction yield is high and be convenient to advantage such as scale operation because microbial fermentation is produced polyglutamic acid, so the microorganism synthesis method main method that progressively becomes research and produce gamma-polyglutamic acid-.So far the gamma-polyglutamic acid-generation bacterium of finding mainly concentrates between the different strains of Bacillus, comprises Bacillus subtillis (Bacillus subtilis), bacillus licheniformis (Bacillus licheniformis) and bacillus pumilus (Bacillus pumilus).Since the nineties in 20th century, the research of the synthetic gamma-polyglutamic acid-of related microorganism is very active always.
Bacillus licheniformis (Bacillus lichemiformis) ATCC 9945 is one of more bacterial classifications of research, this bacterium passes through 4 days cultivation in the optimum medium of L-glutamic acid 20g/L, glycerine 80g/L, citric acid 12g/L, can obtain gamma-polyglutamic acid-(the Cromwick A M of 17~23g/L, Gross R A.Biotechnol Bioeng, 1996,50:222~227).Japan has carried out big quantity research to Bacillus subtillis (Bacillussubtilis) IFO 3335 synthetic gamma-polyglutamic acid-s, this bacterial strain can synthesize gamma-polyglutamic acid-(the Kunioka M of 10-20g/L by 2 days cultivation in the substratum of paddy nitronic acid 30g/L, citric acid 20g/L, Goto A.Appl Microbiol Biotechnol, 1994,40:867~872).
Reports such as Kubota et al in 1993, F-2-01 is a bacterial strain with Bacillus subtillis (Bacillus subtilis), in the liquid culture that contains 70g/L L-glutamic acid, obtained the gamma-polyglutamic acid-of 48g/L by 4 days fermentation, this is production peak (the Kubota H of external report, et al.Biosci Biotech Biochem, 1993,57 (7): 1212~1213).Also have part unit to carry out the research work of gamma-polyglutamic acid-microorganism aspect synthetic in succession in China, the throughput of wherein related bacterial classification is not quite similar.CN200410010509.0 has introduced Zhejiang University's isolation and selection from the soy sauce production waste material and has obtained the Bacillus subtillis Bacillus subtilis zju-7 that a plant height produces gamma-polyglutamic acid-, and it cultivates the gamma-polyglutamic acid-that can synthesize 54g/L in 24 hours in the substratum that contains 150g/L L-glutamic acid, 80g/L peptone.
At present, though the throughput of bacterial classification improves a lot, its cost height, cycle length and production efficiency are low all to be obstacles of commercial scale production gamma-polyglutamic acid-.Therefore seek production bacterial strain and processing condition efficient, low-cost synthetic gamma-polyglutamic acid-and be still the direction of research from now on.
Summary of the invention
The object of the present invention is to provide a kind of Bacillus subtillis and prepare the method for gamma-polyglutamic acid-with this bacterial strain, it can be at efficient synthetic gamma-polyglutamic acid-in the liquid nutrient medium cheaply, and method is simple, and is workable.
Technical solution of the present invention is:
A kind of bacterial strain that produces gamma-polyglutamic acid-, it is characterized in that described bacterial strain is Bacillus subtillis (Bacillus substilis) XN01, described Bacillus subtillis is separating obtained from bean food, described Bacillus subtillis is preserved in Chinese typical culture collection center (CCTCC) on March 18th, 2008, is numbered: CCTCC NO:M 208044.
Its 16s rRNA gene is checked order this bacterial strain for accurately identifying.Wherein amplimer is respectively 27F (5 '-AGAGTTTGATCCTGGCTCAG-3 ') and 1527R (5 '-AGAAAGGAGGTGATCCAGCC-3 ').Gene order comparison with the sequence that records and GenBank are provided confirms that this microorganism is Bacillus subtillis (Bacillus substilis).
The physio-biochemical characteristics of above-mentioned Bacillus subtillis (Bacillus subtilis) XN01 are as shown in the table.
"+" in the wherein said tabulation is positive, and "-" is negative.
A kind ofly prepare the method for gamma-polyglutamic acid-with Bacillus subtillis (Bacillus subtilis) XN01, its special character is that this method may further comprise the steps:
1) spawn culture preservation
With the bacterial classification of Bacillus subtillis (Bacillus subtilis) XN01 at solid slant culture based on 25~40 ℃ of constant temperature culture, then in 2~4 ℃ of short term storages; The composition of described solid slant culture base, in percent weight in volume, unit is g/100ml, wherein peptone 0.8~1.0%, yeast extract paste 0.5~1.0%, NaCl 1.0~2.0% and agar 1.5~2.0%, its pH value is 6.0~8.0;
2) preparation seed liquor
With about 1cm on the above-mentioned inclined-plane
2Bacterial strain insert in the triangular flask of the 250ml that liquid nutrient medium is housed, liquid amount 50ml/250ml shakes bottle, in 25~40 ℃ of following constant temperature culture 10~24 hours, rotating speed was 180~220rpm; The composition of described liquid nutrient medium, in percent weight in volume, unit is g/100ml, wherein glucose 0.1~1.0%, peptone 0.8~1.0%, yeast extract paste 0.5~1.0%, NaCl 1.0~2.0%, and the pH value is 6.5~8.0;
3) shake a bottle liquid fermenting
Seed liquor is changed in the liquid fermentation and culture after the sterilization, and inoculum size is 1~10%, and liquid amount 50ml/250ml shakes bottle, in 30~40 ℃ of constant temperature culture 12~48 hours, and rotating speed 180~220rpm; The composition of described fermention medium, in percent weight in volume, unit is g/100ml, wherein the saccharic carbon source 1~10%, nitrogenous source 0.4~3%, L-L-glutamic acid or Sodium Glutamate 5~12%, NaCl 0~5%, MgSO
47H
2O 0.01~0.1%, MnSO
4H
2O 0.005~0.05%, K
2HPO
40.05~0.5%, CaCl
20.02~0.2%, the pH value is 6.5~8.0;
4) extract gamma-polyglutamic acid-
The centrifugal thalline of removing in the fermented liquid adds the dehydrated alcohol of 3~4 times of volumes in supernatant liquor, refrigerate centrifugal and collecting precipitation after 24 hours, and after small molecules was removed in dialysis, lyophilize obtained gamma-polyglutamic acid-.
Above-mentioned steps 3) the saccharic carbon source is that glucose, sucrose or maltose are advisable in.
Above-mentioned steps 3) content of nitrogenous source is 0.8~1.5% to be advisable.
Above-mentioned steps 3) content 8~12% of L-L-glutamic acid or Sodium Glutamate is advisable.
Above-mentioned steps 3) fermented liquid pH value is 7.0~7.5 to be advisable.
Above-mentioned steps 3) the saccharic carbon source in is glucose, sucrose, maltose, lactose, fructose or is its mixture.
Above-mentioned steps 3) nitrogenous source in is yeast extract paste, peptone, beef extract, corn steep liquor, (NH
4)
2SO
4, nitrate or be its mixture.
Wherein carbon source is advisable with glucose, sucrose and maltose, considers that from the cost angle glucose is better; The mixed nitrogen that nitrogenous source is formed with peptone or the white peptone of peptone and other nitrogenous source is advisable, if when being nitrogenous source with the peptone, preferred content is 0.8~1.5%; Consider that from cost and productive rate angle Sodium Glutamate is better than L-L-glutamic acid, preferred content is 8~12%; Fermented liquid pH value is advisable with 7.0~7.5, exceeds productive rate and output that this scope can influence gamma-polyglutamic acid-.
The present invention has the following advantages:
1) Bacillus subtillis (Bacillus subtilis) the XN01 microorganism strains of the present invention's use can efficiently synthesize gamma-polyglutamic acid-in the liquid state fermentation substratum, the about 2.0g/Lh of its throughput rate, output and throughput rate that it is higher satisfy industrial production requirement.
2) bacterial strain that uses of the present invention to carbon source and nitrogenous source require extensively, carbon source can be single carbon source or mixed carbon source, nitrogenous source can be single nitrogenous source or mixed nitrogen.
3) an amount of NaCl that adds can improve gamma-polyglutamic acid-output in fermented liquid, reduces fermentation broth viscosity simultaneously, can reduce when improving the fermented liquid dissolved oxygen level and stir in the production process and the power consumption when centrifugal.
4) in the synthetic gamma-polyglutamic acid-process of liquid fermenting, used carbon source, nitrogenous source and L-glutamic acid (or Sodium Glutamate) concentration is low, and particularly nitrogen concentration is lower, thereby has saved production cost greatly.
Embodiment
The following examples will the present invention is further illustrated, but to the present invention without limits.
% unit all represents percent weight in volume in following examples, and unit is g/100ml.
Embodiment 1
With the bacterial classification of Bacillus subtillis (Bacillus subtilis) XN01 at solid slant culture based on 37 ℃ of constant temperature culture 24 hours, then in 2~4 ℃ of preservations.Wherein said slant medium consists of: peptone 1%, and yeast extract paste 0.5%, NaCl 1%, agar 2.0%, the pH value is 6.0.
Get about 1cm that the inclined-plane is preserved
2Bacillus subtillis (Bacillus subtilis) XN01 insert seed culture medium, through 37 ℃ of shake-flask culture 20 hours, rotating speed 210rpm.Wherein said seed culture medium consists of: glucose 1%, and peptone 1%, yeast extract paste 0.5%, NaCl 1%, and pH 7.1~7.2, and liquid amount 50ml/250ml shakes bottle.
Inoculum size by 5% inserts seed culture medium in the fermention medium, through 37 ℃ of shake-flask culture 24 hours, and rotating speed 210rpm.Wherein said fermention medium consists of: sucrose 3%, L-L-glutamic acid 5%, yeast extract paste 1.0%, MgSO
47H
2O 0.05%, (NH
4)
2SO
41.0%, MnSO
4H
2O 0.01%, K
2HPO
40.1%, pH7.5, liquid amount 50ml/250ml shakes bottle.
After the fermentation ends, the centrifugal thalline of removing, the cold ethanol of 3 times of volumes of adding in supernatant liquor, centrifugal and the supernatant liquor that inclines after refrigeration is spent the night, to precipitate and be dissolved in again in isopyknic deionized water, hydrolysis is after Paper Chromatography detects, and gamma-polyglutamic acid-content is 24.5g/L.
Wherein said Paper Chromatography solvent for use system is a propyl carbinol: 80% formic acid (V/V): water=15:3:2, developer are 0.5% triketohydrindene hydrate acetone soln, through 0.1% copper sulfate (CuSO
45H
2O): measure absorbancy in 520nm behind the eluant solution of 75% ethanol (V/V)=2:38, calculate the content of gamma-polyglutamic acid-by the L-glutamic acid typical curve.
Embodiment 2
With example 1, carbon source is wherein changed into the maltose of equal in quality concentration, analytical results shows that the content of gamma-polyglutamic acid-in the fermented liquid is 30.1g/L.
Embodiment 3
XN01 activates 15 hours in the seed culture medium of example 1 with Bacillus subtillis (Bacillus subtilis), be pressed in the shake flask fermentation substratum by 5% inoculum size then, and through 37 ℃ of shake-flask culture 24 hours, rotating speed 210rpm.Wherein said fermention medium consists of: glucose 3%, and L-L-glutamic acid 5%, peptone 0.8%, NaCl 1%, MgSO
47H
2O 0.05%, MnSO
4H
2O 0.01%, K
2HPO
40.1%, the pH value is 7.4~7.5, and liquid amount 50ml/250ml shakes bottle.
After the fermentation ends, according to the content of gamma-polyglutamic acid-in the Paper Chromatography detection fermented liquid of embodiment 1, the result shows that its content is 48.0g/L.
Embodiment 4
XN01 activates 15 hours in the seed culture medium of example 1 with Bacillus subtillis (Bacillus subtilis), be pressed in the shake flask fermentation substratum by 5% inoculum size then, and through 37 ℃ of shake-flask culture 24 hours, rotating speed 210rpm.Wherein said fermention medium consists of: glucose 3%, L-L-glutamic acid 5%, nitrogenous source 0.8%, MgSO
47H
2O 0.05%, MnSO
4H
2O 0.01%, K
2HPO
40.1%, the pH value is 7.4~7.5, and liquid amount 50ml/250ml shakes bottle.
Wherein said nitrogenous source is yeast extract paste, peptone, (NH
4)
2SO
4, beef extract, yeast extract paste+(NH
4)
2SO
4(etc. quality mixed nitrogen) or peptone+(NH
4)
2SO
4(etc. quality mixed nitrogen), behind shake flask fermentation, it is as shown in the table that the gained fermented liquid adopts example 1 described Paper Chromatography to detect the content of gamma-polyglutamic acid-by above-mentioned fermention medium.
Embodiment 5
With the bacterial classification of Bacillus subtillis (Bacillus subtilis) XN01 at solid slant culture based on 37 ℃ of constant temperature culture 24 hours, then in 2~4 ℃ of short term storages.Wherein the solid slant culture base is composed as follows: peptone 1%, and yeast extract paste 0.5%, NaCl 1%, agar 2.0%, pH 7.0.
Bacillus subtillis (Bacillus subtilis) XN01 of about 1cm2 inserted be equipped with in the 250ml triangular flask of liquid nutrient medium, in 37 ℃ of constant temperature culture 18 hours under rotating speed 180-220rpm.Wherein the seed liquid nutrient medium is composed as follows: glucose 1%, and peptone 1%, yeast extract paste 0.5%, NaCl 1%, and pH 7.1~7.2, and liquid amount 50ml/250ml shakes bottle.
Inoculum size by 5% inserts seed culture medium in the fermention medium, through 37 ℃ of shake-flask culture 24 hours, and rotating speed 210rpm.Wherein said fermention medium consists of: glucose 2%, and Sodium Glutamate 5%, peptone 0.8%, NaCl 1%, MgSO
47H
2O 0.05%, MnSO
4H
2O 0.01%, K
2HPO
40.1%, pH 7.5, and liquid amount 50ml/250ml shakes bottle.
After the fermentation ends, according to the content of gamma-polyglutamic acid-in the Paper Chromatography detection fermented liquid of embodiment 1, the result shows that its content is 21.3g/L.
Embodiment 6
Method by example 5 prepares seed liquor, and by 5% inoculum size seed culture medium is inserted in the fermention medium, through 37 ℃ of shake-flask culture 24 hours, and rotating speed 210rpm.Wherein said fermention medium consists of: glucose 4%, and Sodium Glutamate 10%, peptone 1.2%, NaCl 1.25%, MgSO
47H
2O 0.05%, MnSO
4H
2O0.01%, K
2HPO
40.15%, CaCl
20.04%, pH 7.5, and liquid amount 50ml/250ml shakes bottle.
After the fermentation ends, according to the content of gamma-polyglutamic acid-in the Paper Chromatography detection fermented liquid of embodiment 1, the result shows that its content is 56.3g/L.
Embodiment 7
Method by example 5 prepares seed liquor, and by 1% inoculum size seed culture medium is inserted in the fermention medium, through 37 ℃ of shake-flask culture 29 hours, and rotating speed 210rpm.Wherein said fermention medium consists of: glucose 4%, and Sodium Glutamate 8%, peptone 0.8%, NaCl 1.5%, MgSO
47H
2O 0.05%, MnSO
4H
2O0.01%, K
2HPO
40.1%, pH 7.5, and liquid amount 50ml/250ml shakes bottle.
After the fermentation ends, detect the content of gamma-polyglutamic acid-according to the Paper Chromatography of embodiment 1, the result shows that its content is 36.9g/L.
Embodiment 8
With example 7, change wherein inoculum size into 2.5%, the fermention medium initial pH value is 6.5, and fermentation culture detects the content of gamma-polyglutamic acid-according to the Paper Chromatography of embodiment 1 after 29 hours, and the result shows that its content is 39.8g/L.
Embodiment 9
Method by example 5 prepares seed liquor, and by 5% inoculum size seed culture medium is inserted in the fermention medium, 37 ℃ of culture temperature, rotating speed 210rpm.Wherein said fermention medium consists of: glucose 4%, and Sodium Glutamate 8%, peptone 0.8%, NaCl 1.5%, MgSO
47H
2O 0.025%, MnSO
4H
2O0.005%, K
2HPO
40.15%, CaCl
20.04%, pH 7.5, and liquid amount 50ml/250ml shakes bottle.
Ferment after 24 hours, detect the content of gamma-polyglutamic acid-according to the Paper Chromatography of embodiment 1, the result shows that its content is 37.2g/L.After fermentation time increased to 32 hours and 46 hours, gamma-polyglutamic acid-content was increased to 40.1g/L and 42.7g/L respectively in the fermented liquid.
Embodiment 10
With example 9, with MgSO wherein
47H
2O, MnSO
4H
2O and CaCl
2After content is adjusted into 0.05%, 0.015% and 0.02% respectively, ferment after 24 hours, gamma-polyglutamic acid-content is 40.3g/L in the fermented liquid.
Claims (8)
1. Bacillus subtillis, it is characterized in that: described bacterium is Bacillus subtillis (Bacillussubstilis) XN01, is preserved in Chinese typical culture collection center (CCTCC) on March 18th, 2008, is numbered CCTCC NO:M 208044.
2. one kind prepares the method for gamma-polyglutamic acid-with the described Bacillus subtillis of claim 1 (Bacillus subtilis) XN01, it is characterized in that this method may further comprise the steps:
1) spawn culture preservation
With the bacterial classification of Bacillus subtillis (Bacillus subtilis) XN01 at solid slant culture based on 25~40 ℃ of constant temperature culture, then in 2~4 ℃ of short term storages; The composition of described solid slant culture base, in percent weight in volume, unit is g/100ml, wherein peptone 0.8~1.0%, yeast extract paste 0.5~1.0%, NaCl 1.0~2.0% and agar 1.5~2.0%, its pH value is 6.0~8.0;
2) preparation seed liquor
With about 1cm on the above-mentioned inclined-plane
2Bacterial strain insert in the triangular flask of the 250ml that liquid nutrient medium is housed, liquid amount 50ml/250ml shakes bottle, in 25~40 ℃ of following constant temperature culture 10~24 hours, rotating speed was 180~220rpm; The composition of described liquid nutrient medium, in percent weight in volume, unit is g/100ml, wherein glucose 0.1~1.0%, peptone 0.8~1.0%, yeast extract paste 0.5~1.0%, NaCl 1.0~2.0%, and the pH value is 6.5~8.0;
3) shake a bottle liquid fermenting
Seed liquor is changed in the liquid fermentation and culture after the sterilization, and inoculum size is 1~10%, and liquid amount 50ml/250ml shakes bottle, in 30~40 ℃ of constant temperature culture 12~48 hours, and rotating speed 180~220rpm; The composition of described fermention medium, in percent weight in volume, unit is g/100ml, wherein the saccharic carbon source 1~10%, nitrogenous source 0.4~3%, L-L-glutamic acid or Sodium Glutamate 5~12%, NaCl 0~5%, MgSO
47H
2O 0.01~0.1%, MnSO
4H
2O 0.005~0.05%, K
2HPO
40.05~0.5%, CaCl
20.02~0.2%, the pH value is 6.5~8.0;
4) extract gamma-polyglutamic acid-
The centrifugal thalline of removing in the fermented liquid adds the dehydrated alcohol of 3~4 times of volumes in supernatant liquor, refrigerate centrifugal and collecting precipitation after 24 hours, and after small molecules was removed in dialysis, lyophilize obtained gamma-polyglutamic acid-.
3. the method for preparing gamma-polyglutamic acid-according to claim 2 is characterized in that: the saccharic carbon source is glucose, sucrose or maltose in the described step 3).
4. the method for preparing gamma-polyglutamic acid-according to claim 2 is characterized in that: the content of described step 3) nitrogenous source is 0.8~1.5%.
5. the method for preparing gamma-polyglutamic acid-according to claim 2 is characterized in that: the content of described step 3) L-L-glutamic acid or Sodium Glutamate is 8~12%.
6. the method for preparing gamma-polyglutamic acid-according to claim 2 is characterized in that: described step 3) fermented liquid pH value is 7.0~7.5.
7. according to the arbitrary described method for preparing gamma-polyglutamic acid-of claim 2~6, it is characterized in that: the saccharic carbon source in the described step 3) is glucose, sucrose, maltose, lactose, fructose or is its mixture.
8. the method for preparing gamma-polyglutamic acid-according to claim 7 is characterized in that: the nitrogenous source in the described step 3) is yeast extract paste, peptone, beef extract, corn steep liquor, (NH
4)
2SO
4, nitrate or be its mixture.
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