CN101486977B - Bacillus subtilis and method for preparing gamma-polyglutamic acid by using the same - Google Patents
Bacillus subtilis and method for preparing gamma-polyglutamic acid by using the same Download PDFInfo
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Abstract
The invention discloses a bacillus subtilis and a Gamma-polyglutamic acid preparation method that uses the bacillus subtilis strain. The strain is bacillus subtilis XN01 and is preserved in China Center for Type Culture Collection (CCTCC) on March 18th, 2008 with CCTCC NO: M 208044; the Gamma-polyglutamic acid can be prepared with the strain through the steps of strain cultivation and preservation, seed fluid preparation, shaking fluid fermentation and Gamma-polyglutamic acid extraction. The Gamma-polyglutamic acid preparation method can achieve high synthesis efficiency of Gamma-polyglutamic acid in a low-cost fluid culture medium, is simple and has strong operability.
Description
Technical field
The present invention relates to a kind of genus bacillus and prepare the method for gamma-polyglutamic acid-, be specifically related to a kind of subtilis 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 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 subtilis (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 subtilis (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 subtilis (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 subtilis 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 subtilis 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 subtilis (Bacillus substilis) XN01, described subtilis is separating obtained from bean food, described subtilis 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 subtilis (Bacillus substilis).
The physio-biochemical characteristics of above-mentioned subtilis (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 subtilis (Bacillus subtilis) XN01, its special character is that this method may further comprise the steps:
1) spawn culture preservation
With the bacterial classification of subtilis (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 50m1/250ml 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) subtilis (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 subtilis (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
2Subtilis (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 subtilis (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 subtilis (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 subtilis (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.
Will about 1cm
2Subtilis (Bacillus subtilis) XN01 insert and to 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 (7)
1. subtilis, it is characterized in that: described bacterium is subtilis (Bacillus substilis) 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 subtilis 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 subtilis (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 content of described step 3) nitrogenous source is 0.8～1.5%.
4. 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%.
5. 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.
6. according to the arbitrary described method for preparing gamma-polyglutamic acid-of claim 2～5, it is characterized in that: the saccharic carbon source in the described step 3) is glucose, sucrose, maltose, lactose, fructose or is its mixture.
7. the method for preparing gamma-polyglutamic acid-according to claim 6 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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| TWI381049B (en) * | 2010-07-08 | 2013-01-01 | Meiho Inst Of Technology | Liquid medium for bacillus subtilis and method for manufacturing of the liquid medium |
| CN101948785B (en) * | 2010-08-31 | 2012-06-13 | 南京医科大学 | Gamma-polyglutamic acid producing bacterium and method for preparing gamma-polyglutamic acid and salts thereof by using gamma-polyglutamic acid producing bacterium |
| CN103214101B (en) * | 2012-01-19 | 2014-05-07 | 中国中化股份有限公司 | Microbial flocculating agent, and preparation and use thereof |
| CN102827890A (en) * | 2012-09-07 | 2012-12-19 | 吉林中粮生化科技有限公司 | Method for fermentation production of gamma-polyglutamic acid by corn soaking water |
| CN102911974B (en) * | 2012-09-28 | 2014-06-11 | 广西大学 | Method for producing gamma-polyglutamic acid though hot fermentation of bacillus subtilis |
| CN102965311B (en) * | 2012-11-16 | 2015-03-25 | 南京轩凯生物科技有限公司 | Bacillus subtilis and application thereof in preparation of gamma-D-polyglutamic acid |
| CN104164376B (en) * | 2013-05-20 | 2016-05-11 | 中国中化股份有限公司 | A large amount of mutagenic strain bacillus subtilis and cultural methods thereof of producing gamma-polyglutamic acid- |
| CN103497914B (en) * | 2013-09-17 | 2015-04-15 | 河南大学 | Bacillus subtilis strain and method for gamma-PGA (poly-glutamic acid) by utilizing same |
| CN106834190A (en) * | 2017-03-11 | 2017-06-13 | 鲁东大学 | Culture medium, bacterial strain and the method for polyglutamic acid are produced with pea protein wastewater fermentation |
| CN115058352B (en) * | 2022-03-28 | 2023-08-11 | 四川师范大学 | Bacillus subtilis and method for producing agricultural gamma-polyglutamic acid by using same |
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