CN1231575C - Poly-gamma-glutamic acid generation bacteria and method for producing poly-gamma-glutamic acid - Google Patents

Poly-gamma-glutamic acid generation bacteria and method for producing poly-gamma-glutamic acid Download PDF

Info

Publication number
CN1231575C
CN1231575C CN 03118908 CN03118908A CN1231575C CN 1231575 C CN1231575 C CN 1231575C CN 03118908 CN03118908 CN 03118908 CN 03118908 A CN03118908 A CN 03118908A CN 1231575 C CN1231575 C CN 1231575C
Authority
CN
China
Prior art keywords
poly
gamma
glutamic acid
grams
gram
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Expired - Fee Related
Application number
CN 03118908
Other languages
Chinese (zh)
Other versions
CN1536071A (en
Inventor
陈守文
喻子牛
江昊
孙明
何进
蔡皓
李林
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Huazhong Agricultural University
Original Assignee
Huazhong Agricultural University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Huazhong Agricultural University filed Critical Huazhong Agricultural University
Priority to CN 03118908 priority Critical patent/CN1231575C/en
Publication of CN1536071A publication Critical patent/CN1536071A/en
Application granted granted Critical
Publication of CN1231575C publication Critical patent/CN1231575C/en
Anticipated expiration legal-status Critical
Expired - Fee Related legal-status Critical Current

Links

Images

Landscapes

  • Preparation Of Compounds By Using Micro-Organisms (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

The present invention discloses a bacterial strain which can generate poly-gamma-glutamate, and a method for producing the poly-gamma-glutamate by the bacterial strain. The present invention is characterized in that the bacterial strain is X-003 which is bacillus subtilis, CCTCC, NO: M202048. The poly-gamma-glutamate product is produced and obtained by the technology of strain liquid preparation and liquid submerged fermentation. The bacterial strain has stable genetic properties and a strong capability of synthesizing the poly-gamma-glutamate, and the molecular weight of the synthesized poly-gamma-glutamate reaches more than 10000KD. The shake flask fermentation level of the poly-gamma-glutamate produced by the bacterial strain and the fermentation method of the present invention reaches 10 mg/ml, and the fermentation level of a 80L fermentation tank is 30 mg/ml.

Description

The method that poly-gamma-glutamic acid produces bacterium and produces poly-gamma-glutamic acid
Technical field
The invention belongs to the using microbe technical field, relate in particular to seed selection and produce bacterium and utilize this bacterial strain to produce the method for poly-gamma-glutamic acid to a strain poly-gamma-glutamic acid.
Background technology
Poly-gamma-glutamic acid (poly-γ-glutamic acid) is the biodegradable high molecular polymer that is formed by connecting by peptide bond by D type and L type L-glutamic acid, molecular-weight average is from 10,000 to 2,000,000 dalton (.1997 Hydrophilic surface treating aqueous solution and hydrophilic surfacetreating method such as Matsukawa, U.S.Patents 5.616,585.Ito, Y., Tamaka etc., 1996.Glutamic acidIndependent Production of poly (glutamic acid) by Bacillus subtilis TAM-4, Biosci.Biotechnol.Biochen.60:1239-1242; Cromwick etc., Effect of magnese (II) on Bacilluslicheniformis ATCC9945A physiology and poly-(glutamic acid) formation.Int.J.Biol.Macromol.17:259-267; Cromwick etc., 1995 b.Investigation by NMR of metabolicroutes to bacterial poly-(glutamic acid) using C13 labeled citrate and glutamateas media carbon sources, An, J.Microbiol.41:902-909; Birrer etc., 1994 γ-poly (glutamic acid) formation of Bacillus lecheniformis 9945A:Physiology andbiochemical studies.Int.J.Biol.Micromol.16:265-275.).Purified poly-gamma-glutamic acid under different pH conditions, the physicochemical characteristic significant difference, under slightly acidic, neutrality or alkaline condition, the side chain of poly-gamma-glutamic acid is electronegative, is stretch-like; At the next positively charged of acidic conditions, conformation is spherical closely (He, L.M etc., 2000.Bacillus lichenformis γ-glutamyl exopolymer:physiochemicalcharacterization and U (VI) interaction, JP Patent 11240827).Ca is being arranged 2+Under the condition that exists, poly-gamma-glutamic acid also can be spherical closely structure phase in neutral environment.Poly-gamma-glutamic acid and derivative thereof are as a kind of environment protection biological polymer, because of it has good water-solubility, can be thoroughly by biological degradation, to people's toxicological harmless even advantage such as edible, have broad application prospects in fields such as agricultural, food, medicine, makeup, fiber light industries.
Poly-gamma-glutamic acid has extremely good water-absorbent, can be used for the development of water-retaining agent.At present, a lot of units develop the water absorption Humectant of absorptive macromolecular compound as plant.But the macromolecular compound of these synthetic can not be degraded at nature, has caused environmental pollution again in suction.In this respect, poly-gamma-glutamic acid has advantageous advantage: water-absorbent is strong, but natural degradation.
Aspect the animals and plants breed, poly-gamma-glutamic acid contains a large amount of free α-carboxyls materials such as divalent-metal ion, agricultural chemicals, amino acid is had sequestering action, can these ions of enrichment, and the effective supply animals and plants utilize.Reports such as Kinnersley are used the Ca through the poly-gamma-glutamic acid enrichment 2+, Mg 2+Deng divalent cation, plant can more effective utilization (Kinnersley etc., 1994, Compisition and method for enhanced fertilizer uptake by plants, pateneWO9409628).On the other hand, add 0.1～3.0% poly-gamma-glutamic acid in the feed, can reduce the fat accumulation of poultry and livestock, can be used for raising (Tanimot etc. such as lean meat species poultry, domestic animal and laying hen, 2000, Feed compositioncontaining poly-γ-glutamic acid.JP Patent WO9635339).
Aspect food applications, discoveries such as Konno, poly-gamma-glutamic acid can increase the elasticity of bread, make bread, the particle of cake more careful (Konno etc., 1989, Bakery products and hoodles containing polyglutamicacid.U.S.Patents.4,8888,193.).Poly-gamma-glutamic acid can also strengthen the toughness of noodles, prevents that the solid matter in the noodles is dissolved in the boiling water.Mitsuikzi etc. found in 1998: molecular weight is lower than 200,000 poly-gamma-glutamic acid to have than the better frost resistance of glucose, so frost resistance of poly-gamma-glutamic acid, edibility makes poly-gamma-glutamic acid can be widely used in food processing field and to the enzyme of deep refrigeration sensitivity or the freezing (Mitsuiki etc. of culture, 1998, Relationship between the antifreeze activities and the chemical structures ofoligo-and poly (glutamic acid) s.J.Agric.Food.Chem.46:891-895); Poly-gamma-glutamic acid can also help the absorption of human body to mineral element as the mineral absorption enhancer.(CPP) compares with phosphopeptide caseinate, and because of it has good water-solublely, assimilation effect is better; And poly-gamma-glutamic acid can cover the palatability problems such as pungency, convergency and harsh feeling that the high density mineral element brings, and is particularly useful for replenishing the calcium and mends the healthcare products of iron.
Because poly-gamma-glutamic acid has good water-solubility, to people's toxicological harmless, himself no antigen, thereby attract tremendous attention in the conveying of medicine and the application aspect the slowly-releasing.Sakurai has developed a kind of composition polymer that can be used for embedding medicinal, poly-gamma-glutamic acid is wherein main water-soluble substances (Sakurai etc., 1995, Water soluble high molecularweight polymerized drug preparation, U.S.Patent, 5,693,751.).A large amount of free α-carboxyls of poly-gamma-glutamic acid can affine antigen presenting cell, and adsorbable multiple medicine.With the poly-gamma-glutamic acid skeleton is bridge, antigen presenting cell and certain drug to target site, can reduce the usage quantity of medicine with drug conveying in conjunction with forming conjugated body significantly, so not only reduced the use cost of medicine, reduced the murder by poisoning of medicine simultaneously human body.This scheme is mainly used in treatment lupus erythematosus, rheumatic arthritis and type i diabetes etc.
Poly-gamma-glutamic acid also is used as medicine and is directly used in some treatment of diseases, as: Kato etc. have antineoplastic activity after just reporting 1-beta-D-arabinofuranosylcyutosine and poly-gamma-glutamic acid combines in 1984.In addition, Otani equals report in 1998: can be as the xanthan gum of soft tissue after hydrogel of being made up of gelatin and poly-gamma-glutamic acid and water miscible carbodimides are crosslinked, this material is than the effective more (Markland etc. of traditional fibrin glue, 1999.Modified polypeptides containing γ-benzyl glutamic acid asdrug delivery platforms.Int.J.Pharm.178,183-192; Otani γ etc., 1998, Effectof additives on gelatin and tissue adhesion of gelatin-poly (L-glutamicacid) mixture, Biomaterials, 19,2167-2173.).
At present, the biosynthesizing of poly-gamma-glutamic acid mainly obtains product by the bacillus licheniformis (Bacilluslicheniformis) and Bacillus subtillis (Bacillus subtilis) fermentation of Bacillus.It is reported Bacilluslicheniformis 9945a bacterial strain, in the shake flask fermentation that contains the E substratum, the output of poly-gamma-glutamic acid can reach 17g/L, molecular weight is (Cromwick below 3000kDa, Deng, 1995a.Effect of magnese (II) on Bacilluslicheniformis ATCC9945A physiology and γ-poly (glutamic acid) formation.Int.J.Biol.Macromol.17:259-267.).Ko and Gross were in (Ko in 1997, Deng, 1998.Effects of glucoseand glycerol on gamma-poly (glutamic acid) formation by Bacillus licheniformis ATCC9945a.Biotechnology and Bioengineering.57 (4) 430-436.) find: utilize the mixed carbon source of glucose and glycerine, can improve the output of poly-gamma-glutamic acid.They think for Bacillus licheniformis ATCC9945a, and glucose is the carbon source that more helps the cell growth than glycerine, and somatic cells is also fast than glycerine, citric acid, L-glutamic acid to the utilization of glucose simultaneously.But, lack the rapid decline that glycerine will cause the poly-gamma-glutamic acid productive rate in the fermention medium.The content of glycerine in the substratum is reduced to 0 from 40 grams per liters, the cultivation through 96 hours, the concentration of poly-gamma-glutamic acid is reduced to 5.7 grams per liters from 20.5 grams per liters of contrast.This synergism for glycerine does not also have good explanation now.In addition, the glucose of high density can make bacillus licheniformis can produce the by product of polyose.Ito etc. found in 1996: glucose, fructose, maltose, sucrose in water ratio lactose, semi-lactosi and glycerine are more suitable for the synthetic poly-gamma-glutamic acid of Bacillus subtilis TAM-4, under the condition of being fit to, the highest 22.1 grams per liter poly-gamma-glutamic acids that produce of this bacterial strain.Bacillus subtillis IFO3335 needs L-L-glutamic acid, and citric acid and ammonium sulfate synthesize poly-gamma-glutamic acid as carbon source, and output reaches 20 grams per liters.Bacillus subtillis (natto) can produce 35 grams per liter poly-gamma-glutamic acids (Ogawa etc., 1991 Purification and properties of γ-glutamyltranspeptidasefrom Bacillus subtilis (natto) .Agric.Biol.Chem.55 (12): 2971-2977.) in containing the MSG substratum of maltose, soya-bean oil and glycerine.
At the bacterial classification Bacillus licheniformis ATCC 9945a that generally uses at present, in the process of the synthetic poly-gamma-glutamic acid of Bacillussubtilis (natto), exist bacterial strain to synthesize the unsettled phenomenon of poly-gamma-glutamic acid proterties, after preservation or switching, most bacterium colony has been lost the performance (Ashiuchi etc. of synthetic poly-gamma-glutamic acid, 2001, Isolation of Bacillus subtilis (chungkookjang), a poly-γ-glutamate producer withhigh genetic competence.Appl.Microbiol.Biotechnol., 57:764-769.), this situation requires in the experimentation purifying bacterial classification repeatedly; Simultaneously, adopt the synthetic poly-gamma-glutamic acid of Bacillus licheniformis ATCC 9945a, need to add the glycerine (to 80g/L) of high density in the substratum, thereby cause fermentation costs too high, exist fermentation period to grow problem (Soon etc. such as (2 days to 5 days) in addition, 2000, Biotech.Lett., 22:585-588.; Borbely, 2001, Polymeric product, United States Patent6,326,511); More than these problems seriously restricted the poly-gamma-glutamic acid industrialization process.In addition, molecular weight ((Cromwick below 3000KD of Bacillus licheniformis ATCC 9945a and the Bacillus subtilis biosynthetic poly-gamma-glutamic acids of bacterial strain such as (natto), Deng, 1995a.Effect of magnese (II) on Bacillus licheniformis ATCC9945Aphysiology and γ-poly (glutamic acid) formation.Int.J.Biol.Macromol.17:259-267.)), when utilizing poly-gamma-glutamic acid synthetic macromolecule biomaterial, be subjected to very big restriction on the performance He on the range of application.At present, only report that the molecular weight of the biosynthetic poly-gamma-glutamic acid of Bacillus subtilis (chungkookjang) can reach 10000KD, whether can screen the higher bacterial strain of generation poly-gamma-glutamic acid molecular weight, urgently remain to be developed.
Summary of the invention
The objective of the invention is to overcome the defective that prior art exists, seed selection one strain product poly-gamma-glutamic acid ability is strong, the stability of characteristics of synthetic poly-gamma-glutamic acid; Adapt to liquid fermentation condition, generation bacterium that can synthetic macromolecule amount poly-gamma-glutamic acid and the method for producing poly-gamma-glutamic acid thereof.
The present invention is achieved in that
A kind of bacterial strain that can produce poly-gamma-glutamic acid is characterized in that, described bacterial strain is X-003, a kind of subtilis Bacillus subtilis, be deposited in Chinese typical thing preservation center (CCTCC), the preservation time: on December 25th, 2002, deposit number: NO:M202048.
The separation of bacterial classification and screening:
Get the soil of the Chinese Wuhan City, Hubei Province Hua Zhong Agriculture University experimental farm following 5cm in water paddy soil top layer, add an amount of sterilized water, shake up, 100 times of gradient dilutions.With suspension 80 ℃ of thermal treatments 15 minutes, be coated with the LB solid plate, through the cultivation of spending the night, the picking slime bacteria falls within 5 milliliters of poly-gamma-glutamic acid screening culture medium (E substratum: see document: Cromwick etc., Effect of magnese (II) on Bacillus licheniformis ATCC9945A physiology andpoly-(glutamic acid) formation.Int.J.Biol.Macromol.17:259-267) and cultivated 2～3 days.Medium centrifugal is got supernatant, and with 3 times of volume of ethanol precipitations, collecting precipitation thing, frost drying adds an amount of distilled water dissolving, utilizes HPLC to detect product.The bacterial strain method in accordance with regulations that screens is carried out identification of strains.
Poly-gamma-glutamic acid produces bacterium Bacillus subtilis, the biology of X-003 and hereditary property thereof:
Biological characteristics: the thalline direct rod shape, Gram-positive, brood cell's sub-circular, end is given birth to partially.Bacterium colony circle on the LB solid medium, edge-smoothing, lawn is plentiful, thickness.Optimum growth temperature 30-37 ℃, appropriate pH 6.8-7.2.The catalase experiment is negative, the indoles feminine gender, and nitrate utilizes positive, and Citrate trianion utilizes positive, and the gelatine liquefication experiment is positive.
Hereditary property: this bacterial strain contains two plasmids (as shown in Figure 1), and the poly-gamma-glutamic acid synthase gene is positioned on the karyomit(e).The stable performance of the synthetic poly-gamma-glutamic acid of this bacterial strain, continuous passage are more than 20 times, and the ability of the synthetic poly-gamma-glutamic acid of this bacterial strain is constant.
Spawn culture, go down to posterity and preservation;
On LB substratum (culture medium prescription as previously mentioned) solid inclined-plane in 37 ℃ of cultivations, can be in 4 ℃ of short term storages, with freezing glycerine pipe range phase preservation, as original strain; Producing bacterial classification needs to transfer from original strain.
2, utilize described bacterial strain to be X-003, a kind of subtilis X-003, Bacillus subtilis, CCTCC, NO:M202048 produce the method for poly-gamma-glutamic acid, according to the following step production:
Bacterial strain of the present invention (bacterial strain is X-003, a kind of subtilis Bacillus subtilis, and CCTCC, NO:M202048) preservation follows these steps to:
A, Bacillus subtilis X-003 rule 37 ℃ of cultivations on LB (culture medium prescription as previously mentioned) solid plate;
B, draw together and get lawn to the 20% aseptic glycerine preservation pipe, 65 ℃ of water bath processing 15 minutes;
C ,-70 ℃ of freezings.
The seed liquor preparation:
A, with the bacterial classification inoculation of freezing on the inclined-plane of the LB solid medium that contains peptone 10 grams per liters, NaCl 10 grams per liters, yeast extract 5 grams per liters, agar 20 grams per liters, pH7.0, cultivated 24～48 hours, and made its activation for 37 ℃;
B, above-mentioned activatory seed is changed in the LB triangular flask liquid seed culture medium that contains peptone 10 grams per liters, NaCl 10 grams per liters, yeast extract 5 grams per liters, pH7.0,37 ℃ of 200rpm cultivate 5～6 hours to logarithmic growth mid-term;
C, according to the add-on of per 1 liter of substratum preparation seed culture medium: L-glutamic acid 15 grams; Glucose 40 grams; Citric acid 10 grams, ammonium chloride 7 grams; K 2HPO 40.5 gram; MgSO 47H 2O 0.5 gram; FeCl 36H 2O 0.04 gram; CaCl 22H 2The O0.5 gram; MnSO 4H 2O 0.104 gram, peptone 10 grams; Replenish distilled water to 1000ml, pH6.5～6.8;
D, press the substratum that seeding tank volumetrical 60～80% drops into the preparation of C steps,, kept 30 minutes, be cooled to 37 ℃ of seed liquor that insert the preparation of B steps with the 0.1Mpa high pressure steam sterilization;
Fermentative production:
A, according to the add-on of per 1 liter of substratum preparation fermention medium: L-glutamic acid 15 grams; Glucose 40～100 grams; Citric acid 10 grams, ammonium chloride 7 grams; K 2HPO 40.5 gram; MgSO 47H 2O 0.5 gram; FeCl 36H 2O 0.04 gram; CaCl 22H 2O 0.5 gram; MnSO 4H 2O 0.104 gram, peptone 10 grams; Bubble enemy 0.5～2ml replenishes distilled water to 1000ml, pH6.5～6.8;
B, press fermentor tank volumetrical 60～80% and drop into above-mentioned fermention medium, kept 30 minutes, be cooled to 37 ℃ and insert seed liquor with 0.07～0.1Mpa high pressure steam sterilization; Under 37 ℃ of conditions, cultivate tank pressure 0.3～0.5Kg/cm 2, air flow 1: 0.6～1.2 (v/v);
Extract with refining:
Described bacterial strain is carried out liquid submerged fermentation, and 28～36 hours finish to ferment; Centrifugal then removal thalline is collected supernatant liquor, the ultrafiltration and concentration supernatant liquor, and with 95% ethanol sedimentation poly-gamma-glutamic acid, reclaim and obtain poly-gamma-glutamic acid.
Effect of the present invention
Produce bacterium with the external poly-gamma-glutamic acid that adopts and compare, poly-gamma-glutamic acid of the present invention produces bacterium Bacillussubtilis X-003 and has following characteristics:
(1) strain growth speed is fast: bacterial strain of the present invention is in 10 liters of fermentor tanks, and fermentation time is 28 hours; Other Bacillus subtillis is more than 36 hours; Bacillus licheniformis needs more than 48 hours.
(2) institute's synthetic poly-gamma-glutamic acid molecular weight height (more than the molecular weight 10000KD, is seen accompanying drawing: 2,3,4).Poly-gamma-glutamic acid output height: adopt liquid triangular flask shake-flask culture method (250ml triangular flask shake flask fermentation 48 hours), the concentration of poly-gamma-glutamic acid reaches 10mg/ml in the fermented liquid, the range of molecular weight distributions 1000～20 of poly-gamma-glutamic acid, 000kDa.
(3) do not need glycerine in the fermention medium, thereby can reduce production costs.
(4) also by product such as synthetic polysaccharide under the glucose of high density.
(5) stabilization characteristics of genetics of bacterial strain of the present invention, the phenomenon that the ability that does not all have generation to synthesize poly-gamma-glutamic acid after preservation, the switching reduces; Do not need the separation of ruling again before the fermentation inoculation.
Accompanying drawing and explanation thereof:
Fig. 1: be subtilis Bacillus subtilis of the present invention, the plasmid map of X-003 bacterial strain (1, be the plasmid of Bacillus subtilis X-003,2, be λ/HindIII)
Fig. 2: the HPLC that is the pure product of poly-gamma-glutamic acid analyzes (retention time is that the chromatographic peak of 14min is a tunning, molecular weight 200KD).
Fig. 3: be Bacillus subtilis, the HPLC of the poly-gamma-glutamic acid fermented liquid of X-003 bacterial strain analyzes (retention time is that the chromatographic peak of 10min is a tunning, more than the molecular weight 10000KD).
Fig. 4: the HPLC that is the poly-gamma-glutamic acid fermented liquid analyzes (retention time is that the chromatographic peak of 10min is a tunning, molecular weight 10000KD)
Embodiment
Embodiment 1: poly-gamma-glutamic acid produces separation, the evaluation of bacterium X-003:
Get the soil of the Hua Zhong Agriculture University experimental farm following 5cm in water paddy soil top layer, add an amount of sterilized water, shake up, 100 times of gradient dilutions.With suspension 80 ℃ of thermal treatments 15 minutes, (the LB substratum is as follows by its prescription of the amount that should add in per 1 liter of distilled water: peptone 10 grams per liters to be coated with LB, NaCl 10 grams per liters, yeast extract 5 grams per liters, agar 20 grams per liters, pH7.0) solid plate, through the cultivation of spending the night, the picking slime bacteria falls within 5 milliliters of poly-gamma-glutamic acid screening culture medium E (E culture medium prescription and making: see document: Cromwick etc., Effect ofmagnese (II) on Bacillus licheniformis ATCC9945A physiology and poly-(glutamicacid) formation.Int.J.Biol.Macromol.17:259-267) and cultivated 2-3 days.Medium centrifugal is got supernatant, and with 3 times of volume of ethanol precipitations, collecting precipitation thing, frost drying adds an amount of distilled water dissolving, utilizes HPLC (high performance liquid chromatography) to detect product.The bacterial strain method in accordance with regulations that screens is carried out identification of strains, determine that this bacterial strain is a subtilis: Bacillus subtilis.
This bacterial strain is done the shake flask fermentation test, and product detects with HPLC through purifying, and detects its molecular weight more than 10000KD.The bacterial strain of molecular weight more than 10000KD of report has only Bacillus subtilis chungkookjang both at home and abroad at present, under identical HPLC analysis condition, its retention time is 11min, the molecular weight that its product is described is less than Bacillussubtilis, the poly-gamma-glutamic acid molecular weight of X-003; And Bacillus subtilis chungkookjang bacterial strain exists without any plasmid; The generation of its product poly-gamma-glutamic acid needs glycerine.So the present invention separates the bacterial strain that obtains and belongs to a strain novel bacterial through being accredited as Bacillus subtilis X-003.
Embodiment 2: the HPLC analytical procedure of poly-gamma-glutamic acid
The quantitative analysis of poly-gamma-glutamic acid and the mensuration of molecular weight are finished chromatographic column by HPLC: G6000PW XL, moving phase is 25mM Na 2SO 4Solution and acetonitrile are in the mixed solution of 4: 1 ratios.Poly-gamma-glutamic acid through purifying is as the quantitative standards thing, and the standard substance of molecular weight is TSK standard Poly (ETHYLENE OXIDE).
The output of poly-gamma-glutamic acid and the determination step of molecular weight are as follows: fermented liquid is regulated pH value to 3.0 after quantitatively diluting, 10000rpm, and 30 minutes are centrifugal, collect supernatant.Fermented liquid after dilution is used for detectable level and molecular weight.The retention time of tunning poly-gamma-glutamic acid is 10min, molecular weight (is seen Fig. 2,3) more than 10000KD shown in.
Embodiment 3: poly-gamma-glutamic acid produces the liquid batch fermentation of bacterium and produces
1. use the bacterial classification of freeze pipe preservation as original strain.
2. slant strains
The bacterial classification Bacillus subtilis x-003 of freeze pipe preservation is inoculated into LB solid inclined-plane (adding peptone 10 grams per liters, NaCl 10 grams per liters, yeast extract 5 grams per liters, agar 20 grams per liters, pH7.0 in the substratum of per 1 liter of distillation water yield), 37 ℃ of cultivations with transfering loop.
3. seed liquor is cultivated
With activatory bacterium colony on LB (medium component is same as above) the solid inclined-plane, be inoculated in substratum and add in 250 milliliters of triangular flasks of peptone 10 grams per liters, NaCl 10 grams per liters, yeast extract 5 grams per liters, pH7.0 by 1 liter of distillation water yield, the liquid amount of every flask culture liquid is 20～40 milliliters, and 37 ℃ of 200rpm cultivate 5～6 hours to logarithmic growth mid-term.
4. fermentor cultivation:
With 560g glucose and 3.5g MnSO 47H 2O mixes, and is made into the 1.5L mother liquor, sterilizes 20 minutes for 115 ℃.All the other compositions (prescription: L-glutamic acid 15 grams; Citric acid 10 grams, ammonium chloride 7 grams; K 2HPO 40.5 gram; FeCl 36H 2O 0.04 gram; CaCl 22H 2O 0.5 gram; MnSO 4H 2O 0.104 gram, peptone: 10 grams; Bubble enemy 0.5～2ml, water 1000ml, pH6.5～6.8) directly add in the 10L fermentor tank, adding water to culture volume is 5.5L, regulating the medium pH value is 6.5～6.8,121 ℃ of sterilizations 30 minutes.Sterilization adds glucose mother liquid in the fermentor tank after finishing, and with the inoculation of 1% inoculum size, fermentation mixing speed 300～800rpm, air flow 1: 0.6～1.2 (v/v), 37 ± 2 ℃ of leavening temperatures.Fermentation to 28 hour end, the concentration that detects, analyzes resulting poly-gamma-glutamic acid (molecular weight is more than 10000KD) is 12g/L.
5, poly-gamma-glutamic acid fermented liquid aftertreatment:
Get 5 liters of above-mentioned fermented liquids, directly regulate fermented liquid pH value to 3.0, under 10000rpm, centrifugal 30 minutes, collect supernatant liquor; Small molecular weight material is removed in ultrafiltration, and is concentrated into 1 liter; Add 3 liter of 95% ethanol in concentrated solution, precipitation is spent the night.Under 10000rpm, 30 minutes centrifugal collecting precipitations, vacuum-drying obtains 52 gram drying products.The poly-gamma-glutamic acid extraction recovery is 86.7% in the present embodiment.
Embodiment 4: poly-gamma-glutamic acid produces the liquid fed-batch fermentation of bacterium and produces
1. use the bacterial classification of freeze pipe preservation as original strain.
2. slant strains:
The bacterial classification Bacillus subtilis x-003 of freeze pipe preservation is inoculated into LB solid inclined-plane (adding peptone 10 grams per liters, NaCl 10 grams per liters, yeast extract 5 grams per liters, agar 20 grams per liters, pH7.0 in the substratum of per 1 liter of distillation water yield), 37 ℃ of cultivations with transfering loop.
3. seed liquor is cultivated:
With activatory bacterium colony on LB (medium component is same as above) the solid inclined-plane, be inoculated in substratum and add in 250 milliliters of triangular flasks of peptone 10 grams per liters, NaCl 10 grams per liters, yeast extract 5 grams per liters, pH7.0 by 1 liter of distillation water yield, the liquid amount of every flask culture liquid is 20～40 milliliters, and 37 ℃ of 200rpm cultivate 5～6 hours to logarithmic growth mid-term.
4. fermentor cultivation:
With 1120g glucose and 28g MgSO 47H 2O mixes, and is made into the 3L mother liquor, sterilizes 20 minutes for 115 ℃; Other gets 4480g glucose, is made into the 15L glucose mother liquid, sterilizes 20 minutes for 115 ℃.All the other compositions (prescription: L-glutamic acid 15 grams; Citric acid 10 grams, ammonium chloride 7 grams; K 2HPO 40.5 gram; FeCl 36H 2O 0.04 gram; CaCl 22H 2O 0.5 gram; MnSO 4H 2O 0.104 gram, peptone: 10 grams; Bubble enemy 0.5～2ml, water 1000ml, pH6.5～6.8) directly add in the 80L fermentor tank, adding water to culture volume is 38L, regulating the medium pH value is 6.5～6.8,121 ℃ of sterilizations 30 minutes.After sterilization finishes, 3L glucosamine salt mother liquor is added in the fermentor tank, and with the inoculation of 1% inoculum size, fermentation mixing speed 200～400rpm, air flow 1: 0.6～1.2 (v/v), 37 ± 2 ℃ of leavening temperatures.In the fermenting process, stream adds glucose mother liquid.Fermentation to 36 hour end, the concentration that detects, analyzes resulting poly-gamma-glutamic acid (molecular weight is more than 10000KD) is 30g/L.
5, poly-gamma-glutamic acid fermented liquid aftertreatment:
Get 30 liters of above-mentioned fermented liquids, after diluting, regulate fermented liquid pH value to 3.0, under 10000rpm, centrifugal 30 minutes, collect supernatant liquor; Small molecular weight material is removed in ultrafiltration, and is concentrated into 15 liters; Add 45 liter of 95% ethanol in concentrated solution, precipitation is spent the night.Under 10000rpm, 30 minutes centrifugal collecting precipitations, vacuum-drying obtains 819 gram drying products.The poly-gamma-glutamic acid extraction recovery is 91% in the present embodiment.

Claims (2)

1, a kind of bacterial strain that can produce poly-gamma-glutamic acid is characterized in that, described bacterial strain is a kind of subtilis (Bacillus subtilis) X-003, is deposited in CCTCC, and deposit number is CCTCCNO:M202048.
2, a kind of method of producing poly-gamma-glutamic acid is characterized in that, adopts subtilis (Bacillussubtilis) X-003, is deposited in CCTCC, deposit number be the bacterial strain of CCTCC NO:M202048 as producing bacterial strain, concrete steps are as follows:
(1) seed liquor preparation
A, the bacterial classification X-003 of freezing is inoculated on the inclined-plane of the LB solid medium that contains peptone 10 grams per liters, NaCl 10 grams per liters, yeast extract 5 grams per liters, agar 20 grams per liters, pH7.0, cultivated 24～48 hours, and made its activation for 37 ℃;
B, above-mentioned activatory seed is changed in the LB triangular flask liquid seed culture medium that contains peptone 10 grams per liters, NaCl 10 grams per liters, yeast extract 5 grams per liters, pH7.0,37 ℃ of 200rpm cultivate 5～6 hours to logarithmic growth mid-term;
C, according to the add-on of per 1 liter of substratum preparation seed culture medium: L-glutamic acid 15 grams; Glucose 40 grams; Citric acid 10 grams, ammonium chloride 7 grams; K 2HPO 40.5 gram; MgSO 47H 2O 0.5 gram; FeCl 36H 2O 0.04 gram; CaCl 22H 2O 0.5 gram; MnSO 4H 2O 0.104 gram, peptone 10 grams; Replenish distilled water to 1000ml, pH6.5～6.8;
D, press the substratum that seeding tank volumetrical 60～80% drops into the preparation of C steps,, kept 30 minutes, be cooled to 37 ℃ of seed liquor that insert the preparation of B steps with the 0.1Mpa high pressure steam sterilization;
(2) fermentative production
A, according to the add-on of per 1 liter of substratum preparation fermention medium: L-glutamic acid 15 grams; Glucose 40～100 grams; Citric acid 10 grams, ammonium chloride 7 grams; K 2HPO 40.5 gram; MgSO 47H 2O 0.5 gram; FeCl 36H 2O 0.04 gram; CaCl 22H 2O 0.5 gram; MnSO 4H 2O 0.104 gram, peptone 10 grams; Bubble enemy 0.5～2ml replenishes distilled water to 1000ml, pH6.5～6.8;
B, press fermentor tank volumetrical 60～80% and drop into above-mentioned fermention medium, kept 30 minutes, be cooled to 37 ℃ and insert seed liquor with 0.07～0.1Mpa high pressure steam sterilization; Under 37 ℃ of conditions, cultivate tank pressure 0.3～0.5Kg/cm 2, air flow 1: 0.6～1.2 (v/v);
(3) extract with refining
Described bacterial strain is carried out liquid submerged fermentation, and 28～36 hours finish to ferment; Centrifugal then removal thalline is collected supernatant liquor, the ultrafiltration and concentration supernatant liquor, and with 95% ethanol sedimentation poly-gamma-glutamic acid, reclaim and obtain poly-gamma-glutamic acid.
CN 03118908 2003-04-10 2003-04-10 Poly-gamma-glutamic acid generation bacteria and method for producing poly-gamma-glutamic acid Expired - Fee Related CN1231575C (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN 03118908 CN1231575C (en) 2003-04-10 2003-04-10 Poly-gamma-glutamic acid generation bacteria and method for producing poly-gamma-glutamic acid

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN 03118908 CN1231575C (en) 2003-04-10 2003-04-10 Poly-gamma-glutamic acid generation bacteria and method for producing poly-gamma-glutamic acid

Publications (2)

Publication Number Publication Date
CN1536071A CN1536071A (en) 2004-10-13
CN1231575C true CN1231575C (en) 2005-12-14

Family

ID=34320809

Family Applications (1)

Application Number Title Priority Date Filing Date
CN 03118908 Expired - Fee Related CN1231575C (en) 2003-04-10 2003-04-10 Poly-gamma-glutamic acid generation bacteria and method for producing poly-gamma-glutamic acid

Country Status (1)

Country Link
CN (1) CN1231575C (en)

Families Citing this family (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1300304C (en) * 2005-04-28 2007-02-14 广东省微生物研究所 5-amino-4-carbamyl imidazole nucleoside producing bacteria and preparing method thereof
CN100383094C (en) * 2006-02-22 2008-04-23 华中农业大学 Use of poly-gamma-glutamic acid as fertilizer absorbefacient in agricultural planting
CN101709283B (en) * 2009-12-18 2012-05-30 南京华洲药业有限公司 Bacillus subtilis and application thereof in preparation of niacin by biocatalysis
CN102763684A (en) * 2011-05-01 2012-11-07 华中农业大学 Microcapsule preparation, preparation method and application
CN102249753B (en) * 2011-05-16 2013-03-13 华中农业大学 Method for producing multifunctional bioorganic fertilizer and application of multifunctional bioorganic fertilizer
CN103373870B (en) * 2012-04-12 2015-02-04 武汉瑞阳生物科技有限公司 Composite microbial fertilizer and preparation method therefor
CN102719501A (en) * 2012-06-29 2012-10-10 天津北洋百川生物技术有限公司 Method for producing polyglutamic acid
CN104561159A (en) * 2013-10-25 2015-04-29 武汉骏安生物科技有限公司 Fermentation method for efficient coproduction of poly-gamma-glutamic acid and 2,3-butanediol by virtue of bacillus
CN107022581B (en) * 2017-05-22 2020-08-14 东莞理工学院 Method for producing gamma-polyglutamic acid by air pressure pulsation solid fermentation
CN110734938A (en) * 2019-11-11 2020-01-31 四川轻化工大学 Bacillus subtilis YB18 and application thereof in fermentation production of high molecular weight poly-gamma-glutamic acid

Also Published As

Publication number Publication date
CN1536071A (en) 2004-10-13

Similar Documents

Publication Publication Date Title
CN1324143C (en) Poly-gamma-glutamate having ultra high molecular weight and method for using the same
CN108865951B (en) A kind of general bacterium of camel thorn and its microbial inoculum and application
CN1830917A (en) Method. for preparing quick-acting compound organic fertilizer
CN101037661A (en) Pseudoalteromonas and its usage
CN1231575C (en) Poly-gamma-glutamic acid generation bacteria and method for producing poly-gamma-glutamic acid
CN111635284B (en) Preparation method of polyglutamic acid charcoal-based organic fertilizer
CN101503716B (en) Method for preparing poly-gamma-glutamic acid by fermenting maize raw material Bacillus subtilis
CN102367431A (en) Bacillus licheniformis and applications thereof
CN110423718B (en) Method for producing trichoderma chlamydospore by using bacillus fermentation liquor and application
CN112359002A (en) Streptomyces albus and application thereof in production of epsilon-polylysine
CN1957711A (en) Biologic bactericide and preparation method
CN107354190A (en) The process of antibacterial peptide is prepared using bacillus licheniformis
CN1908178A (en) Method of low cost preparing gamma-polyglutamic acid utilizing farm and side line products solid fermentation
CN111500653B (en) Production process of polyglutamic acid
CN103820370A (en) Pseudomanas aeruginosa and application thereof
CN1699585A (en) Process for preparing biological antibacterial agent
CN1908155A (en) Microbial viable bacteria combination and preparation method thereof
CN103451116B (en) Pseudomonas fluorescens Y13, and preparation method and applications thereof
CN108546663A (en) One boar source book song lactobacillus and its application
CN1117855C (en) Method of producing beta-1,3-glucan
CN1948463A (en) Application of poly gamma glutamic acid as bacillus thuringiensis agent protector
CN1911043A (en) Method for preparing biological type seed coating agent for anti-cucumber powdery mildew of system induction type
CN115232766B (en) Halophilic microorganism sewage cleaning agent and preparation method thereof
CN1724646A (en) Bacillus subtilis mutant strain, breeding method and application in fermentation method of producing adenosin
CN106957804A (en) Prevent and treat the microbe controlling method that drip emitter is blocked

Legal Events

Date Code Title Description
C06 Publication
PB01 Publication
C10 Entry into substantive examination
SE01 Entry into force of request for substantive examination
C14 Grant of patent or utility model
GR01 Patent grant
C19 Lapse of patent right due to non-payment of the annual fee
CF01 Termination of patent right due to non-payment of annual fee