CN110628688A - Bacillus subtilis for producing gamma-polyglutamic acid and application thereof - Google Patents
Bacillus subtilis for producing gamma-polyglutamic acid and application thereof Download PDFInfo
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Abstract
The invention discloses Bacillus Subtilis for producing gamma-polyglutamic acid, which is classified and named as Bacillus Subtilis, has a strain number of XK, is preserved in China center for type culture collection, has a preservation number of CCTCC M2016605, and has a preservation date of 2016, 11 and 1 days. The invention also discloses application of the bacillus subtilis XK in production of gamma-polyglutamic acid. Compared with the prior art, the method takes the bacillus subtilis XK as a starting strain, obtains the gamma-polyglutamic acid with three different molecular weight regions of less than 100kDa, 100-700 kDa and more than 700kDa by adjusting the concentrations of manganese sulfate and magnesium sulfate in a fermentation medium, has the yield of more than 35g/L, and has good application in the fields of fertilizer, cosmetics, sewage treatment and the like. Meanwhile, the method is simple in process, suitable for industrial popularization, and good in market prospect and economic benefit.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to bacillus subtilis for producing gamma-polyglutamic acid and application thereof.
Background
Gamma-polyglutamic acid is an anionic high polymer formed by connecting D-glutamic acid monomer and L-glutamic acid monomer through an amido bond formed by alpha-amino and gamma-carboxyl. Because of its water solubility, biodegradability, edibility, and non-toxicity to humans and the environment, gamma-polyglutamic acid and its derivatives have been widely used in various fields in recent years, as flocculants in water treatment, ingredients in cosmetics and medicines, and as fertilizer additives, etc.
At present, the commercial gamma-polyglutamic acid is mainly produced by submerged liquid fermentation of microorganisms, and the commonly used production strain bacillus subtilis generates the gamma-polyglutamic acid extracellularly by taking monosodium glutamate as a main substrate. In general, gamma-polyglutamic acid produced by Bacillus subtilis has an average molecular weight of 105～8×106The larger the molecular weight between Da and the larger the rheological property, the more difficult the modification by chemical agents, and the limited the application of gamma-polyglutamic acid. The molecular weight of the gamma-polyglutamic acid is required to be different in different industry fields, the molecular weight of the gamma-polyglutamic acid is required to be more than 1000kDa by a flocculating agent in sewage treatment, more anions on the gamma-polyglutamic acid are used for attracting colloidal particles, and larger micelles are formed through adhesion and crosslinking so as to achieve the aim of flocculation; the molecular weight of gamma-polyglutamic acid is required to be about 700kDa in the cosmetic industry, and the gamma-polyglutamic acid is used as a humectant in a product to reduce the loss rate of moisture on the surface of skin; some products such as fertilizer additives require that the molecular weight of the gamma-polyglutamic acid is below 80kDa, and the gamma-polyglutamic acid is applied by being matched with common fertilizers, so that the utilization rate of the fertilizers is higher while a protective film is formed at the roots of plants.
The acid/base hydrolysis method is commonly used in industrial production to obtain low molecular weight gamma-polyglutamic acid. When the influence of pH on the structure of polyglutamic acid is studied, it is found that gamma-polyglutamic acid is a low molecular mass fragment at a low pH value, and after the acidic solution is neutralized to neutral pH, the fragment-like gamma-polyglutamic acid cannot return to a long-chain regular structure in a neutral state. Chinese patent CN 105296558A discloses a method for producing low molecular weight gamma-polyglutamic acid by adjusting and optimizing fermentation process, wherein in different fermentation stages, different culture temperatures, aeration flow rates and stirring speeds are controlled to obtain low molecular weight (35-260kDa) gamma-polyglutamic acid fermentation liquor. Chinese patent CN 105385717A discloses an application of iron ions in Bacillus subtilis fermentation to produce low molecular weight gamma-polyglutamic acid, so that the content of gamma-polyglutamic acid higher than 245kD in the total gamma-polyglutamic acid is reduced to 27.38% -4.01% from the original 66.16%, and the content of gamma-polyglutamic acid lower than 100kD in the total gamma-polyglutamic acid is increased to 33.13% -81.03% from the original 4.59%.
At present, the method for obtaining the low molecular weight gamma-polyglutamic acid is not complicated in subsequent process and has higher loss rate of the gamma-polyglutamic acid, but is low in proportion of the low molecular weight gamma-polyglutamic acid in the total polyglutamic acid. Therefore, there is a need for a method of obtaining gamma-polyglutamic acid having various molecular weights by adjusting the process during fermentation, and also having a high yield of low molecular weight gamma-polyglutamic acid.
Disclosure of Invention
The invention aims to solve the technical problem of providing bacillus subtilis for producing gamma-polyglutamic acid so as to solve the problems of low yield and the like in the prior art.
The technical problem to be solved by the invention is to provide the application of the bacillus subtilis for producing the gamma-polyglutamic acid.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
the Bacillus Subtilis for producing the gamma-polyglutamic acid is classified and named as Bacillus Subtilis, the strain number is XK, the Bacillus Subtilis is preserved in China center for type culture Collection, the address is Wuhan university, Wuhan City, China, the postal code is 430072, the preservation number is CCTCC NO: m2016605, preservation date 2016, 11/1/11/2016.
Most of the 16S rDNA sequence of the strain XK is 1420bp, which is shown in a nucleotide or amino acid sequence table. The sequences tested were compared from the correlations in the GenBank database to construct phylogenetic trees based on the full 16S rDNA sequence. The results show that: the homology of strain XK and Bacillus subtilis (Bacillus Subtilis) is 99%. The bacillus subtilis can be classified and positioned by combining the morphology, the cytochemical composition and the phylogenetic research result of 16S rDNA full-series analysis.
The application of the bacillus subtilis for producing the gamma-polyglutamic acid in producing the gamma-polyglutamic acid.
The application method comprises the following steps: sequentially inoculating bacillus subtilis for producing the gamma-polyglutamic acid into a seed culture medium and a fermentation culture medium for aerobic culture, and fermenting to obtain the gamma-polyglutamic acid.
The method for producing the gamma-polyglutamic acid by fermenting the bacillus subtilis XK for producing the gamma-polyglutamic acid comprises the following steps:
(1) preparing a seed solution: inoculating bacillus subtilis XK into a seed culture medium, and culturing at the rotating speed of 120-200 rpm of a shaking table at the temperature of 28-36 ℃ for 10-18 h until OD660 is greater than 5.0 to obtain a seed solution;
(2) fermentation culture: inoculating the seed solution obtained in the step (1) into a fermentation culture medium in an inoculation amount of 2-10%, wherein the liquid loading capacity of a fermentation culture medium of a 7.5L fermentation tank is 4L, the aeration ratio is controlled to be 1.0-1.5 VVM, the initial pH is 6.5-7.2, and the seed solution is cultured at 28-36 ℃ for 24-72 hours to obtain a fermentation liquid;
(3) diluting the fermentation liquor obtained in the step (2) (the concentration is below 1g/L), and detecting the molecular weight and the yield of the gamma-polyglutamic acid in the fermentation liquor by high performance liquid chromatography.
Wherein, the formula of the seed culture medium is as follows: 10-95 g/L of glutamic acid or glutamate, 15-100 g/L of carbon source, 5-50 g/L of nitrogen source, 0.05-35 g/L of other inorganic salts, and the balance of water, wherein the pH value is 6.5-7.2.
Wherein the formula of the fermentation medium is as follows: 10-95 g/L of glutamic acid or glutamate, 15-100 g/L of carbon source, 5-50 g/L of nitrogen source, 0-1 g/L of manganese sulfate, 0.05-2 g/L of magnesium sulfate, 0.05-35 g/L of other inorganic salts, the balance of water, and the pH value of the mixture is 6.5-7.2.
Wherein, in the seed culture medium and the fermentation culture medium,
the carbon source is any one or combination of more of glucose, sucrose, maltose, lactose, xylose, fructose, lactic acid, citric acid, glycerol and molasses, preferably the carbon source is glucose and/or citric acid, and further preferably citric acid;
the nitrogen source is beef extract, peptone, yeast extract, corn steep liquor, bean cake powder, cottonseed cake powder, urea, (NH)4)2SO4、NH4Cl and NH4NO3Preferably the nitrogen source is bean cake powder, yeast extract and (NH)4)2SO4Any one or a combination of several of the above, more preferably bean cake powder;
the other inorganic salt is any one or combination of more of phosphate, dihydric phosphate and hydrochloride.
Wherein the content of the first and second substances,
when the concentration of manganese sulfate is more than or equal to 0 and less than 0.1g/L and the concentration of magnesium sulfate is more than or equal to 1 and less than 2g/L, the molecular weight of the gamma-polyglutamic acid obtained by fermentation is less than 100kDa, and the optimal yield reaches 46.5 g/L; the gamma-polyglutamic acid with the molecular weight is matched with agricultural fertilizer for use, and has obvious yield increasing effect.
When the concentration of manganese sulfate is more than or equal to 0.1 and less than 0.5g/L and the concentration of magnesium sulfate is more than or equal to 0.5 and less than 1g/L, the molecular weight of the gamma-polyglutamic acid obtained by fermentation is more than or equal to 100 and less than 700kDa, and the optimal yield reaches 43.8 g/L; the gamma-polyglutamic acid with the molecular weight can be applied to cosmetics and has better moisturizing performance.
When the concentration of manganese sulfate is more than or equal to 0.5 and less than 1g/L and the concentration of magnesium sulfate is more than or equal to 0.05 and less than 0.5g/L, the molecular weight of the gamma-polyglutamic acid obtained by fermentation is more than 700kDa, and the optimal yield reaches 39.5 g/L; the gamma-polyglutamic acid with the molecular weight is used as a flocculating agent in sewage treatment, and has good effect.
Has the advantages that:
compared with the prior art, the method takes the bacillus subtilis XK as an initial strain, obtains the gamma-polyglutamic acid with three different molecular weight regions of less than 100kDa, 100-700 kDa and more than 700kDa by adjusting the concentrations of manganese sulfate and magnesium sulfate in a fermentation medium, and the yield reaches more than 35 g/L. Meanwhile, the method is simple in process, suitable for industrial popularization, and good in market prospect and economic benefit.
Detailed Description
The invention will be better understood from the following examples. However, those skilled in the art will readily appreciate that the description of the embodiments is only for illustrating the present invention and should not be taken as limiting the invention as detailed in the claims.
Example 1:
the method for producing the gamma-polyglutamic acid by fermenting the bacillus subtilis XK for producing the gamma-polyglutamic acid comprises the following specific steps:
(1) preparing a seed solution: inoculating bacillus subtilis to a fermentation medium containing sodium glutamate (45g/L), citric acid (50g/L), bean cake powder (40g/L), potassium dihydrogen phosphate (1g/L) and water, and culturing at a shaker rotation speed of 120-200 rpm and a temperature of 28-36 ℃ for 10-18 h to OD660Greater than 5.0;
(2) fermentation culture: inoculating the seed solution obtained in the step (1) into a fermentation medium containing sodium glutamate (45g/L), citric acid (50g/L), bean cake powder (40g/L), potassium dihydrogen phosphate (1g/L), manganese sulfate (0.05g/L) and magnesium sulfate (1.2g/L) in an inoculation amount of 3%, wherein the liquid loading amount of the fermentation medium in a 7.5L fermentation tank is 4L, the aeration ratio is controlled at 1.0vvm, the initial pH is 6.5, and the fermentation medium is cultured at 28 ℃ for 48 h;
(3) diluting the fermentation liquor (the concentration is below 1g/L), and detecting the molecular weight and the yield of the gamma-polyglutamic acid in the fermentation liquor by high performance liquid chromatography. The produced gamma-polyglutamic acid has a molecular weight of about 50kDa, and the yield of gamma-polyglutamic acid is 46.5 g/L.
Example 2:
the seed liquid was prepared as in example 1.
(1) Fermentation culture: inoculating the seed solution into a fermentation medium with the inoculation amount of 4%, wherein the fermentation medium contains sodium glutamate (65g/L), citric acid (34g/L), bean cake powder (20g/L), potassium dihydrogen phosphate (1g/L), manganese sulfate (0.09g/L) and magnesium sulfate (1.8g/L), the liquid loading amount of the fermentation medium in a 7.5L fermentation tank is 4L, the aeration ratio is controlled at 1.5vvm, the initial pH is 6.9, and the fermentation medium is cultured at 32 ℃ for 52 h;
(2) diluting the fermentation liquor (the concentration is below 1g/L), and detecting the molecular weight and the yield of the gamma-polyglutamic acid in the fermentation liquor by high performance liquid chromatography. The produced gamma-polyglutamic acid has a molecular weight of about 89kDa, and the yield of gamma-polyglutamic acid is 42.7 g/L.
Example 3:
the seed liquid was prepared as in example 1.
(1) Fermentation culture: inoculating the seed solution into a fermentation medium with the inoculation amount of 3%, wherein the fermentation medium contains sodium glutamate (51g/L), citric acid (29g/L), bean cake powder (37g/L), potassium dihydrogen phosphate (1g/L), manganese sulfate (0.02g/L) and magnesium sulfate (1.1g/L), the liquid loading amount of the fermentation medium in a 7.5L fermentation tank is 4L, the aeration ratio is controlled at 1.2vvm, the initial pH is 6.5, and the fermentation medium is cultured at 34 ℃ for 36 h;
(2) diluting the fermentation liquor (the concentration is below 1g/L), and detecting the molecular weight and the yield of the gamma-polyglutamic acid in the fermentation liquor by high performance liquid chromatography. The produced gamma-polyglutamic acid has a molecular weight of about 38kDa and a yield of 36.8 g/L.
Example 4:
the seed liquid was prepared as in example 1.
(1) Fermentation culture: inoculating the seed solution into a fermentation medium with the inoculation amount of 2%, wherein the fermentation medium contains sodium glutamate (45g/L), citric acid (50g/L), bean cake powder (40g/L), potassium dihydrogen phosphate (1g/L), manganese sulfate (0.2g/L) and magnesium sulfate (0.8g/L), the liquid loading amount of the fermentation medium in a 7.5L fermentation tank is 4L, the aeration ratio is controlled at 1.3vvm, the initial pH is 7.1, and the fermentation medium is cultured at 30 ℃ for 48 h;
(2) diluting the fermentation liquor (the concentration is below 1g/L), and detecting the molecular weight and the yield of the gamma-polyglutamic acid in the fermentation liquor by high performance liquid chromatography. The produced gamma-polyglutamic acid has a molecular weight of about 560kDa and a yield of 43.8 g/L.
Example 5:
the seed liquid was prepared as in example 1.
(1) Fermentation culture: inoculating the seed liquid into a fermentation medium with the inoculation amount of 2%, wherein the fermentation medium contains sodium glutamate (95g/L), citric acid (89g/L), bean cake powder (50g/L), potassium dihydrogen phosphate (0.5g/L), manganese sulfate (0.5g/L) and magnesium sulfate (1.0g/L), the liquid loading amount of the fermentation medium in a 7.5L fermentation tank is 4L, the aeration ratio is controlled at 1.3vvm, the initial pH is 7.0, and the fermentation medium is cultured at 30 ℃ for 52 h;
(2) diluting the fermentation liquor (the concentration is below 1g/L), and detecting the molecular weight and the yield of the gamma-polyglutamic acid in the fermentation liquor by high performance liquid chromatography. The produced gamma-polyglutamic acid has a molecular weight of about 340kDa, and the yield of gamma-polyglutamic acid is 40.9 g/L.
Example 6:
the seed liquid was prepared as in example 1.
(1) Fermentation culture: inoculating the seed solution into a fermentation medium with the inoculation amount of 8%, wherein the fermentation medium contains sodium glutamate (39g/L), citric acid (30g/L), bean cake powder (50g/L), potassium dihydrogen phosphate (1g/L), manganese sulfate (0.1g/L) and magnesium sulfate (0.5g/L), the liquid loading amount of the fermentation medium in a 7.5L fermentation tank is 4L, the aeration ratio is controlled at 1.0vvm, the initial pH is 7.1, and the fermentation medium is cultured at 30 ℃ for 72 h;
(2) diluting the fermentation liquor (the concentration is below 1g/L), and detecting the molecular weight and the yield of the gamma-polyglutamic acid in the fermentation liquor by high performance liquid chromatography. The produced gamma-polyglutamic acid has a molecular weight of about 190kDa, and the yield of gamma-polyglutamic acid is 35.2 g/L.
Example 7:
the seed liquid was prepared as in example 1.
(1) Fermentation culture: inoculating the seed solution into a fermentation medium containing sodium glutamate (45g/L), citric acid (50g/L), bean cake powder (40g/L), potassium dihydrogen phosphate (1g/L), manganese sulfate (0.6g/L) and magnesium sulfate (0.2g/L) in an inoculation amount of 5%, wherein the liquid loading amount of the fermentation medium in a 7.5L fermentation tank is 4L, the aeration ratio is controlled at 1.5vvm, the initial pH is 7.2, and the fermentation medium is cultured at 33 ℃ for 46 h;
(2) diluting the fermentation liquor (the concentration is below 1g/L), and detecting the molecular weight and the yield of the gamma-polyglutamic acid in the fermentation liquor by high performance liquid chromatography. The produced gamma-polyglutamic acid has a molecular weight of about 1190kDa, and the yield of gamma-polyglutamic acid is 39.5 g/L.
Example 8:
the seed liquid was prepared as in example 1.
(1) Fermentation culture: inoculating the seed solution into a fermentation medium containing sodium glutamate (83g/L), citric acid (51g/L), bean cake powder (39g/L), potassium dihydrogen phosphate (1g/L), manganese sulfate (1.0g/L) and magnesium sulfate (0.05g/L) in an inoculation amount of 2%, wherein the liquid loading amount of the fermentation medium in a 7.5L fermentation tank is 4L, the aeration ratio is controlled at 1.1vvm, the initial pH is 7.1, and the fermentation medium is cultured at 35 ℃ for 68 h;
(2) diluting the fermentation liquor (the concentration is below 1g/L), and detecting the molecular weight and the yield of the gamma-polyglutamic acid in the fermentation liquor by high performance liquid chromatography. The produced gamma-polyglutamic acid has a molecular weight of about 960kDa, and the yield of gamma-polyglutamic acid is 38.1 g/L.
Example 9:
the seed liquid was prepared as in example 1.
(1) Fermentation culture: inoculating the seed solution into a fermentation medium containing sodium glutamate (49g/L), citric acid (59g/L), bean cake powder (43g/L), potassium dihydrogen phosphate (1g/L), manganese sulfate (0.5g/L) and magnesium sulfate (0.1g/L) at an inoculation amount of 5%, wherein the liquid loading amount of the fermentation medium in a 7.5L fermentation tank is 4L, the aeration ratio is controlled at 1.5vvm, the initial pH is 7.2, and the fermentation medium is cultured at 33 ℃ for 46 h;
(2) diluting the fermentation liquor (the concentration is below 1g/L), and detecting the molecular weight and the yield of the gamma-polyglutamic acid in the fermentation liquor by high performance liquid chromatography. The molecular weight of the produced gamma-polyglutamic acid is about 780kDa, and the yield of the gamma-polyglutamic acid is 35.4 g/L.
Example 10: utilization of gamma-polyglutamic acid in fertilizer
The experiment is set as 3 treatments with potted brassica chinensis as an experimental object, wherein the treatment 1 is not treated as a blank control, the treatment 2 is applied with a common fertilizer, and the treatment 3 is applied with a common fertilizer containing 0.2% of gamma-polyglutamic acid fermentation liquor, wherein the added gamma-polyglutamic acid fermentation liquor is the gamma-polyglutamic acid with the molecular weight of 50kDa obtained in the example 1.
TABLE 1 Effect of different fertilizers on growth characteristics and yield of potted Brassica chinensis
| Treatment of | Number of leaves | Specific gravity (g) |
| 1 | 13±1 | 130±4 |
| 2 | 15±1 | 139±5 |
| 3 | 17±1 | 148±4 |
The growth of the small green vegetables is obviously influenced by applying the common fertilizer, the leaf number and the particle weight of the small green vegetables can be increased, and the effect of the fertilizer can be exerted by matching the gamma-polyglutamic acid with the fertilizer, so that the yield increasing effect of the small green vegetables is more obvious.
Example 11: gamma-polyglutamic acid used as moisturizer in cosmetics
The gamma-polyglutamic acid used in this example was the gamma-polyglutamic acid having a molecular weight of 560kDa obtained in example 4. The fermentation broth of example 4 was subjected to half-frame filtration, alcohol precipitation, and vacuum drying to obtain gamma-polyglutamic acid powder, which was then compared with the water retentivity of hyaluronic acid and collagen.
Anhydrous cosmetic formulation according to patent CN 105411975 a: 1-6% of gamma-polyglutamic acid, 11-15% of dispersing agent, 29-32% of stabilizing agent and 50-58% of oily matrix. Specifically, the gamma-polyglutamic acid group cosmetic uses 4% of the gamma-polyglutamic acid with the molecular weight of 560kDa obtained in example 4 as a humectant, 13% of caprylin as a dispersant, 30% of polydimethylsiloxane as a stabilizer and 53% of glycerol as an oily substrate; the specific cosmetic for the hyaluronic acid group uses 4% of hyaluronic acid as a humectant, 13% of caprylin as a dispersant, 30% of polydimethylsiloxane as a stabilizer and 53% of glycerol as an oily matrix; the specific collagen group cosmetic uses 4% of collagen as a humectant, 13% of caprylin as a dispersant, 30% of polydimethylsiloxane as a stabilizer and 53% of glycerol as an oily matrix. The three groups of cosmetics were equally applied to 3 pieces of skin of the same size on the arms of the experimenters, respectively, and the moisture content was measured using a skin moisture tester SK-08, and the ratio of the measured moisture content to the moisture content of the skin was P% as shown in table 2:
TABLE 2 Effect of three groups of cosmetics on skin moisturizing Properties
| Group of | Gamma-polyglutamic acid | Hyaluronic acid | Collagen protein |
| P | 59.7±2.5 | 51.3±1.9 | 48.5±2.3 |
Note: values are expressed as mean. + -. standard deviation
As can be seen from Table 2, the cosmetic composition containing 560kDa gamma-polyglutamic acid as humectant has the best moisturizing effect on skin, which indicates that the molecular weight gamma-polyglutamic acid has the potential to replace hyaluronic acid and collagen in cosmetic aspect.
Example 12: gamma-polyglutamic acid is used as flocculant in sewage treatment
The gamma-polyglutamic acid used in this example was the 1190kDa molecular weight gamma-polyglutamic acid obtained in example 7.
495mL of distilled water and 5mL of the gamma-polyglutamic acid fermentation broth obtained in example 7 were added to a 1L beaker, and 2.0g of calcium hydroxide was precisely weighed, poured into the beaker, adjusted to pH 7.4 while stirring for 5 minutes, and left to stand for 10 minutes. A liquid layer of half the height of the liquid was sucked up with a plastic dropper, the absorbance at 570nm was measured, the flocculation activity of the flocculant was determined with a calcium hydroxide suspension without addition of a γ -polyglutamic acid solution as a control, and the flocculation rate calculated from the measured absorbance was 64.5% in terms of the flocculation rate. The specific calculation method is shown in the reference (the university of Zhejiang university of Industrial science, vol.36, 2008, pp.647-650, 4 pages in total).
Sequence listing
<110> Nanjing Xuan Kai Biotech Co., Ltd
<120> bacillus subtilis for producing gamma-polyglutamic acid and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
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<212> DNA
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tgcaagtcga gcggacagat gggagcttgc tccctgatgt tagcggcgga cgggtgagta 60
acacgtgggt aacctgcctg taagactggg ataactccgg gaaaccgggg ctaataccgg 120
atggttgttt gaaccgcatg gttcaaacat aaaaggtggc ttcggctacc acttacagat 180
ggacccgcgg cgcattagct agttggtgag gtaacggctc accaaggcaa cgatgcgtag 240
ccgacctgag agggtgatcg gccacactgg gactgagaca cggcccagac tcctacggga 300
ggcagcagta gggaatcttc cgcaatggac gaaagtctga cggagcaacg ccgcgtgagt 360
gatgaaggtt ttcggatcgt aaagctctgt tgttagggaa gaacaagtac cgttcgaata 420
gggcggtacc ttgacggtac ctaaccagaa agccacggct aactacgtgc cagcagccgc 480
ggtaatacgt aggtggcaag cgttgtccgg aattattggg cgtaaagggc tcgcaggcgg 540
tttcttaagt ctgatgtgaa agcccccggc tcaaccgggg agggtcattg gaaactgggg 600
aacttgagtg cagaagagga gagtggaatt ccacgtgtag cggtgaaatg cgtagagatg 660
tggaggaaca ccagtggcga aggcgactct ctggtctgta actgacgctg aggagcgaaa 720
gcgtggggag cgaacaggat tagataccct ggtagtccac gccgtaaacg atgagtgcta 780
agtgttaggg ggtttccgcc ccttagtgct gcagctaacg cattaagcac tccgcctggg 840
gagtacggtc gcaagactga aactcaaagg aattgacggg ggcccgcaca agcggtggag 900
catgtggttt aattcgaagc aacgcgaaga accttaccag gtcttgacat cctctgacaa 960
tcctagagat aggacgtccc cttcgggggc agagtgacag gtggtgcatg gttgtcgtca 1020
gctcgtgtcg tgagatgttg ggttaagtcc cgcaacgagc gcaacccttg atcttagttg 1080
ccagcattca gttgggcact ctaaggtgac tgccggtgac aaaccggagg aaggtgggga 1140
tgacgtcaaa tcatcatgcc ccttatgacc tgggctacac acgtgctaca atggacagaa 1200
caaagggcag cgaaaccgcg aggttaagcc aatcccacaa atctgttctc agttcggatc 1260
gcagtctgca actcgactgc gtgaagctgg aatcgctagt aatcgcggat cagcatgccg 1320
cggtgaatac gttcccgggc cttgtacaca ccgcccgtca caccacgaga gtttgtaaca 1380
cccgaagtcg gtgaggtaac cttttaggag ccagccgccg 1420
Claims (10)
1. The Bacillus Subtilis for producing the gamma-polyglutamic acid is classified and named as Bacillus Subtilis, has a strain number of XK, is preserved in China center for type culture collection, and has a preservation number of CCTCC NO: m2016605 with a collection date of 2016, 11, 1.
2. Use of the Bacillus subtilis for producing gamma-polyglutamic acid according to claim 1 for producing gamma-polyglutamic acid.
3. The application of claim 2, wherein the application method comprises: sequentially inoculating bacillus subtilis for producing the gamma-polyglutamic acid into a seed culture medium and a fermentation culture medium for aerobic culture, and fermenting to obtain the gamma-polyglutamic acid.
4. The use according to claim 3, wherein the seed medium is formulated as: 10-95 g/L of glutamic acid or glutamate, 15-100 g/L of carbon source, 5-50 g/L of nitrogen source, 0.05-35 g/L of other inorganic salts, and the balance of water, wherein the pH value is 6.5-7.2.
5. The use according to claim 3, wherein the fermentation medium is formulated as: 10-95 g/L of glutamic acid or glutamate, 15-100 g/L of carbon source, 5-50 g/L of nitrogen source, 0-1 g/L of manganese sulfate, 0.05-2 g/L of magnesium sulfate, 0.05-35 g/L of other inorganic salts, the balance of water, and the pH value of the mixture is 6.5-7.2.
6. The use according to claim 4 or 5, wherein the carbon source is any one or a combination of glucose, sucrose, maltose, lactose, xylose, fructose, lactic acid, citric acid, glycerol and molasses.
7. The use according to claim 4 or 5, wherein the nitrogen source is beef extract, peptone, yeast extract, corn steep liquor, soybean meal, cottonseed meal, urea, (NH)4)2SO4、NH4Cl and NH4NO3Any one or a combination of several of them.
8. The use according to claim 4 or 5, wherein the other inorganic salt is any one or a combination of phosphate, dihydrogen phosphate and hydrochloride.
9. The use according to claim 5,
when the concentration of manganese sulfate is more than or equal to 0 and less than 0.1g/L and the concentration of magnesium sulfate is more than or equal to 1 and less than 2g/L, the molecular weight of the gamma-polyglutamic acid obtained by fermentation is less than 100 kDa;
when the concentration of manganese sulfate is more than or equal to 0.1 and less than 0.5g/L and the concentration of magnesium sulfate is more than or equal to 0.5 and less than 1g/L, the molecular weight of the gamma-polyglutamic acid obtained by fermentation is more than or equal to 100 and less than 700 kDa;
when the concentration of manganese sulfate is more than or equal to 0.5 and less than 1g/L and the concentration of magnesium sulfate is more than or equal to 0.05 and less than 0.5g/L, the molecular weight of the gamma-polyglutamic acid obtained by fermentation is more than 700 kDa.
10. The use according to claim 3, wherein the culture temperature is 28-36 ℃ and the culture time is 10-18 h in the seed culture medium; and (3) culturing in a fermentation medium at the temperature of 28-36 ℃ for 24-72 h.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111876364A (en) * | 2020-09-10 | 2020-11-03 | 南京轩凯生物科技有限公司 | Bacillus subtilis and method for regulating and controlling molecular weight of fermentation product gamma-polyglutamic acid |
| CN114634894A (en) * | 2022-03-31 | 2022-06-17 | 安徽粤智徽源生物科技有限公司 | Bacillus subtilis for producing gamma-polyglutamic acid by salt-tolerant fermentation and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109929778B (en) * | 2019-03-08 | 2020-11-17 | 湖北中烟工业有限责任公司 | Efficient aroma-enhancing strain and application thereof in improving tobacco quality |
| CN110373346B (en) * | 2019-06-10 | 2023-05-23 | 上海市农业科学院 | Bacillus subtilis sp.A-5 and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1644677A (en) * | 2004-12-29 | 2005-07-27 | 浙江大学 | Bacillus and its use of preparation of gama-polycysteine |
| 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 |
| CN104232504A (en) * | 2014-07-10 | 2014-12-24 | 樟树市狮王生物科技有限公司 | Strain for synthesizing gamma-polyglutamic acid and method for efficiently preparing gamma-polyglutamic acid by using strain |
| CN104830713A (en) * | 2015-04-09 | 2015-08-12 | 河南工业大学 | Bacillus thuringiensis for combined production of antimicrobial lipopeptide and gamma-polyglutamic acid |
| CN105385718A (en) * | 2014-05-21 | 2016-03-09 | 武汉慧宝康源医学研究有限责任公司 | Fermentation preparation method of gamma-polyglutamic acid and strain |
-
2016
- 2016-12-23 CN CN201911094540.XA patent/CN110628688A/en active Pending
- 2016-12-23 CN CN201611205616.8A patent/CN107881124A/en active Pending
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1644677A (en) * | 2004-12-29 | 2005-07-27 | 浙江大学 | Bacillus and its use of preparation of gama-polycysteine |
| 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 |
| CN105385718A (en) * | 2014-05-21 | 2016-03-09 | 武汉慧宝康源医学研究有限责任公司 | Fermentation preparation method of gamma-polyglutamic acid and strain |
| CN104232504A (en) * | 2014-07-10 | 2014-12-24 | 樟树市狮王生物科技有限公司 | Strain for synthesizing gamma-polyglutamic acid and method for efficiently preparing gamma-polyglutamic acid by using strain |
| CN104830713A (en) * | 2015-04-09 | 2015-08-12 | 河南工业大学 | Bacillus thuringiensis for combined production of antimicrobial lipopeptide and gamma-polyglutamic acid |
Non-Patent Citations (2)
| Title |
|---|
| JIE CHEN ET AL.: "Effects of Cultivation Conditions on the Production of γ-PGA with Bacillus subtilis ZJU-7", 《APPLY BIOCHEMICAL BIOTECHNOLOGY》 * |
| 王卫国等: "γ-聚谷氨酸的研究及应用进展", 《河南工业大学学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111876364A (en) * | 2020-09-10 | 2020-11-03 | 南京轩凯生物科技有限公司 | Bacillus subtilis and method for regulating and controlling molecular weight of fermentation product gamma-polyglutamic acid |
| CN111876364B (en) * | 2020-09-10 | 2021-07-23 | 南京轩凯生物科技股份有限公司 | Bacillus subtilis and method for regulating and controlling molecular weight of fermentation product gamma-polyglutamic acid |
| CN114634894A (en) * | 2022-03-31 | 2022-06-17 | 安徽粤智徽源生物科技有限公司 | Bacillus subtilis for producing gamma-polyglutamic acid by salt-tolerant fermentation and application thereof |
| CN114634894B (en) * | 2022-03-31 | 2024-04-16 | 安徽粤智徽源生物科技有限公司 | Bacillus subtilis for realizing salt-tolerant fermentation production of gamma-polyglutamic acid and application thereof |
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