CN106701644A - Bacillus subtilis and application thereof in producing gama-polyglutamic acid - Google Patents
Bacillus subtilis and application thereof in producing gama-polyglutamic acid Download PDFInfo
- Publication number
- CN106701644A CN106701644A CN201710158450.7A CN201710158450A CN106701644A CN 106701644 A CN106701644 A CN 106701644A CN 201710158450 A CN201710158450 A CN 201710158450A CN 106701644 A CN106701644 A CN 106701644A
- Authority
- CN
- China
- Prior art keywords
- bacillus subtilis
- fermentation medium
- pga
- culture
- solute
- 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.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/07—Bacillus
- C12R2001/125—Bacillus subtilis ; Hay bacillus; Grass bacillus
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
- C12N1/205—Bacterial isolates
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P13/00—Preparation of nitrogen-containing organic compounds
- C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Biomedical Technology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Medicinal Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses bacillus subtilis and application thereof in producing gama-polyglutamic acid. The invention provides the bacillus subtilis KH2 with the preservation number being CGMCC NO.12426. The invention also discloses the application of the bacillus subtilis KH2in producing the gama-polyglutamic acid. The bacillus subtilis KH2 is utilized to produce the gama-PGA, industrial glucose is adopted as a substrate, the gama-PGA is efficiently fermented and produced with the conversion rate being 0.96-1.20g/L/h, the concentration of the gama-PGA can reach to 46.0g/L maximally, the proportion of the gama-D-PGA is 84 percent to 86 percent, the production capacity is high, the medium components are simple, and meanwhile, the production cost of the gama-PGA can be also controlled.
Description
Technical field
The present invention relates to a kind of bacillus subtilis and its application in gamma-polyglutamic acid is produced.
Background technology
Gamma-polyglutamic acid (γ-PGA) is by passing through alpha-amido and β-carboxyl shape between D-Glu and Pidolidone monomer
The same polyamide (γ-L-PGA, γ-D-PGA, γ-DL-PGA) generated after into peptide bond, generally by the paddy ammonia of 5000 or so
Acid monomers are constituted, and molecular mass is typically in 100~1000kD.γ-PGA are a kind of water-soluble and biodegradable biological high scores
Sub- material, acts on human body and environment nonhazardous, has widely in fields such as food, cosmetics, medicine and water process
Purposes.
The production method of γ-PGA mainly has chemical synthesis, extraction method and fermentation method etc..Chemical method synthetic route is long, secondary
Product is more, and chemical synthesis γ-PGA in soil decomposition rate it is slow, therefore one of the reason for be listed in environmental pollution.It is early
Using extraction method phase Japan's production γ-PGA more, with ethanol by the γ-PGA separation and Extractions in natto out.What extraction method was obtained
γ-PGA concentration is relatively low, and has fluctuation, and complex process, production cost is high, therefore can not be widely accepted.It is main at present to use
Production by Microorganism Fermentation γ-PGA.There is Production by Microorganism Fermentation γ-PGA production process to be easily controlled, and fermentation yield is steady
Fixed, recovery rate is high, and target product yield is higher, and molecular weight of product is suitable, advantages of environment protection, is large-scale production
The main method of γ-PGA, just as the study hotspot of domestic and international γ-PGA industries.
Production cost is to restrict one of key factor that γ-PGA are applied.Nitrogen source and various biologies in existing production technology
Element substantially increases the production cost of γ-PGA.Two approach of reduces cost, one to reduce culture medium cost, and two is raising bacterium
The production capacity of strain.Although the production capacity of current strain improves a lot, its production cost is high, the cycle is long, production effect
Rate ground, is the obstacle of industrial-scale production γ-PGA.Therefore, find efficiently, low cost γ-PGA production bacterial strain and its
Process conditions are still the direction studied from now on.
The content of the invention
It is an object of the invention to provide a kind of bacillus subtilis and its application in gamma-polyglutamic acid is produced.
Bacillus subtilis (Bacillus subtilis) KH2 that the present invention is provided, in preservation on the 10th in 05 month in 2016
In China Committee for Culture Collection of Microorganisms's common micro-organisms center (abbreviation CGMCC；Address：The Chaoyang District, Beijing City North Star
The institute 3 of West Road 1, Institute of Microorganism, Academia Sinica；Postcode：100101), deposit number is CGMCC NO.12426.It is withered
Careless bacillus (Bacillus subtilis) KH2 is referred to as bacillus subtilis KH2.
Applications of the present invention protection bacillus subtilis KH2 in gamma-polyglutamic acid is produced.
The present invention also protects a kind of method for producing gamma-polyglutamic acid, comprises the following steps：Culture bacillus subtilis
KH2。
The present invention also protects a kind of method for producing gamma-polyglutamic acid, comprises the following steps：Using fermentation medium culture
Bacillus subtilis KH2.
" using the fermentation medium culture bacillus subtilis KH2 " can specifically be carried out in 7L fermentation tanks.The training
Foster condition of culture is：(rotating speed is 500rpm within 0-24 hours, and rotating speed is within 25-48 hours within 48 hours for 37 DEG C of cultures
700rpm), whole process throughput is 1L/min.
" using the fermentation medium culture bacillus subtilis KH2 " can specifically be carried out in 5L fermentation tanks.The training
Foster condition of culture is：(rotating speed is 500rpm within 0-24 hours, and rotating speed is within 25-48 hours within 48 hours for 37 DEG C of cultures
300rpm), whole process throughput is 1L/min, and radius of turn is 33mm.
" using the fermentation medium culture bacillus subtilis KH2 " can specifically be carried out in triangular flask.The culture
Condition of culture be：32.5-37 DEG C, 220rpm is cultivated 48 hours.
" using the fermentation medium culture bacillus subtilis KH2 " can specifically be carried out in triangular flask.The culture
Condition of culture be：32.5 DEG C, 220rpm is cultivated 48 hours.
" using the fermentation medium culture bacillus subtilis KH2 " can specifically be carried out in triangular flask.The culture
Condition of culture be：37 DEG C, 220rpm is cultivated 48 hours.
The present invention also protects a kind of method for producing gamma-polyglutamic acid, comprises the following steps (a) and step (b)：
Step (a)：Using seed culture medium culture bacillus subtilis KH2, seed liquor is obtained；
Step (b)：The seed liquor of step (a) is seeded into fermentation medium to be cultivated.
The present invention also protects a kind of method for cultivating bacillus subtilis KH2, comprises the following steps：Using fermentation medium
Culture bacillus subtilis KH2.
The present invention also protects a kind of method of bacillus subtilis KH2, and in turn include the following steps (a) and step (b)：
Step (a)：Using seed culture medium culture bacillus subtilis KH2, seed liquor is obtained；
Step (b)：The seed liquor of step (a) is seeded into fermentation medium to be cultivated.
Step (b) described in any of the above can be carried out in 7L fermentation tanks.In the step (b), condition of culture is：37 DEG C of trainings
48 hours (0-24 hours rotating speed is 500rpm, and 25-48 hours rotating speed is 700rpm) is supported, whole process throughput is 1L/
min.In the step (b), the seed liquor OD600nm=10-14.The volume ratio tool of the seed liquor and the fermentation medium
Body can be 1: 5.
Step (b) described in any of the above can be carried out in 5L fermentation tanks.In the step (b), condition of culture is：37 DEG C of trainings
48 hours (0-24 hours rotating speed is 500rpm, and 25-48 hours rotating speed is 300rpm) is supported, whole process throughput is 1L/
Min, radius of turn is 33mm.In the step (b), the seed liquor OD600nm=10-14.The seed liquor and the fermentation
The volume ratio of culture medium concretely 1: 5.
Step (b) described in any of the above can be carried out in triangular flask.In the step (b), condition of culture is：32.5-37
DEG C, 220rpm is cultivated 48 hours.In the step (b), the seed liquor OD600nm=7-12.The seed liquor and the fermentation
The volume ratio of culture medium concretely 1: 100.
Step (b) described in any of the above can be carried out in triangular flask.In the step (b), condition of culture is：37 DEG C,
220rpm is cultivated 48 hours.In the step (b), the seed liquor OD600nm=7-12.The seed liquor and the fermented and cultured
The volume ratio of base concretely 1: 100.
Step (b) described in any of the above can be carried out in triangular flask.In the step (b), condition of culture is：32.5 DEG C,
220rpm is cultivated 48 hours.In the step (b), the seed liquor OD600nm=7-12.The seed liquor and the fermented and cultured
The volume ratio of base concretely 1: 100.
In step (a) described in any of the above, the condition of culture is concretely：37 DEG C, 220rpm cultures.
Seed culture medium described in any of the above is made up of solute and solvent；The solute and its in the seed culture medium
Concentration it is as follows：Glucose 10-40g/L, dusty yeast 2-7g/L, sodium glutamate 5-15g/L, dipotassium hydrogen phosphate 1-3g/L, sulfuric acid
Magnesium 0.1-0.3g/L；The solvent is water.
The pH of the seed culture medium is 6.5-7.0.The pH of the seed culture medium is specially 7.0.
The solute and its concentration in the seed culture medium are concretely：Glucose 20g/L, dusty yeast
5g/L, sodium glutamate 10g/L, dipotassium hydrogen phosphate 2g/L, magnesium sulfate 0.25g/L.
Fermentation medium described in any of the above is made up of solute and solvent；The solute and its in the liquid fermentation and culture
Concentration in base is as follows：Carbon source 20-80g/L, sodium glutamate 20-50g/L, yeast extract 2-15g/L, magnesium sulfate 0.1-
0.3g/L, dipotassium hydrogen phosphate 1-3g/L；The solvent is water.
The carbon source is cerelose or glycerine.
The pH of the fermentation medium is 6.5-7.5.
The pH of the fermentation medium is 7.0.
Solute described in any of the above and its concentration in the liquid fermentation medium are specially：Cerelose 60g/
L, sodium glutamate 30g/L, yeast extract 5g/L, magnesium sulfate 0.25g/L, dipotassium hydrogen phosphate 2g/L.
Solute described in any of the above and its concentration in the liquid fermentation medium are specially：Cerelose 60g/
L, sodium glutamate 30g/L, yeast extract 2g/L, magnesium sulfate 0.25g/L, dipotassium hydrogen phosphate 2g/L.
Solute described in any of the above and its concentration in the liquid fermentation medium are specially：Cerelose 40g/
L, sodium glutamate 40g/L, yeast extract 5g/L, magnesium sulfate 0.25g/L, dipotassium hydrogen phosphate 2g/L.
Solute described in any of the above and its concentration in the liquid fermentation medium are specially：Glycerine 40g/L, paddy ammonia
Sour sodium 40g/L, yeast extract 5g/L, magnesium sulfate 0.25g/L, dipotassium hydrogen phosphate 2g/L.
Solute described in any of the above and its concentration in the liquid fermentation medium are specially：Cerelose 20g/
L, sodium glutamate 40g/L, yeast extract 5g/L, magnesium sulfate 0.25g/L, dipotassium hydrogen phosphate 2g/L.
Solute described in any of the above and its concentration in the liquid fermentation medium are specially：Cerelose 60g/
L, sodium glutamate 40g/L, yeast extract 5g/L, magnesium sulfate 0.25g/L, dipotassium hydrogen phosphate 2g/L.
Solute described in any of the above and its concentration in the liquid fermentation medium are specially：Cerelose 80g/
L, sodium glutamate 40g/L, yeast extract 5g/L, magnesium sulfate 0.25g/L, dipotassium hydrogen phosphate 2g/L.
Solute described in any of the above and its concentration in the liquid fermentation medium are specially：Cerelose 40g/
L, sodium glutamate 20g/L, yeast extract 5g/L, magnesium sulfate 0.25g/L, dipotassium hydrogen phosphate 2g/L.
Solute described in any of the above and its concentration in the liquid fermentation medium are specially：Cerelose 40g/
L, sodium glutamate 30g/L, yeast extract 5g/L, magnesium sulfate 0.25g/L, dipotassium hydrogen phosphate 2g/L.
Solute described in any of the above and its concentration in the liquid fermentation medium are specially：Cerelose 40g/
L, sodium glutamate 50g/L, yeast extract 5g/L, magnesium sulfate 0.25g/L, dipotassium hydrogen phosphate 2g/L.
Solute described in any of the above and its concentration in the liquid fermentation medium are specially：Cerelose 40g/
L, sodium glutamate 40g/L, yeast extract 7g/L, magnesium sulfate 0.25g/L, dipotassium hydrogen phosphate 2g/L.
Solute described in any of the above and its concentration in the liquid fermentation medium are specially：Cerelose 40g/
L, sodium glutamate 40g/L, yeast extract 10g/L, magnesium sulfate 0.25g/L, dipotassium hydrogen phosphate 2g/L.
Solute described in any of the above and its concentration in the liquid fermentation medium are specially：Cerelose 40g/
L, sodium glutamate 40g/L, yeast extract 15g/L, magnesium sulfate 0.25g/L, dipotassium hydrogen phosphate 2g/L.
The present invention also protects a kind of kit for cultivating bacillus subtilis KH2, including is fermented described in any of the above
Culture medium.
The present invention also protects a kind of kit for producing gamma-polyglutamic acid, including fermented and cultured described in any of the above
Base.
Kit described in any of the above also includes seed culture medium described in any of the above.
The invention provides a kind of bacillus subtilis (Bacillus subtilis) KH2.The present invention utilizes withered grass gemma
Bacillus (Bacillus subtilis) KH2 produces γ-PGA, with cerelose as substrate, with 0.96-1.20 g/l/hour
Efficient fermenting and producing γ-PGA, the γ-PGA concentration of conversion ratio reach as high as 46.0 g/l, wherein γ-D-PGA proportions are
84%-86%.The present invention produces γ-PGA using bacillus subtilis (Bacillus subtilis) KH2, and production capacity is high,
Medium component is simple, while the production cost of γ-PGA can also be controlled.
Brief description of the drawings
Fig. 1 is bacillus subtilis (Bacillus subtilis) KH2 production γ-PGA configurations liquid phase detection figures.Figure 1A
It is standard items high-efficient liquid phase chromatogram.Figure 1B is the high-efficient liquid phase chromatogram after the hydrolysis of bacillus subtilis KH2 zymotic fluids.
Fig. 2 is that bacillus subtilis (Bacillus subtilis) KH2 produces γ-PGA fermentation diagrams.
Specific embodiment
Following embodiment facilitates a better understanding of the present invention, but does not limit the present invention.Experiment in following embodiments
Method, unless otherwise specified, is conventional method.Test material used in following embodiments, unless otherwise specified, is certainly
What routine biochemistry reagent shop was commercially available.Quantitative test in following examples, is respectively provided with three repetitions and tests, and as a result makes even
Average.
γ-PGA standard items：Ka Sima stores, article No.：94791.
Pidolidone standard items：Ka Sima stores, article No.：LGC.MM133300.
D-Glu standard items：Ka Sima stores, article No.：D810325.
Cerelose：Zibo rainbow space industry sugar Co., Ltd.
Glycerine：Shandong Yu Ling Chemical Co., Ltd.s.
Slant medium is made up of solute and solvent；The solute and its concentration in slant medium are as follows：Albumen
10 g/l of peptone, 5 g/l of yeast extract, 10 g/l of sodium chloride, 15 g/l of agar powder；The solvent is water.Inclined-plane culture
The pH of base is 7.0.121 DEG C sterilize 15 minutes.
Seed culture medium is made up of solute and solvent；The solute and its concentration in seed culture medium are as follows：Grape
20 g/l of sugar, 5 g/l of dusty yeast, 10 g/l of sodium glutamate, 2 g/l of dipotassium hydrogen phosphate, 0.25 g/l of magnesium sulfate；It is described
Solvent is water.The pH of the seed culture medium is 7.0.Sterilized 20 minutes under the conditions of 115 DEG C.
Glucose content and content of glutamic acid use bio-sensing analyzer SBA-40C (sections of Shandong Province in following examples
Biological study institute of institute) determine.
The detection method of the γ-PGA contents in following examples in sample solution is：2mL sample solutions are taken, 2mL is added
CTAB solution, fully shaking simultaneously avoids reaction solution from producing foam as far as possible, then stands 3 minutes, and absorbance is detected under 250nm.
Prepare various concentrations γ-PGA standard solutions, detected using same procedure, according to standard curve calculate sample in γ-
The content of PGA.
The assay method of γ-L-PGA and γ-D-PGA purity is in sample solution in following examples：
(1) sample solution, deionized water 24 hours (changing a water in every 4 hours) of dialysis are taken.
(2) the solution freeze-drying after step (1) is dialysed, then obtains powder 6M HCl solutions molten by freeze-drying
Solution, 105 DEG C hydrolyze 10 hours, are subsequently adding the neutralization of 6M NaOH solutions, obtain hydrolysate.
(3) hydrolysate for obtaining step (2) detects the content of Pidolidone and D-Glu with HPLC.HPLC detector bars
Part is：Chiral column, mobile phase is 2mmol/L CuSO4The aqueous solution, flow velocity 0.5mL/min, 25 DEG C of column temperature, the μ L of sample introduction 5, ultraviolet inspection
Survey device detection, Detection wavelength 254nm.
The retention time of Pidolidone standard items is 23.05min, and peak position is gone out under the same conditions within ± 1min,
It can be assumed that being same substance.
The retention time of D-Glu standard items is 15.31min, and peak position is gone out under the same conditions within ± 1min,
It can be assumed that being same substance.
γ-L-PGA accountings are calculated as follows：Pidolidone ÷ (Pidolidone+D-Glu) × 100%.
γ-D-PGA accountings are calculated as follows：D-Glu ÷ (Pidolidone+D-Glu) × 100%.
The separation of embodiment 1, strain, mutagenesis screening and identification
First, the separation of strain
Soil sample is obtained from screening near the bean product factory of Beijing, 5 grams of soil samples is weighed in 250ml triangular flasks, added
100ml physiological saline, dilutes 10000 times and coats on LB solid mediums after being fully mixed with sterilized water, and 37 DEG C of cultures are waited to grow
After going out single bacterium colony, the single bacterium colony that form is big, viscosity is high is selected, be inoculated in 10m1 seed culture mediums, 37 DEG C, 220 revs/min of shakes
Culture 48 hours is swung, γ-PGA concentration in zymotic fluid is determined, one plant of γ-PGA yield highest bacterial strain of picking is inoculated in LB cultures
Refrigerated on base inclined-plane standby.
2nd, ion beam mutagenesis screening
The inoculation that step one is obtained is cultivated to mid-log phase (OD in LB fluid nutrient mediums600nm=0.5-0.6),
Bacterial sediment physiological saline is suspended after cultivating system is centrifuged, is made bacteria suspension.Draw 100 μ L bacteria suspensions and coat diameter
It is (coating diameter about 1.5cm) on the aseptic blank culture dish of 3.5cm, sterile wind drying carries out ion implanting after being made mycoderm,
Power is 120W, and throughput is 8slpm, and duration of ventilation is respectively 1 minute, 2 minutes, 2.5 minutes, 3 minutes, 4 minutes.Ion is noted
Plate after entering is eluted with 1mL physiological saline.Variant implantation dosage draws 100 μ L respectively, and the training of LB solids is coated on after dilution
Support on base, after single bacterium colony is grown, select the larger bacterium colony of viscosity, be inoculated in 10ml fermentation mediums, 37 DEG C, 200 revs/min
Clock concussion and cultivate 48 hours, determines γ-PGA concentration in zymotic fluid.By repeatedly screening, obtain one plant of γ-PGA yield and significantly carry
Bacterial strain high, is named as bacterial strain KH2.
3rd, the identification of bacterial strain KH2
According to the method described in " uncle outstanding Bacteria Identification handbook " (the 9th edition) bacterial strain KH2 is carried out morphological feature observation and
Physio-biochemical characteristics are identified.
Bacterial strain KH2 is gram-positive bacteria, there is gemma, cell direct rod shape, and size is (0.7-0.9) μ m (1.5-3.0) μ
m.Bacterial strain KH2 can utilize maltose, glucose, sucrose, fructose.Lactose, xylose, arabinose, arabinose, lemon can not be utilized
Lemon hydrochlorate negative.
It is precise Identification bacterial strain KH2, its 16S rDNA gene is sequenced, in the sequence that will be measured and ncbi database
The 16S rDNA sequences of many bacillus subtilis contrasted, it was demonstrated that bacterial strain KH2 is bacillus subtilis, is named as
Bacillus subtilis KH2.
Bacillus subtilis KH2 physiological and biochemical property comparative results are shown in Table 1.In table 1 ,+represent that experiment is positive or grows ,-
Represent that experiment is negative or does not grow.
The bacillus subtilis KH2 physiological and biochemical properties of table 1
| Physiological and biochemical property | Bacillus subtilis KH2 |
| Starch Hydrolysis are tested | + |
| Gelatin hydrolysis are tested | + |
| Litmus milk | + |
| Glucose | + |
| Fructose | + |
| Xylose | - |
| Sucrose | + |
| Lactose | - |
| Maltose | + |
| Arabinose | - |
| Catalase | + |
| Nitrate reduction | + |
| Citrate | - |
4th, the preservation of bacillus subtilis KH2
Bacillus subtilis (Bacillus subtilis) KH2, was preserved in China Microbiological on 05 10th, 2016
Culture presevation administration committee common micro-organisms center (abbreviation CGMCC；Address：Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3,
Institute of Microorganism, Academia Sinica；Postcode：100101), deposit number is CGMCC NO.12426.Bacillus subtilis
(Bacillus subtilis) KH2 is referred to as bacillus subtilis KH2.
5th, the genetic stability of bacillus subtilis KH2
Bacillus subtilis KH2 is carried out into Secondary Culture to investigate its genetic stability, passage in every 24 hours once, is passed on
In 15 generations, shake flask fermentation measure γ-PGA yield is carried out per a generation and is had no significant change, with good genetic stability.
Embodiment 2, using bacillus subtilis KH2 in triangular flask fermenting and producing γ-PGA
First, one is tested
1st, bacillus subtilis KH2 is seeded in slant medium, 37 DEG C of quiescent cultures 24 hours.
2nd, after completing step 1,2 rings aseptically are connect with oese, is placed in the 100ml equipped with 20m1 seed culture mediums
In triangular flask, 37 DEG C, 220rpm is cultivated 12 hours, obtains seed culture fluid (OD600nm=7-12).
3rd, the seed culture fluid that 0.5ml steps 2 are obtained is seeded to the 250ml triangles equipped with 50ml fermentation mediums (1)
In bottle, 32.5 DEG C, 220rpm is cultivated 48 hours.
Fermentation medium (1) is made up of solute and solvent；The solute and its concentration in fermentation medium (1) are such as
Under：40 g/l of cerelose, 40 g/l of sodium glutamate, 5 g/l of yeast extract, 0.25 g/l of magnesium sulfate, phosphoric acid hydrogen
2 g/l of dipotassium；The solvent is water.The pH of fermentation medium (1) is 7.0.Sterilized 10 minutes using first 115 DEG C.
4th, after completing step 3, zymotic fluid is taken, 6000rpm centrifugation 5min take supernatant.To 4 times of supernatants of addition in supernatant
The ice ethanol of liquid product, 4 DEG C stand overnight.Then 8000rpm, 4 DEG C of centrifugation 12min collect precipitation, and precipitation is re-dissolved in
In the deionized water isometric with zymotic fluid, sample solution is obtained.γ-PGA contents and γ-D-PGA in detection sample solution
Proportion.By determining, γ-PGA contents are 23.4 g/l, and the ratio shared by γ-D-PGA is 84%-86% (Fig. 1).
2nd, two are tested
1st, with experiment one the step of 1.
2nd, with experiment one the step of 2.
3rd, the seed culture fluid that 0.5ml steps 2 are obtained is seeded to the 250ml triangles equipped with 50ml fermentation mediums (2)
In bottle, 32.5 DEG C, 220rpm is cultivated 48 hours.
Fermentation medium (2) is made up of solute and solvent；The solute and its concentration in fermentation medium (2) are such as
Under：40 g/l of glycerine, 40 g/l of sodium glutamate, 5 g/l of yeast extract, 0.25 g/l of magnesium sulfate, dipotassium hydrogen phosphate 2
G/l；The solvent is water.The pH of fermentation medium (2) is 7.0.Sterilized 10 minutes using first 115 DEG C.
4th, with experiment one the step of 4.By determining, γ-PGA contents are 15.1/ liter.
3rd, three are tested
1st, with experiment one the step of 1.
2nd, with experiment one the step of 2.
3rd, the seed culture fluid that 0.5ml steps 2 are obtained is seeded to the 250ml triangles equipped with 50ml fermentation mediums (3)
In bottle, 32.5 DEG C, 220rpm is cultivated 48 hours.
Fermentation medium (3) is made up of solute and solvent；The solute and its concentration in fermentation medium (3) are such as
Under：40 g/l of cerelose, 40 g/l of sodium glutamate, 5 g/l of yeast extract, 0.25 g/l of magnesium sulfate, phosphoric acid hydrogen
2 g/l of dipotassium, 13.5 g/l of citric acid, 6.8 g/l of ammonium chloride, 0.03 g/l of manganese sulfate；The solvent is water.Fermentation training
The pH for supporting base (3) is 7.0.Sterilized 10 minutes using first 115 DEG C.
4th, with experiment one the step of 4.By determining, γ-PGA contents are 8/ liter.
4th, four are tested
1st, with experiment one the step of 1.
2nd, with experiment one the step of 2.
3rd, the seed culture fluid for obtaining 0.5ml steps 2 is seeded to equipped with 50ml fermentation mediums (4)-(7) respectively
In 250ml triangular flasks, 32.5 DEG C, 220rpm is cultivated 48 hours.
Fermentation medium (4) is made up of solute and solvent；The solute and its concentration in fermentation medium (4) are such as
Under：20 g/l of cerelose, 40 g/l of sodium glutamate, 5 g/l of yeast extract, 0.25 g/l of magnesium sulfate, phosphoric acid hydrogen
2 g/l of dipotassium；The solvent is water.The pH of fermentation medium (4) is 7.0.Sterilized 10 minutes using first 115 DEG C.
Fermentation medium (5)：Cerelose concentration in fermentation medium (4) is replaced with 40 g/l, remaining component
It is constant.
Fermentation medium (6)：Cerelose concentration in fermentation medium (4) is replaced with 60 g/l, remaining component
It is constant.
Fermentation medium (7)：Cerelose concentration in fermentation medium (4) is replaced with 80 g/l, remaining component
It is constant.
4th, with experiment one the step of 4.By determining, γ-PGA contents are as shown in table 2.
γ-PGA the contents of table 2
5th, five are tested
1st, with experiment one the step of 1.
2nd, with experiment one the step of 2.
3rd, the seed culture fluid for obtaining 0.5ml steps 2 is seeded to equipped with 50ml fermentation mediums (8)-(11) respectively
In 250ml triangular flasks, 32.5 DEG C, 220rpm is cultivated 48 hours.
Fermentation medium (8) is made up of solute and solvent；The solute and its concentration in fermentation medium (8) are such as
Under：40 g/l of cerelose, 20 g/l of sodium glutamate, 5 g/l of yeast extract, 0.25 g/l of magnesium sulfate, phosphoric acid hydrogen
2 g/l of dipotassium；The solvent is water.The pH of fermentation medium (8) is 7.0.Sterilized 10 minutes using first 115 DEG C.
Fermentation medium (9)：Concentration of sodium glutamate in fermentation medium (8) is replaced with 30 g/l, remaining component is not
Become.
Fermentation medium (10)：Concentration of sodium glutamate in fermentation medium (8) is replaced with 40 g/l, remaining component
It is constant.
Fermentation medium (11)：Concentration of sodium glutamate in fermentation medium (8) is replaced with 50 g/l, remaining component
It is constant.
4th, with experiment one the step of 4.By determining, γ-PGA contents are as shown in table 3.
γ-PGA the contents of table 3
6th, six are tested
1st, with experiment one the step of 1.
2nd, with experiment one the step of 2.
3rd, the seed culture fluid for obtaining 0.5ml steps 2 is seeded to equipped with 50ml fermentation mediums (12)-(15) respectively
In 250ml triangular flasks, 32.5 DEG C, 220rpm is cultivated 48 hours.
Fermentation medium (12) is made up of solute and solvent；The solute and its concentration in fermentation medium (12) are such as
Under：40 g/l of cerelose, 40 g/l of sodium glutamate, 5 g/l of yeast extract, 0.25 g/l of magnesium sulfate, phosphoric acid hydrogen
2 g/l of dipotassium；The solvent is water.The pH of fermentation medium (12) is 7.0.Sterilized 10 minutes using first 115 DEG C.
Fermentation medium (13)：Yeast extract concentration in fermentation medium (12) is replaced with 7 g/l, remaining group
Divide constant.
Fermentation medium (14)：Yeast extract concentration in fermentation medium (12) is replaced with 10 g/l, remaining group
Divide constant.
Fermentation medium (15)：Yeast extract concentration in fermentation medium (12) is replaced with 15 g/l, remaining group
Divide constant.
4th, with experiment one the step of 4.By determining, γ-PGA contents are as shown in table 4.
γ-PGA the contents of table 4
7th, seven are tested
1st, with experiment one the step of 1.
2nd, with experiment one the step of 2.
3rd, the seed culture fluid for obtaining 0.5ml steps 2 is seeded to the 250ml equipped with 50ml fermentation mediums (16) respectively
In triangular flask, 37 DEG C, 220rpm is cultivated 48 hours.
Fermentation medium (16) is made up of solute and solvent；The solute and its concentration in fermentation medium (16) are such as
Under：60 g/l of cerelose, 30 g/l of sodium glutamate, 2 g/l of yeast extract, 0.25 g/l of magnesium sulfate, phosphoric acid hydrogen
2 g/l of dipotassium；The solvent is water.The pH of fermentation medium (16) is 7.0.Sterilized 10 minutes using first 115 DEG C.
4th, with experiment one the step of 4.By determining, γ-PGA contents are 41.4 g/l.
Embodiment 3, using bacillus subtilis KH2 in 5L fermentation cylinder for fermentation production γ-PGA
1st, bacillus subtilis KH2 is seeded in the 100ml triangular flasks containing 20ml seed culture mediums, 37 DEG C,
220rpm concussion and cultivates 12 hours, take 4ml and are seeded in the 1L triangular flasks equipped with 400ml seed culture mediums, 37 DEG C, 220rpm trainings
Support 12 hours, obtain seed liquor (OD600nm=10-14).
2nd, by the seed culture fluid that 400ml steps 1 are obtained be seeded to the 5L fermentation tanks equipped with 2L fermentation mediums (16) (on
5 liters of fermentation tanks of Hai Baoxing BIOTECH) in, 37 DEG C are cultivated 48 hours, and preceding 24 hours rotating speeds are 500rpm, and 24 hours rotating speeds are afterwards
300rpm, whole process throughput is 1L/min.Radius of turn is 33mm.
Fermentation medium (16) is made up of solute and solvent；The solute and its concentration in fermentation medium (16) are such as
Under：60 g/l of cerelose, 30 g/l of sodium glutamate, 2 g/l of yeast extract, 0.25 g/l of magnesium sulfate, phosphoric acid hydrogen
2 g/l of dipotassium；The solvent is water.The pH of fermentation medium (16) is 7.0.Sterilized 10 minutes using first 115 DEG C.
3rd, after completing step 2, zymotic fluid is taken, 6000rpm centrifugation 5min take supernatant.To 4 times of supernatants of addition in supernatant
The ice ethanol of liquid product, 4 DEG C stand overnight.Then 8000rpm, 4 DEG C of centrifugation 12min collect precipitation, and precipitation is re-dissolved in
In the deionized water isometric with zymotic fluid, sample solution is obtained.γ-PGA contents in detection sample solution.
Result shows that γ-PGA yield reaches 32.5 g/l.Throughput rate is 0.68g/L × h.
Embodiment 4, using bacillus subtilis KH2 in 7L fermentation cylinder for fermentation production γ-PGA
1st, bacillus subtilis KH2 is seeded in the 100ml triangular flasks of 20m1 seed culture mediums, 37 DEG C, 220 revs/min
Concussion and cultivate 12 hours, then takes 6ml and is seeded in the 1L triangular flasks equipped with 600m1 seed culture mediums, 37 DEG C, 220rpm cultures
Obtain seed liquor (OD within 12 hours600nm=10-14).
2nd, the seed culture fluid that 600ml steps 1 are obtained is seeded to the 7L fermentation tank (morals equipped with 3L fermentation mediums (17)
State Ai Bende Eppendorf7 rise fermentation tank) in, 37 DEG C are cultivated 48 hours, and preceding 24 hours rotating speeds are 500rpm, are turned within 24 hours afterwards
Speed is 700rpm, and whole process throughput is 1L/min.
Fermentation medium (17) is made up of solute and solvent；The solute and its concentration in fermentation medium (17) are such as
Under：60 g/l of cerelose, 30 g/l of sodium glutamate, 5 g/l of yeast extract, 0.25 g/l of magnesium sulfate, phosphoric acid hydrogen
2 g/l of dipotassium；The solvent is water.The pH of fermentation medium (17) is 7.0.Sterilized 10 minutes using first 115 DEG C.
3rd, after completing step 2, zymotic fluid is taken, 6000rpm centrifugation 5min take supernatant.Glucose in detection supernatant
And content of glutamic acid.To 4 times of ice ethanol of supernatant volume are added in supernatant, 4 DEG C stand overnight.Then 8000rpm, 4 DEG C
Centrifugation 12min collects precipitation, and precipitation is re-dissolved in the deionized water isometric with zymotic fluid, obtains sample solution.Inspection
γ-PGA the contents surveyed in sample solution.
Result is as shown in Figure 2.Result shows that γ-PGA yield reaches 46.0 g/l.Throughput rate is 1.20g/L × h.
Claims (10)
1. bacillus subtilis (Bacillus subtilis) KH2, its deposit number is CGMCC NO.12426.
2. bacillus subtilis (Bacillus subtilis) KH2 described in claim 1 is in gamma-polyglutamic acid is produced
Using.
3. a kind of method for producing gamma-polyglutamic acid, comprises the following steps：Bacillus subtilis described in culture claim 1
(Bacillus subtilis)KH2。
4. a kind of method for producing gamma-polyglutamic acid, comprises the following steps：Using described in fermentation medium culture claim 1
Bacillus subtilis (Bacillus subtilis) KH2；
The fermentation medium is made up of solute and solvent；The solute and its concentration in the liquid fermentation medium are such as
Under：Carbon source 20-80g/L, sodium glutamate 20-50g/L, yeast extract 2-15g/L, magnesium sulfate 0.1-0.3g/L, phosphoric acid hydrogen two
Potassium 1-3g/L；The solvent is water；
The carbon source is cerelose or glycerine.
5. a kind of method for producing gamma-polyglutamic acid, comprises the following steps (a) and step (b)：
Step (a)：Using the bacillus subtilis (Bacillus subtilis) described in seed culture medium culture claim 1
KH2, obtains seed liquor；
Step (b)：The seed liquor of step (a) is seeded to the fermentation medium described in claim 4 to be cultivated；
The seed culture medium is made up of solute and solvent；The solute and its concentration in the seed culture medium are as follows：
Glucose 10-40g/L, dusty yeast 2-7g/L, 5-15g/L of sodium glutamate, dipotassium hydrogen phosphate 1-3g/L, magnesium sulfate 0.1-0.3g/
L；The solvent is water.
6. the method that one kind cultivates bacillus subtilis (Bacillus subtilis) KH2 described in claim 1, including it is as follows
Step：Using bacillus subtilis (Bacillus subtilis) described in the fermentation medium culture described in claim 4
KH2。
7. a kind of method of bacillus subtilis (Bacillus subtilis) KH2 described in culture claim 1, includes successively
Following steps (a) and step (b)：
Step (a)：Using the seed culture medium culture bacillus subtilis (Bacillus described in claim 5
Subtilis) KH2, obtains seed liquor；
Step (b)：The seed liquor of step (a) is seeded to the fermentation medium described in claim 4 to be cultivated.
8. a kind of kit for cultivating bacillus subtilis (Bacillussubtilis) KH2 described in claim 1, wraps
Include the fermentation medium described in claim 4.
9. a kind of kit for producing gamma-polyglutamic acid, including the fermentation medium described in claim 4.
10. kit as claimed in claim 8 or 9, it is characterised in that：The kit is also included described in claim 5
Seed culture medium.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710158450.7A CN106701644A (en) | 2017-03-16 | 2017-03-16 | Bacillus subtilis and application thereof in producing gama-polyglutamic acid |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710158450.7A CN106701644A (en) | 2017-03-16 | 2017-03-16 | Bacillus subtilis and application thereof in producing gama-polyglutamic acid |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN106701644A true CN106701644A (en) | 2017-05-24 |
Family
ID=58886985
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710158450.7A Pending CN106701644A (en) | 2017-03-16 | 2017-03-16 | Bacillus subtilis and application thereof in producing gama-polyglutamic acid |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106701644A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111990397A (en) * | 2020-08-13 | 2020-11-27 | 山东省药学科学院 | Fresh cut flower preservation material and preparation method thereof |
| CN112410253A (en) * | 2020-11-23 | 2021-02-26 | 中国农业科学院农产品加工研究所 | Bacillus subtilis, application and method for resisting aspergillus flavus and producing polyglutamic acid with high yield |
| CN114854635A (en) * | 2022-05-18 | 2022-08-05 | 四川生力源生物工程有限公司 | Fermentation medium of bacillus subtilis and method for producing gamma-polyglutamic acid |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101109010A (en) * | 2007-07-17 | 2008-01-23 | 秦皇岛领先科技发展有限公司 | Mycopremna generating gamma- polyglutamic acid and culturing method thereof |
| CN101948785A (en) * | 2010-08-31 | 2011-01-19 | 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 |
| CN102965311A (en) * | 2012-11-16 | 2013-03-13 | 南京轩凯生物科技有限公司 | Bacillus subtilis and application thereof in preparation of gamma-D-polyglutamic acid |
| CN103497914A (en) * | 2013-09-17 | 2014-01-08 | 河南大学 | Bacillus subtilis strain and method for gamma-PGA (poly-glutamic acid) by utilizing same |
-
2017
- 2017-03-16 CN CN201710158450.7A patent/CN106701644A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101109010A (en) * | 2007-07-17 | 2008-01-23 | 秦皇岛领先科技发展有限公司 | Mycopremna generating gamma- polyglutamic acid and culturing method thereof |
| CN101948785A (en) * | 2010-08-31 | 2011-01-19 | 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 |
| CN102965311A (en) * | 2012-11-16 | 2013-03-13 | 南京轩凯生物科技有限公司 | Bacillus subtilis and application thereof in preparation of gamma-D-polyglutamic acid |
| CN103497914A (en) * | 2013-09-17 | 2014-01-08 | 河南大学 | Bacillus subtilis strain and method for gamma-PGA (poly-glutamic acid) by utilizing same |
Non-Patent Citations (2)
| Title |
|---|
| 刘洁: "枯草芽孢杆菌发酵制备γ-聚谷氨酸的研究", 《万方学位论文数据库》 * |
| 朱丽娟: "枯草芽孢杆菌HD11发酵生产γ-聚谷氨酸的研究", 《万方学位论文数据库》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111990397A (en) * | 2020-08-13 | 2020-11-27 | 山东省药学科学院 | Fresh cut flower preservation material and preparation method thereof |
| WO2022032980A1 (en) * | 2020-08-13 | 2022-02-17 | 山东省药学科学院 | Fresh-cut flower fresh-keeping material and preparation method therefor |
| CN111990397B (en) * | 2020-08-13 | 2022-04-29 | 山东省药学科学院 | Fresh cut flower preservation material and preparation method thereof |
| CN112410253A (en) * | 2020-11-23 | 2021-02-26 | 中国农业科学院农产品加工研究所 | Bacillus subtilis, application and method for resisting aspergillus flavus and producing polyglutamic acid with high yield |
| CN112410253B (en) * | 2020-11-23 | 2022-08-09 | 中国农业科学院农产品加工研究所 | Bacillus subtilis, application and method for resisting aspergillus flavus and producing polyglutamic acid with high yield |
| CN114854635A (en) * | 2022-05-18 | 2022-08-05 | 四川生力源生物工程有限公司 | Fermentation medium of bacillus subtilis and method for producing gamma-polyglutamic acid |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN113801810B (en) | Halomonas strain and application thereof | |
| CN104694437B (en) | One bacillus licheniformis and its purposes in gamma-polyglutamic acid is produced | |
| CN100554405C (en) | A kind of method and special-purpose lactobacillus rhamnosus thereof that produces L-lactic acid | |
| CN100410364C (en) | Gamma-polyglutamic acid generating bacterium and process of preparing gamma-polyglutamic acid therewith | |
| CN100485027C (en) | Method for producing D-lactic acid and spore lactobacillus special for the same | |
| CN104342390B (en) | A kind of Sinorhizobium meliloti strain and combinations thereof and application | |
| CN104762238B (en) | A kind of lactic acid bacteria for not producing the high urease-producing of amino acid decarboxylases and its application | |
| CN104593442A (en) | A method of producing ectoine by high-density culture of recombinant escherichia coli | |
| CN101457211B (en) | Klebsiella pneumoniae and its application in preparing 2,3-butanediol | |
| CN106701644A (en) | Bacillus subtilis and application thereof in producing gama-polyglutamic acid | |
| CN106947724B (en) | Method for increasing dissolved oxygen of gamma-polyglutamic acid fermentation liquor | |
| CN110317744B (en) | Marseillea fungus for producing blue-violet pigment and method for producing blue-violet pigment by using Marseillea fungus | |
| CN112410253B (en) | Bacillus subtilis, application and method for resisting aspergillus flavus and producing polyglutamic acid with high yield | |
| CN102586151B (en) | High-yield polysaccharide strain and method for producing polysaccharide by utilizing strain through fermentation | |
| CN109880758A (en) | A kind of lactobacillus plantarum mutagenic strain and its method of mutagenesis and application | |
| CN106755186A (en) | A kind of middle anthropi exocellular polysaccharide and its application in terms of soil improvement | |
| CN102226159A (en) | Strain of Enterobacter cloacae and its application in the preparation of 2,3-butylene glycol | |
| CN106011002A (en) | Bacillus megatherium T317, microbial agent and preparation method of microbial agent | |
| CN107674841A (en) | A kind of Aureobasidium pullulans of high yield polymalic acid and application thereof | |
| CN101845401A (en) | Rhodotorula mucilaginose strain and application thereof in degradation of butachlor | |
| CN103146595B (en) | Bacillus subtilis and method for fermentation production of D- ribose | |
| CN106148220B (en) | A kind of method preparing Nahsi peptide and its production bacterial strain | |
| CN101701243B (en) | Method for producing R-mandelic acid and derivates thereof by biocatalysis | |
| CN114214235B (en) | Efficient flocculating bacterium and application thereof in sewage treatment | |
| CN109652340A (en) | A kind of soybean special-purpose multifunctional growth promoting bacteria agent and its preparation method and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20170524 |