CN107267411B - Glutamic acid-independent producing strain for producing gamma-polyglutamic acid by high-temperature fermentation and fermentation method thereof - Google Patents

Glutamic acid-independent producing strain for producing gamma-polyglutamic acid by high-temperature fermentation and fermentation method thereof Download PDF

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CN107267411B
CN107267411B CN201710364518.7A CN201710364518A CN107267411B CN 107267411 B CN107267411 B CN 107267411B CN 201710364518 A CN201710364518 A CN 201710364518A CN 107267411 B CN107267411 B CN 107267411B
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曾伟
梁智群
陈桂光
郭烨
董梦娜
张斌
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Abstract

The invention discloses a glutamic acid-independent producing strain for producing gamma-polyglutamic acid by high-temperature fermentation, which is classified and named as bacillus subtilis (Bacillus subtilis)Bacillus subtilis) GXG-5, the preservation number is CCTCC NO: m2017083, the preservation date is 3/6 in 2017, and the preservation unit: china center for type culture Collection. The Bacillus subtilis (A), (B) and (C)Bacillus subtilis) GXG-5 can use sugar raw material as substrate to ferment and produce gamma-polyglutamic acid at higher temperature, the highest fermentation temperature can be 55 ℃, the fermentation time is 48-72 h, and the yield can reach more than 15 g/L. The bacillus subtilis provided by the invention can be used for producing gamma-polyglutamic acid without depending on exogenous glutamic acid fermentation, can effectively reduce the fermentation production cost compared with the conventional glutamic acid-dependent strains, has the advantages of high fermentation temperature, simple nutrition requirement, simple and easy culture method and the like, and provides favorable conditions for industrial production due to the excellent characteristics.

Description

Glutamic acid-independent producing strain for producing gamma-polyglutamic acid by high-temperature fermentation and fermentation method thereof
Technical Field
The invention belongs to the field of microbial engineering, and particularly relates to a glutamic acid-independent producing strain for producing gamma-polyglutamic acid by high-temperature fermentation and a fermentation method thereof.
Background
The gamma-polyglutamic acid (Poly-gamma-glutamic acid) is a microbial anionic extracellular high molecular compound formed by combining L-glutamic acid and D-glutamic acid through α -amino and gamma-carboxyl by amido bonds, the molecular weight of the gamma-polyglutamic acid can reach more than 1,000kDa, and a large amount of free hydrophilic carboxyl groups on a molecular main chain enable the gamma-polyglutamic acid to have the property of water-soluble polycarboxylic acid and be used as an active site for wide chemical modification, so that the gamma-polyglutamic acid has strong water absorption and moisture retention, flocculation property and excellent plasticity, on the other hand, the amido bonds in the molecular main chain of the gamma-polyglutamic acid are easy to be subjected to enzymolysis, degradation products can be absorbed by organisms, so that the gamma-polyglutamic acid has excellent biodegradability and biocompatibility and is harmless to human bodies and the environment.
Gamma-polyglutamic acid was first found in 1937 in the capsular component of Bacillus anthracis (Bacillus anthracis), and this substance was released into the culture broth with lysis of the bacterial cells. In 1942, gamma-polyglutamic acid was found to be freely secreted into a culture solution as a fermentation product of Bacillus subtilis, and subsequently, a strain of Bacillus was reported to synthesize extracellular gamma-polyglutamic acid. The gamma-polyglutamic acid producing strains are divided into glutamic acid dependent strains and glutamic acid independent strains according to whether glutamic acid is required to be added into a fermentation culture medium. Among them, the more deeply studied glutamate-dependent strains are: B. subtilis F-2-01, B. subtilis IFO3335, B. subtilis NX-2, B. licheniformis ATCC 9945A, B. licheniformis WX-02, in which glutamic acid is both an inducer of gamma-polyglutamic acid synthesis and a substrate for gamma-polyglutamic acid synthesis. Glutamate independent strains are: B. the substrate glutamic acid in the strains is mainly converted from sugar raw materials such as glucose or sucrose through glycolysis pathway and tricarboxylic acid cycle.
At present, a glutamic acid dependent strain is mainly used in the research of fermentation production of gamma-polyglutamic acid, a synthetic culture medium is adopted, wherein a carbon-nitrogen source and L-glutamic acid are necessary components, the pH value is generally maintained at 6.5-7.5, the temperature is 30-40 ℃, the product yield is closely related to the appropriate carbon-nitrogen ratio, the addition amount of glutamic acid and biomass, and the ratio of various inorganic salts is also important for the efficient production of the gamma-polyglutamic acid. Glutamate independent strains have been reported and studied relatively rarely relative to glutamate dependent strains, mainly due to their relatively low gamma-polyglutamic acid production. However, from the viewpoint of fermentation raw material cost, the glutamic acid-independent strain can produce gamma-polyglutamic acid using an inexpensive saccharine raw material as a substrate without using relatively expensive glutamic acid or glutamate as a substrate. Therefore, if a glutamic acid independent strain with high yield of the gamma-polyglutamic acid can be bred and obtained, another choice is provided for the industrial large-scale production of the gamma-polyglutamic acid from the aspect of saving raw material cost.
In addition, the fermentation temperature of the gamma-polyglutamic acid producing bacteria is generally 30-40 ℃, however, a large amount of heat is generated due to the growth and metabolism of microorganisms in the fermentation process, and particularly in summer or southern areas, a large amount of cooling water needs to be provided for heat exchange of the fermentation tank so as to maintain the specific temperature required by the fermentation process. Therefore, if a strain capable of producing gamma-polyglutamic acid by fermentation at a higher temperature can be obtained by breeding from the perspective of strains, another choice is provided for the industrial large-scale production of gamma-polyglutamic acid from the aspect of saving the cost of the fermentation process.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provide a glutamic acid-independent producing strain for producing gamma-polyglutamic acid by high-temperature fermentation and a fermentation method thereof. The bacillus subtilis can be used for producing gamma-polyglutamic acid by fermenting sugar raw materials serving as substrates at a high temperature, the highest fermentation temperature can be 55 ℃, the fermentation time can be 48-72 hours, and the highest yield can reach more than 15 g/L. The bacillus subtilis provided by the invention can be used for producing gamma-polyglutamic acid without depending on exogenous glutamic acid fermentation, can effectively reduce the fermentation production cost compared with the conventional glutamic acid-dependent strains, has the advantages of high fermentation temperature, simple nutrition requirement, simple and easy culture method and the like, and provides favorable conditions for industrial production due to the excellent characteristics.
In order to achieve the purpose, the invention is realized by the following technical scheme:
the glutamic acid independent producing strain is classified and named as Bacillus subtilis GXG-5 with the preservation number of CCTCC NO: m2017083, the preservation date is 3/6 in 2017, and the preservation unit: china center for type culture Collection, the preservation address is: wuhan university in Wuhan, China. The culture was tested as viable by the culture collection on day 16 at 3 months 2017.
The Bacillus subtilis GXG-5 has the following characteristics:
morphological characteristics of the thallus: culturing on solid slant culture medium at 50 deg.C for 16 hr, observing with scanning electron microscope, making the thallus be rod-shaped, with size of 0.7-0.9 × 2.0-3.0 μm, movable, and gram-positive. Spore staining observation is carried out after 30h of culture on a solid slant culture medium at 50 ℃, and obvious spores can be seen.
And (3) colony morphology characteristics: culturing on solid plate culture medium at 50 deg.C for 16h, wherein the colony is circular, raised, has wet surface, diameter of 0.5-1.0cm, and is in hanging drop shape when the plate is inverted; after incubation for 30h, the colony center began to dry out, flat, and the edges secreted a viscous mass.
The concentration composition of the solid plate medium was: 22.5g/L of glucose, 3g/L of soybean peptone, NH4Cl17g/L, KNO35g/L, 5g/L of NaCl, 42.5g/L of K2HPO42, 15g/L of agar, 7.0 of pH value and preparation by using distilled water.
Physiological and biochemical activities are shown in the following table
Detecting items Results Detecting items Results
Growth conditions Sugar utilization
28℃ + Glucose +
37℃ + Fructose +
50℃ + Sucrose +
55℃ + Maltose +
pH5.0 + Lactose +
pH9.0 + Mannitol +
NaCl5% + Citric acid -
NaCl10% + Indole test -
NaCl15% - VP assay +
Anaerobic growth - Starch hydrolysis +
Catalase enzyme + Casein hydrolysis +
Lecithinase + Liquefaction of gelatin +
Tyrosine hydrolase + Glucose gas production -
Phenylalanine dehydrogenase - Urease hydrolysis +
Note: the "+" in the list is good or positive for growth; "-" is not growing or is negative.
16S rDNA sequence analysis:
the 16S rDNA of the strain is amplified and sequenced by using universal amplification primers 1540r (5'-AGGAGGTGATCCAGCCGCA-3') and 7f (5'-CAGAGTTTGATCCTGGCT-3'), and the sequence length is 1447 bp. Submitting the obtained sequence to a GenBank database to obtain a sequence number GenBank ID: KY711183, and performing Blast comparison analysis on the sequence and a gene sequence provided by the GenBank to construct a phylogenetic tree. The results show that the homology of the strain and bacillus subtilis is 99%. The strain can be classified and identified as Bacillus subtilis by combining thalli, colony morphological characteristics, physiological and biochemical characteristics and 16S rDNA sequence analysis, and the strain is particularly Bacillus subtilis GXG-5.
The invention also provides the bacillus subtilis (A), (B)Bacillus subtilis) GXG-5A method for producing gamma-polyglutamic acid by fermentation under high-temperature fermentation conditions by using a saccharine raw material as a substrate, which comprises the following steps:
(1) activating and preserving strain
Bacillus subtilis (A), (B) and (C)Bacillus subtilis) GXG-5 is inoculated on a solid slant culture medium and cultured for 12-24h at the temperature of 45-50 ℃; the concentration composition of the solid slant culture medium is as follows: 10-15 g/L of glucose, 2-5 g/L of yeast extract, 3-6 g/L of tryptone, 3-6 g/L of NaCl, 15-20 g/L of agar, 6.5-7.5 of pH value and distilled water.
(2) Seed liquid preparation
Inoculating the lawn with the thickness of 1-3 cm2 on the inclined plane into a 250mL triangular flask filled with 30mL of liquid seed culture medium, and culturing at the rotating speed of a shaker of 160-200 rpm at the temperature of 45-50 ℃ for 12-24h until the logarithmic growth middle stage of the strain; the concentration of the liquid seed culture medium is as follows: 10-20 g/L of glucose, 2-5 g/L of yeast extract, 5-10 g/L of tryptone, 5-10 g/L of NaCl, 6.5-7.5 of pH value and distilled water.
(3) Liquid shake flask fermentation
Inoculating the seed solution into a sterilized liquid fermentation culture medium, wherein the inoculation amount is 1-10%, the liquid loading amount of a shake flask is 20-80 mL/250mL, the rotation speed of a shaking table is 160-250 rpm, and the seed solution is cultured for 48-72 h at 40-55 ℃; the concentration of the liquid fermentation culture medium comprises: 10-50 g/L of sugar raw material, 5-30 g/L of nitrogen source and KNO 32~10g/L，NaCl 2~10g/L，K2HPO40.5-5 g/L, pH 6.5-7.5, and distilled water.
The saccharic material in the liquid fermentation medium is any one or a mixture of glucose, sucrose, maltose, fructose and molasses. The nitrogen source in the liquid fermentation medium is soybean peptone, tryptone, yeast extract and NH4Cl、(NH4)2SO4And NH4NO3Any one or a mixture of several of them.
(4) Extracting and purifying gamma-polyglutamic acid
Collecting mature fermentation liquor, adding 2-5 times of volume of distilled water for dilution, removing thalli by using a microfiltration membrane with the aperture of 0.45 mu m, concentrating supernatant by using an ultrafiltration membrane with the molecular weight cutoff of 10-50 KDa to 30-50% of the volume of the original fermentation liquor, adding 2-4 times of volume of industrial grade ethanol for precipitation, collecting precipitate, and freeze-drying to obtain a finished product of the gamma-polyglutamic acid.
The gamma-polyglutamic acid product obtained by the invention has the following physicochemical properties:
(1) the product of the invention is easy to dissolve in water and insoluble in organic solvents such as methanol, ethanol or acetone.
(2) The ninhydrin product of the invention has negative color reaction, and the ninhydrin product of hydrochloric acid hydrolysis has positive reaction; analyzing the hydrochloric acid complete hydrolysate by thin layer chromatography, wherein the amino acid component only contains glutamic acid; the above indicates that the product is a homopolymer of glutamic acid.
(3) The product of the invention has a characteristic absorption peak at 216nm and no absorption peak at 280 nm; the biuret coloration reaction is negative; the above shows that the product has no typical peptide chain structure.
(4) The product of the invention is detected by nuclear magnetic resonance (1H-NMR) and infrared spectrum, and the atlas result shows that the product conforms to the structure of a gamma-polyglutamic acid standard product.
Compared with the prior art, the invention has the beneficial effects that:
1. the invention separates and obtains a glutamic acid independent gamma-polyglutamic acid producing strain, can utilize cheap saccharic raw materials as substrates to produce gamma-polyglutamic acid, does not need to add relatively high-price glutamic acid or glutamate as substrates, and provides another choice for the industrial large-scale production of the gamma-polyglutamic acid from the aspect of saving the cost of the raw materials.
2. The bacillus subtilis can be used for producing the gamma-polyglutamic acid by fermenting the saccharine raw material serving as a substrate at a higher temperature, the highest fermentation temperature can be 55 ℃, the fermentation time can be 48-72 hours, the yield can reach more than 15g/L, the nutrition requirement is simple, the culture method is simple and easy to implement, the consumption of cooling water can be greatly saved in large-scale production, and the practical application value for reducing the cost of the fermentation process is obvious.
Drawings
FIG. 1 shows NMR of gamma-polyglutamic acid standard substance: (1H-NMR) chart.
FIG. 2 shows the NMR of gamma-polyglutamic acid of the present invention1H-NMR) chart.
FIG. 3 is an infrared spectrum of a gamma-polyglutamic acid standard.
FIG. 4 is an infrared spectrum of the gamma-polyglutamic acid product of the present invention.
Detailed Description
The present invention will be described in further detail with reference to examples, but the embodiments of the present invention are not limited to the scope of the examples.
Example 1: bacillus subtilis (A), (B) and (C)Bacillus subtilis) GXG-5 separation
(1) Preparing a bacterial suspension: 1g of a soil sample was weighed into a 250mL triangular flask containing 50mL of sterile physiological saline, and the mixture was magnetically stirred at room temperature for 25 minutes to prepare a bacterial suspension, which was treated at 100 ℃ for 15 minutes.
(2) Diluting the bacterial suspension, and picking bacterial colonies: diluting the obtained bacterial suspension into bacterial suspensions with different concentrations by 10-fold dilution method, selecting 105、106、107Coating 100 mu L of the bacterial suspension on a solid plate culture medium; after culturing at the constant temperature of 50 ℃ for 20h, picking out single bacterial colonies which are viscous on the surfaces of the bacterial colonies and can be pulled up by toothpicks.
(3) And (3) scribing and purifying: and (3) continuously streaking and purifying the obtained single colony on a solid plate culture medium by using a smooth inoculating loop under an aseptic operation condition, culturing at a constant temperature of 50 ℃ for 15 hours, picking the pure single colony on the plate to a test tube inclined plane, and preserving after culturing.
(4) And (3) shaking flask fermentation: picking 1cm above the inclined plane with a smooth inoculating loop2The lawn is inoculated into a 250mL triangular flask filled with 30mL liquid seed culture medium, the rotating speed of a shaking table is 160rpm, the cultivation is carried out for 10h at 50 ℃ until the logarithmic growth middle stage of the strain, the seed liquid is inoculated into the 250mL triangular flask filled with 50mL liquid fermentation culture medium according to the inoculation amount of 2%, the rotating speed of the shaking table is 160rpm, and the cultivation is carried out for 48h at 50 ℃.
(5) Determination of yield of gamma-polyglutamic acid: collecting the shake flask fermentation liquor, adding 3 times of distilled water for dilution, removing thallus by using a microfiltration membrane with the aperture of 0.45 mu m, concentrating the supernatant by using an ultrafiltration membrane with the molecular weight cutoff of 20KDa to 50% of the volume of the original fermentation liquor, adding industrial grade ethanol with the volume of 4 times of the original fermentation liquor for precipitation, collecting the precipitate, and freeze-drying to obtain the finished product of the gamma-polyglutamic acid. And (3) accurately weighing a proper amount of finished product, and converting the weight into the yield of the gamma-polyglutamic acid in the fermentation liquid.
Wherein, the concentration composition of the solid plate culture medium is as follows: 22.5g/L glucose, 3g/L soyabean peptone, NH4Cl17g/L，KNO35g/L，NaCl 5g/L，K2HPO42.5g/L, 15g/L agar, 7.0 pH value and distilled water. The concentration of the liquid seed culture medium is as follows: 10g/L of glucose, 5g/L of yeast extract, 10g/L of tryptone, 10g/L of NaCl, 7.0 of pH value and distilled water. The concentration of the liquid fermentation culture medium comprises: glucose 22.5g/L, SoybeanPeptone 3g/L, NH4Cl 17g/L，KNO35g/L，NaCl 5g/L，K2HPO42.5g/L, pH 7.0, and distilled water.
The invention screens and obtains a glutamic acid independent strain-bacillus subtilis (Bacillus subtilis) for producing gamma-polyglutamic acid by high-temperature fermentationBacillus subtilis）GXG-5。
Example 2: bacillus subtilis (A), (B) and (C)Bacillus subtilis) GXG-5 identification
(1) Morphological characteristics of the cells
Culturing on solid slant culture medium at 50 deg.C for 16 hr, observing with electron microscope, making the thallus be rod-shaped, with size of 0.7-0.9 × 2.0-3.0 μm, movable, and gram-positive. Spore staining observation is carried out after 30h of culture on a solid slant culture medium at 50 ℃, and obvious spores can be seen.
(2) Morphological characteristics of bacterial colony
Culturing on solid plate culture medium at 50 deg.C for 16h, wherein the colony is circular, raised, has wet surface, diameter of 0.5-1.0cm, and is in hanging drop shape when the plate is inverted; after incubation for 30h, the colony center began to dry out, flat, and the edges secreted a viscous mass.
The concentration composition of the solid plate medium was: 22.5g/L glucose, 3g/L soyabean peptone, NH4Cl17g/L，KNO35g/L，NaCl5g/L，K2HPO42.5g/L, 15g/L agar, 7.0 pH value and distilled water.
(3) Physiological and biochemical activities are shown in the following table
Figure DEST_PATH_IMAGE001
(4) 16SrDNA sequence analysis
The 16S rDNA of the strain is amplified and sequenced by using universal amplification primers 1540r (5'-AGGAGGTGATCCAGCCGCA-3') and 7f (5'-CAGAGTTTGATCCTGGCT-3'), and the sequence length is 1447 bp. Submitting the obtained sequence to a GenBank database to obtain a sequence number GenBank ID: KY711183, and performing Blast comparison analysis on the sequence and a gene sequence provided by the GenBank to construct a phylogenetic tree. The results show that the strain and bacillus subtilis (B)Bacillus subtilis) The homology was 99%. Combining with thallus, colony morphological characteristics, physiological and biochemical characteristics and 16S rDNA sequence analysis, the strain can be classified and identified as Bacillus subtilis, specifically Bacillus subtilis (B) ((B))Bacillus subtilis）GXG-5。
The following are Bacillus subtilis (B) of the present inventionBacillus subtilis) GXG-5 nucleotide sequence information of 16S rDNA:
Figure DEST_PATH_IMAGE003
example 3: bacillus subtilis (A), (B) and (C)Bacillus subtilis) GXG-5 fermentation product gamma-polyglutamic acid identification and characterization
(1) Chemical composition analysis: adding 500mg of the product of the invention into a hydrolysis tube, adding 10mL of 6M hydrochloric acid, vacuumizing, hydrolyzing for 8h at 105 ℃, cooling, adjusting the pH to 7.0 by using 6M sodium hydroxide to obtain hydrolysate, fixing the volume to 100mL, and filtering by using a 0.45-micron microporous filter membrane for later use. Performing thin-layer chromatography analysis on the hydrolysate, wherein the developing agent comprises the following components: n-butanol: acetic acid: pyrimidine: water = 4: 1: 1: 2 (v/v), color developer: 0.2% ninhydrin was dissolved in acetone solution. The result shows that the hydrolysate has only one spot on the silica gel plate and is consistent with the glutamic acid standard solution, which indicates that the product is the polymer of the glutamic acid.
(2) Infrared spectrum analysis: mixing a small amount of the product of the invention with dry KBr powder, grinding and tabletting, and using a Fourier infrared spectrometer at 4000-400 cm-1The infrared absorption spectrum is analyzed by scanning within the range. The results show substantial agreement with the standard fuchsin extragram spectrum of gamma-polyglutamic acid.
(3) Nuclear magnetic resonance spectroscopy: by D2Dissolving the product of the invention into O to prepare 0.2mg/mL solution, and performing hydrogen spectrum scanning analysis by using a Fourier nuclear magnetic resonance spectrometer under the condition of 600 MHz. The result shows that the nuclear magnetic spectrum of the gamma-polyglutamic acid standard substance is basically consistent.
(4) Structural analysis of peptide chain: the product is prepared into 1mg/mL aqueous solution by deionized water, and an ultraviolet absorption spectrum of the product is scanned and analyzed by an ultraviolet visible spectrometer in the wavelength range of 190-390 nm, and the result shows that the product has a characteristic absorption peak at 216nm and has no absorption peak at 280 nm. In addition, when 5mg of the sample of the present invention was dissolved in 1mL of deionized water, and the biuret reagent (NaOH solution (0.1 g/mL): CuSO4 solution (0.01 g/mL) = 5: 1, v/v) was slowly added dropwise, the biuret coloration reaction was negative. The above results indicate that the product of the invention does not have a typical peptide chain structure.
(5) And (3) analysis of solubility: the sample of the invention is 5mg, and the result shows that the sample is easy to dissolve in water and insoluble in organic solvents such as methanol, ethanol or acetone.
Example 4: bacillus subtilis (A), (B) and (C)Bacillus subtilis) GXG-5 high-temperature fermentation for producing gamma-polyglutamic acid
(1) Activating and preserving strains: bacillus subtilis (A), (B) and (C)Bacillus subtilis) GXG-5 inoculating on solid slant culture medium, and culturing at 50 deg.C for 16 h; the concentration composition of the solid slant culture medium is as follows: 10g/L glucose, 5g/L yeast extract, 5g/L tryptone, 5g/L NaCl5g/L agar, 15g/L agar, pH 7.0 and distilled water.
(2) Preparing a seed solution: putting the inclined plane 1cm above2The lawn is inoculated into a 250mL triangular flask filled with 30mL liquid seed culture medium, the rotating speed of a shaking table is 180rpm, and the lawn is cultured for 16h at 50 ℃ until the logarithmic growth middle stage of the strain; the concentration of the liquid seed culture medium is as follows: 10g/L of glucose, 5g/L of yeast extract, 10g/L of tryptone, 10g/L of NaCl, 7.0 of pH value and distilled water.
(3) Liquid shake flask fermentation: inoculating the seed solution into a sterilized liquid fermentation culture medium, wherein the inoculation amount is 5%, the liquid loading amount in a shake flask is 50mL/250mL, the rotating speed of a shaking table is 180rpm, and the seed solution is cultured for 48h at 55 ℃; the concentration of the liquid fermentation culture medium comprises: 30g/L glucose, 5g/L soyabean peptone and NH4NO325g/L，KNO35g/L，NaCl 5g/L，K2HPO42.5g/L, pH 7.0, and distilled water.
(4) Determination of yield of gamma-polyglutamic acid: collecting the shake flask fermentation liquor, adding 3 times of distilled water for dilution, removing thallus by using a microfiltration membrane with the aperture of 0.45 mu m, concentrating the supernatant by using an ultrafiltration membrane with the molecular weight cutoff of 20KDa to 50% of the volume of the original fermentation liquor, adding industrial grade ethanol with the volume of 4 times of the original fermentation liquor for precipitation, collecting the precipitate, and freeze-drying to obtain the finished product of the gamma-polyglutamic acid. Weighing appropriate amount of the finished product, and converting into 15.6 + -0.5 g/L of gamma-polyglutamic acid in the fermentation liquid.
Nucleotide sequence listing
<110> Guangxi university
<120> a glutamic acid-independent producing bacterium for producing gamma-polyglutamic acid by high-temperature fermentation and a fermentation method thereof
<160>1
<170>PatentInversion3.5
<210>1
<211>1447
<212>DNA
<213> Bacillus subtilis
<400>1
acttccccca atcatctgtc ccaccttcgg cggctggctc ctaaaaggtt acctcaccga 60
cttcgggtgt tacaaactct cgtggtgtga cgggcggtgt gtacaaggcc cgggaacgta 120
ttcaccgcgg catgctgatc cgcgattact agcgattcca gcttcacgca gtcgagttgc 180
agactgcgat ccgaactgag aacagatttg tgggattggc ttaacctcgc ggtttcgctg 240
ccctttgttc tgtccattgt agcacgtgtg tagcccaggt cataaggggc atgatgattt 300
gacgtcatcc ccaccttcct ccggtttgtc accggcagtc accttagagt gcccaactga 360
atgctggcaa ctaagatcaa gggttgcgct cgttgcggga cttaacccaa catctcacga 420
cacgagctga cgacaaccat gcaccacctg tcactctgcc cccgaagggg acgtcctatc 480
tctaggattg tcagaggatg tcaagacctg gtaaggttct tcgcgttgct tcgaattaaa 540
ccacatgctc caccgcttgt gcgggccccc gtcaattcct ttgagtttca gtcttgcgac 600
cgtactcccc aggcggagtg cttaatgcgt tagctgcagc actaaggggc ggaaaccccc 660
taacacttag cactcatcgt ttacggcgtg gactaccagg gtatctaatc ctgttcgctc 720
cccacgcttt cgctcctcag cgtcagttac agaccagaga gtcgccttcg ccactggtgt780
tcctccacat ctctacgcat ttcaccgcta cacgtggaat tccactctcc tcttctgcac 840
tcaagttccc cagtttccaa tgaccctccc cggttgagcc gggggctttc acatcagact 900
taagaaaccg cctgcgagcc ctttacgccc aataattccg gacaacgctt gccacctacg 960
tattaccgcg gctgctggca cgtagttagc cgtggctttc tggttaggta ccgtcaaggt 1020
accgccctat tcgaacggta cttgttcttc cctaacaaca gagctttacg atccgaaaac 1080
cttcatcact cacgcggcgt tgctccgtca gactttcgtc cattgcggaa gattccctac 1140
tgctgcctcc cgtaggagtc tgggccgtgt ctcagtccca gtgtggccga tcaccctctc 1200
aggtcggcta cgcatcgttg ccttggtgag ccgttacctc accaactagc taatgcgccg 1260
cgggtccatc tgtaagtggt agccgaagcc accttttatg tttgaaccat gcggttcaaa 1320
caaccatccg gtattagccc cggtttcccg gagttatccc agtcttacag gcaggttacc 1380
cacgtgttac tcacccgtcc gccgctaaca tcagggagca agctcccatc tgtccgctcg 1440
acttgca 1447

Claims (7)

1. A glutamic acid-independent producing bacterium for producing gamma-polyglutamic acid by high-temperature fermentation, which is characterized in that: the bacterium is named as: bacillus subtilis (A), (B) and (C)Bacillus subtilis) GXG-5, the preservation number is CCTCC NO: m2017083, the preservation date is 3/6 in 2017, and the preservation unit: china center for type culture Collection, collection address: wuhan university in Wuhan, China.
2. The method for producing gamma-polyglutamic acid by high-temperature fermentation of the glutamic acid-independent producing bacterium as claimed in claim 1, wherein: the method comprises the following steps:
(1) activating strains: bacillus subtilis (A), (B) and (C)Bacillus subtilis) GXG-5 is inoculated on a solid slant culture medium and cultured for 12-24h at the temperature of 45-50 ℃;
(2) preparing a seed solution: 1-3 cm above the slant culture medium in the step (1)2Inoculating the lawn into a 250mL triangular flask filled with 30mL liquid seed culture medium, and culturing at 45-50 ℃ for 12-24h at the rotating speed of a shaker of 160-200 rpm until the logarithmic growth middle stage of the strain;
(3) liquid shake flask fermentation: inoculating the seed solution obtained in the step (2) into a sterilized liquid fermentation culture medium, wherein the inoculation amount is 1-10% (v/v), the liquid loading amount of a triangular flask is 20-80 mL/250mL, the rotating speed of a shaking table is 160-250 rpm, and the seed solution is cultured for 48-72 h at 40-55 ℃ to obtain mature fermentation liquid;
(4) extracting and purifying gamma-polyglutamic acid: and (3) collecting the mature fermentation liquor obtained in the step (3), adding 2-5 times of volume of distilled water for dilution, removing thalli by using a microfiltration membrane with the aperture of 0.45 mu m, concentrating the supernatant by using an ultrafiltration membrane with the molecular weight cutoff of 10-50 KDa until the volume of the supernatant is 30-50% of the volume of the original fermentation liquor, adding 2-4 times of volume of industrial grade ethanol for precipitation, collecting the precipitate, and freeze-drying to obtain the finished product of the gamma-polyglutamic acid.
3. The method for producing gamma-polyglutamic acid by high-temperature fermentation of the glutamic acid-independent producing bacteria according to claim 2, wherein: the concentration of the solid slant culture medium in the step (1) is as follows: 10-15 g/L of glucose, 2-5 g/L of yeast extract, 3-6 g/L of tryptone, 3-6 g/L of NaCl, 15-20 g/L of agar, 6.5-7.5 of pH value and distilled water.
4. The method for producing gamma-polyglutamic acid by high-temperature fermentation of the glutamic acid-independent producing bacteria according to claim 2, wherein: the concentration of the liquid seed culture medium in the step (2) is as follows: 10-20 g/L of glucose, 2-5 g/L of yeast extract, 5-10 g/L of tryptone, 5-10 g/L of NaCl, 6.5-7.5 of pH value and distilled water.
5. The method for producing gamma-polyglutamic acid by high-temperature fermentation of the glutamic acid-independent producing bacteria according to claim 2The method is characterized in that: the concentration of the liquid fermentation medium in the step (3) is composed of: 10-50 g/L of sugar raw material, 5-30 g/L of nitrogen source and KNO32~10g/L，NaCl 2~10g/L，K2HPO40.5-5 g/L, pH 6.5-7.5, and distilled water.
6. The method for producing gamma-polyglutamic acid by high-temperature fermentation of the glutamic acid-independent producing bacteria according to claim 5, wherein: the saccharic material in the liquid fermentation medium is any one or a mixture of glucose, sucrose, maltose, fructose and molasses.
7. The method for producing gamma-polyglutamic acid by high-temperature fermentation of the glutamic acid-independent producing bacteria according to claim 5, wherein: the nitrogen source in the liquid fermentation medium is soybean peptone, tryptone, yeast extract and NH4Cl、(NH4)2SO4And NH4NO3Any one or a mixture of several of them.
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