CN115786186A - Bacillus licheniformis GXU-1 and application thereof in preparation of gamma-polyglutamic acid - Google Patents
Bacillus licheniformis GXU-1 and application thereof in preparation of gamma-polyglutamic acid Download PDFInfo
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Abstract
The invention belongs to the technical field of microbial fermentation, and particularly relates to a bacillus licheniformis GXU-1 and application thereof in preparation of gamma-polyglutamic acid. The invention provides a strain of Bacillus licheniformis GXU-1 which is biologically preserved with the preservation number of CGMCC No.24578. The bacillus licheniformis GXU-1 can produce gamma-polyglutamic acid with high yield, the concentration of the polyglutamic acid produced by fermentation in a 24-hour fermentation period can reach more than 50g/L, and the polyglutamic acid can be produced in a glutamic acid independent mode, so that a strain for efficiently and cheaply preparing the gamma-polyglutamic acid is provided for industrial production.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to a bacillus licheniformis GXU-1 and application thereof in preparation of gamma-polyglutamic acid.
Background
gamma-Polyglutamic acid (gamma-PGA) is a homo-polyamide formed by the gamma-amide bond linkage between alpha-amino and gamma-carboxyl groups in D-glutamic acid and L-glutamic acid, and the relative molecular mass is usually between 100 and 1000 KDa. The gamma-PGA has a large amount of free hydrophilic carboxyl, so that the gamma-PGA has the characteristics of excellent water and fertilizer retention, chelating property and the like, and can be widely applied to the fields of daily chemicals, food, medicine, environmental protection, agriculture and the like as a water and fertilizer retention agent, a flocculating agent, a food additive and the like.
The production method of the gamma-PGA comprises methods of chemical synthesis, enzymatic conversion, microbial fermentation synthesis and the like, the existing production method mainly comprises microbial fermentation, and compared with the chemical synthesis method and the enzymatic conversion method, the microbial fermentation method has the advantages of short production period, mild conditions, simple process, low production cost and the like. Strains for producing γ -PGA are classified into glutamate-dependent strains and glutamate-independent strains according to whether addition of glutamic acid is required in the fermentation medium. The glutamic acid independent type gamma-PGA synthetic strain does not need to add glutamic acid externally, the fermentation cost of the gamma-PGA is reduced to a certain extent, but the yield of the gamma-polyglutamic acid is much smaller than that of the glutamic acid dependent type strain for fermentation production of the polyglutamic acid. Currently, most of the glutamic acid-independent strains studied in gamma-polyglutamic acid producing bacteria are mainly bacillus licheniformis ATCC9945A, but the yield is low. Therefore, in terms of the present situation, glutamate-dependent strains are still a hot spot for the microbial fermentation synthesis of γ -PGA. The patent application number 202010649532.3 discloses a gamma-polyglutamic acid production strain Bacillus bleekensis LT-2, wherein the yield of the gamma-polyglutamic acid synthesized by fermenting with non-grain raw material sugarcane juice as a carbon source, peanut cake powder as an organic nitrogen source and ammonium chloride as an inorganic nitrogen source can reach 47.61g/L. However, the method needs fed-batch, has complex steps, large carbon source consumption and high production cost. The patent publication No. CN107022580A discloses a method for preparing gamma-polyglutamic acid by using gamma-polyglutamic acid producing bacteria, and the yield of the polyglutamic acid produced by using gamma-PGA producing bacteria, namely bacillus licheniformis CICC10099 can reach 40g/L. The patent publication No. CN110016446A discloses a Bacillus belgii PG1-2 and a method for producing polyglutamic acid by using the same, wherein the highest concentration of the produced polyglutamic acid exceeds 40g/L. The patent publication number is CN101603015A, the invention discloses a Bacillus licheniformis WX-02 strain and a method for producing polyglutamic acid by using the same, and the yield of the prepared polyglutamic acid can reach 35.05g/L.
In the invention patents disclosed above, the strains used are mainly Bacillus licheniformis, bacillus subtilis, and Bacillus belgii. Currently, the glutamic acid independent strain produces the polyglutamic acid by bacillus licheniformis in a more reported manner, but the yield is generally lower.
Disclosure of Invention
In order to solve the problems, the invention provides a bacillus licheniformis GXU-1 and application thereof in preparation of gamma-polyglutamic acid. The bacillus licheniformis GXU-1 strain provided by the invention is a glutamic acid independent strain, but can also rely on glutamic acid to produce gamma-polyglutamic acid, the yield of the polyglutamic acid is high, and the concentration of the polyglutamic acid produced by fermentation in a fermentation period of 24h can reach over 50g/L.
In order to achieve the above purpose, the invention provides the following technical scheme:
the invention provides a strain of Bacillus licheniformis (Bacillus Paralicheniformis) GXU-1, wherein the preservation number of the Bacillus licheniformis GXU-1 is CGMCC No.24578.
The invention also provides application of the bacillus licheniformis GXU-1 in the scheme in preparation of gamma-polyglutamic acid.
The invention also provides a method for preparing gamma-polyglutamic acid, which comprises the following steps:
inoculating the seed solution containing the bacillus licheniformis GXU-1 in the scheme into a fermentation culture medium, and performing aerobic fermentation to obtain gamma-polyglutamic acid;
the fermentation medium bagComprises 20 to 150g/L of carbon source, 3 to 15g/L of nitrogen source, 0 to 90g/L of sodium glutamate and K 2 HPO 4 2.5g/L and the balance of distilled water.
Preferably, the aerobic fermentation mode comprises shake flask fermentation or fermentation tank fermentation;
when the aerobic fermentation mode is shake flask fermentation, the fermentation conditions comprise: the temperature is 30-45 ℃, the rotating speed is 150-220 rpm, and the time is 12-36 h;
when the aerobic fermentation mode is fermentation in a fermentation tank, the fermentation conditions comprise: the temperature is 30-45 ℃, the stirring speed is 150-250 rpm, the time is 24-48 h, and the ventilation volume is 0.8-1.2 vvm.
Preferably, the volume ratio of the seed liquid to the fermentation medium in the inoculation is (1-15): 100.
preferably, the initial pH of the fermentation medium is between 6.0 and 7.5.
Preferably, the preparation method of the seed liquid comprises the following steps: inoculating the single colony of the bacillus licheniformis GXU-1 in the scheme into a seed culture medium, and performing shake culture at the temperature of 30-37 ℃ and the rpm of 150-20 for 12-18 h to the middle logarithmic growth phase.
Preferably, the seed culture medium comprises 20g/L glucose, 10g/L peptone, 3g/L beef extract, 5g/L NaCl and the balance of distilled water, and the initial pH is 7.0.
Preferably, the carbon source comprises one or more of glucose, sucrose, maltose, fructose, lactose and starch;
the nitrogen source comprises one or more of peptone, beef extract, yeast extract, ammonium chloride, ammonium sulfate, soybean cake powder and corn steep liquor.
Has the advantages that:
the invention provides a strain of Bacillus licheniformis GXU-1, wherein the preservation number of the Bacillus licheniformis GXU-1 is CGMCC No.24578. The invention separates 1 strain of Bacillus licheniformis GXU-1 from soil, the strain is a glutamic acid independent strain, but can rely on glutamic acid to produce gamma-polyglutamic acid, the yield of the gamma-polyglutamic acid is high, the concentration of the polyglutamic acid produced by fermentation in 24h fermentation period can reach more than 50g/L, and the invention provides a strain for efficiently and cheaply preparing the gamma-polyglutamic acid for industrial production.
Biological preservation information
Bacillus licheniformis (Bacillus paracasei) GXU-1, which was deposited at China general microbiological culture Collection center (CGMCC) at 23/3/2022, accession No.: west road No.1, north chen, chaoyang district, beijing, zip code: 100101, preservation number is CGMCC No.24578.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required in the embodiments will be briefly described below.
FIG. 1 is a diagram showing the morphological identification result of Bacillus licheniformis (Bacillus paraleniformis) GXU-1;
FIG. 2 is a phylogenetic tree of Bacillus licheniformis (Bacillus paracaseformis) GXU-1.
Detailed Description
The invention provides a strain of Bacillus licheniformis GXU-1, wherein the preservation number of the Bacillus licheniformis GXU-1 is CGMCC No.24578.
The bacillus licheniformis GXU-1 is obtained by screening contaminated soil rich in glutamic acid, the 16S rDNA sequence of the bacillus licheniformis GXU-1 is preferably shown in SEQ ID NO.1, and specifically comprises the following steps:
5’-CTGGGGGGGGTGCTATACATGCAGTCGAGCGGACAGATGGGAGCTTGCTCCCTGATGTCAGCGGCGGACGGGTGAGTAACACGTGGGTAACCTGCCTGTAAGACTGGGATAACTCCGGGAAACCGGGGCTAATACCGGATGCTTGATTGAACCGCATGGTTCAATTATAAAAGGTGGCTTTTAGCTACCACTTACAGATGGACCCGCGGCGCATTAGCTAGTTGGTGAGGTAACGGCTCACCAAGGCAACGATGCGTAGCCGACCTGAGAGGGTGATCGGCCACACTGGGACTGAGACACGGCCCAGACTCCTACGGGAGGCAGCAGTAGGGAATCTTCCGCAATGGACGAAAGTCTGACGGAGCAACGCCGCGTGAGTGATGAAGGTTTTCGGATCGTAAAACTCTGTTGTTAGGGAAGAACAAGTACCGTTCGAATAGGGCGGTACCTTGACGGTACCTAACCAGAAAGCCACGGCTAACTACGTGCCAGCAGCCGCGGTAATACGTAGGTGGCAAGCGTTGTCCGGAATTATTGGGCGTAAAGCGCGCGCAGGCGGTTTCTTAAGTCTGATGTGAAAGCCCCCGGCTCAACCGGGGAGGGTCATTGGAAACTGGGGAACTTGAGTGCAGAAGAGGAGAGTGGAATTCCACGTGTAGCGGTGAAATGCGTAGAGATGTGGAGGAACACCAGTGGCGAAGGCGACTCTCTGGTCTGTAACTGACGCTGAGGCGCGAAAGCGTGGGGAGCGAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGAGTGCTAAGTGTTAGAGGGTTTCCGCCCTTTAGTGCTGCAGCAAACGCATTAAGCACTCCGCCTGGGGAGTACGGTCGCAAGACTGAAACTCAAAGGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACGCGAAGAACCTTACCAGGTCTTGACATCCTCTGACAACCCTAGAGATAGGGCTTCCCCTTCGGGGGCAGAGTGACAGGTGGTGCATGGTTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCGCAACGAGCGCAACCCTTGATCTTAGTTGCCAGCATTCAGTTGGGCACTCTAAGGTGACTGCCGGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAATCATCATGCCCCTTATGACCTGGGCTACACACGTGCTACAATGGGCAGAACAAAGGGCAGCGAAGCCGCGAGGCTAAGCCAATCCCACAAATCTGTTCTCAGTTCGGATCGCAGTCTGCAACTCGACTGCGTGAAGCTGGAATCGCTAGTAATCGCGGATCAGCATGCCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCACGAGAGTTTGTAACACCCGAAGTCGGTGAGGTAACCTTTTGGAGCCAGCCGCCGAAGGGGATCG-3’。
the bacterial colony of the bacillus licheniformis GXU-1 on the LB solid culture medium is irregular in shape, irregular in edge, moist in lawn, rough in surface, grey white and opaque (as shown in figure 1). The bacterial cells are short rod-shaped, gram-staining positive, and can decompose and utilize glucose, sucrose, lactose, maltose, fructose and mannitol, can hydrolyze starch, can produce acid by using glucose, has negative oxidase reaction, positive nitrate reduction reaction, negative citrate utilization test and positive gelatin liquefaction test.
The bacillus licheniformis GXU-1 is a glutamic acid independent strain, can rely on glutamic acid to produce gamma-polyglutamic acid at the same time, can produce gamma-polyglutamic acid with high yield, and has wide market application prospect.
According to the physiological and biochemical characteristics of the bacillus parabilis GXU-1, the invention also provides the application of the bacillus parabilis GXU-1 in the technical scheme in the preparation of gamma-polyglutamic acid.
The invention also provides a method for preparing gamma-polyglutamic acid, which comprises the following steps:
inoculating a seed solution containing the bacillus licheniformis GXU-1 of claim 1 into a fermentation culture medium, and performing aerobic fermentation to obtain gamma-polyglutamic acid;
the hairThe fermentation culture medium comprises 20-150 g/L of carbon source, 3-15 g/L of nitrogen source, 0-90 g/L of sodium glutamate and K 2 HPO 4 2.5g/L and the balance of distilled water.
The preparation method of the seed liquid of the bacillus licheniformis GXU-1 preferably comprises the following steps: inoculating the activated and cultured bacillus licheniformis GXU-1 in the technical scheme into a seed culture medium, fermenting and culturing to the middle logarithmic growth phase, and obtaining the seed solution of the bacillus licheniformis GXU-1.
In the present invention, the activation culture preferably comprises: inoculating a single colony of the bacillus licheniformis GXU-1 into an activated culture medium for culture.
In the invention, the culture medium of the activation culture is preferably LB solid culture medium, the components of the culture medium preferably comprise peptone 10g/L, yeast powder 5g/L, naCl 10g/L, agar powder 20g/L and the balance of distilled water, the initial pH value is preferably 7.0, and the culture medium is preferably prepared by sterilizing at 121 ℃ for 30min.
The activation culture method of the bacillus licheniformis GXU-1 preferably comprises the following steps: inoculating single colony of Bacillus licheniformis GXU-1 in slant activating culture medium, and culturing at 37 deg.C for 12 hr.
In the present invention, the fermentation temperature in preparing the seed liquid is preferably 30 to 37 ℃, more preferably 32 to 36 ℃, and still more preferably 35 ℃; the fermentation time for preparing the seed liquid is preferably 12 to 18 hours, more preferably 15 to 18 hours, and even more preferably 18 hours; the fermentation is preferably performed by stirring with shaking at a rotation speed of preferably 150 to 200rpm, more preferably 180 to 200rpm, and still more preferably 200rpm, when the seed liquid is prepared.
In the invention, the seed culture medium preferably comprises 20g/L of glucose, 10g/L of peptone, 3g/L of beef extract, 5g/L of NaCl and the balance of distilled water, the initial pH value of the seed culture medium is preferably 7.0, and the seed culture medium is preferably prepared by sterilizing at 115 ℃ for 20 min. The source of each component in the seed culture medium is not particularly required by the invention, and commercial products well known to those skilled in the art can be adopted.
After the seed solution of the bacillus licheniformis GXU-1 is obtained, the invention preferably inoculates the seed solution of the bacillus licheniformis GXU-1 into a fermentation culture medium for aerobic fermentation to obtain a culture rich in gamma-polyglutamic acid. In the present invention, the volume ratio of the seed liquid to the fermentation medium is preferably (1 to 15): 100, more preferably (5 to 10): 100, more preferably 8:100.
in the present invention, the aerobic fermentation mode preferably comprises shake flask fermentation or fermenter fermentation.
In the present invention, when the aerobic fermentation is shake flask fermentation, the fermentation temperature is preferably 30 to 45 ℃, more preferably 35 to 40 ℃, and even more preferably 37 ℃; the fermentation time is preferably 12 to 36 hours, more preferably 18 to 30 hours, and even more preferably 24 hours; the liquid loading amount of the shake flask fermentation is preferably 1/5-2/5, and more preferably 1/5; the shaking flask fermentation preferably has an oscillation speed of 150 to 220rpm, more preferably 160 to 220rpm, and still more preferably 200rpm.
In the present invention, when the aerobic fermentation is a fermentor fermentation, the fermentation temperature is preferably 30 to 45 ℃, more preferably 35 to 40 ℃, and still more preferably 37 ℃; the fermentation time is preferably 24 to 48 hours, more preferably 30 to 40 hours, and even more preferably 36 hours; the fermentation culture in the fermentation tank preferably also comprises aeration treatment and stirring; the ventilation volume of the ventilation treatment is preferably 0.8 to 1.2vvm, and more preferably 1.0vvm; the stirring speed is preferably 150 to 250rpm, more preferably 160 to 220rpm, still more preferably 170 to 220rpm, and most preferably 220rpm.
In the present invention, the fermentation medium preferably includes a fermentation medium containing sodium glutamate and a fermentation medium containing no sodium glutamate.
In the present invention, the fermentation medium containing sodium glutamate preferably comprises 20 to 150g/L of a carbon source, 3 to 15g/L of a nitrogen source, 10 to 90g/L of sodium glutamate, and K 2 HPO 4 2.5g/L and the balance of distilled water, the initial pH is 6.0-7.5, and the mixture is sterilized for 20min at 115 ℃; the content of the carbon source is preferably 25-75 g/L or 80-140 g/L, more preferably 30-70 g/L or 85-120 g/L, more preferably 40-60 g/L or 90-110 g/L, and most preferably 50g/L or 100g/L; the nitrogen source is preferably 5 to 12g/L, more preferably 6 to 10g/L, and still more preferablyIs 8g/L; the sodium glutamate is preferably 20 to 90g/L, and more preferably 50g/L.
In the present invention, the fermentation medium containing no sodium glutamate preferably comprises 20 to 150g/L of a carbon source, 3 to 15g/L of a nitrogen source, and K 2 HPO 4 2.5g/L and the balance of distilled water, the initial pH value is 6.0-7.5, and the mixture is sterilized for 20min at 115 ℃; the content of the carbon source is preferably 25-75 g/L or 80-140 g/L, more preferably 30-70 g/L or 85-120 g/L, more preferably 40-60 g/L or 90-110 g/L, and most preferably 50g/L or 100g/L; the nitrogen source is preferably 5 to 12g/L, more preferably 6 to 10g/L, and still more preferably 8g/L.
In the present invention, the carbon source preferably includes one or more of glucose, sucrose, maltose, fructose, lactose and starch, and further preferably glucose, or sucrose, glucose and starch, or sucrose and starch, or starch; the nitrogen source preferably comprises one or more of peptone, beef extract, yeast extract, ammonium chloride, ammonium sulfate, soybean cake powder and corn steep liquor, and further preferably ammonium sulfate and corn steep liquor, or ammonium chloride and corn steep liquor, or corn steep liquor.
In the present invention, the initial pH of the fermentation medium is preferably 6.0 to 7.5, more preferably 6.5 to 7.0, and still more preferably 6.8. The invention has no special requirement on the sources of the components in the fermentation medium, and the components can be obtained by adopting commercial products which are well known to the technical personnel in the field.
The bacillus licheniformis GXU-1 is a glutamic acid independent strain, but can also rely on glutamic acid to produce gamma-polyglutamic acid, namely the gamma-polyglutamic acid can be produced in high yield in a fermentation medium containing sodium glutamate and a fermentation medium without sodium glutamate.
After the culture rich in the gamma-polyglutamic acid is obtained, the culture is preferably centrifuged to remove thalli, the supernatant is taken, 4 times volume of precooled absolute ethyl alcohol is added, the mixture is placed in a refrigerator at 4 ℃ for precipitation for 12 hours and then taken out, the centrifugation is carried out for 10min at 4 ℃, the supernatant is removed, the precipitate is washed for 3 times by 75% ethyl alcohol, the precipitate is dried to constant weight at 105 ℃, and the precipitate is taken out and cooled to room temperature to obtain a crude product of the gamma-polyglutamic acid.
The fermentation culture of the bacillus licheniformis obtained by fermenting the seed solution of the bacillus licheniformis GXU-1 is rich in gamma-polyglutamic acid, the yield can reach more than 50g/L, the fermentation process is simple and convenient to operate, the components of the culture medium are simple, the cost is low (the bacillus licheniformis GXU-1 can use various different carbon sources and nitrogen sources, and can use various cheap carbon and nitrogen sources such as starch, corn flour and the like), the fermentation period is short, and the industrial large-scale production can be realized.
In order to further illustrate the present invention, the following detailed description of the technical solutions provided by the present invention is made with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of the present invention.
Unless otherwise indicated, the medium used in the practice and the identification method are conventional. In the following examples and comparative examples, 3 parallel experiments were performed for the determination of the content of gamma-polyglutamic acid, and the determination results were the average of the 3 results.
Example 1
And (3) separating and identifying the bacillus licheniformis:
1. and (3) separating the bacillus parabi:
1.1 sample treatment: weighing 5g of soil sample, placing into a sterilized triangular flask, adding 50mL of sterilized water, shaking by a shaking table at 200rpm at 30 ℃ for 30min, standing for precipitation, taking supernatant, and performing gradient dilution with sterile water with dilution multiple of 10 2 、10 3 、10 4 、10 5 、10 6 、10 7 And 10 8 。
1.2 preliminary screening of the strain producing gamma-polyglutamic acid: selecting seven samples after gradient dilution in the step 1.1, coating 100 mu L of each sample on a primary screening plate culture medium, and performing inverted culture at 37 ℃; the primary screening plate culture medium is an LB solid culture medium and comprises the following components: 10g/L of peptone, 5g/L of yeast powder, 10g/L of NaCl, 20g/L of agar powder and the balance of distilled water, and sterilizing for 30min at the initial pH of 7.0 and 121 ℃ in a primary screening plate culture medium.
1.3 rescreening of the gamma-polyglutamic acid producing strain: selecting a single colony which is in an emulsion state and can be subjected to wire drawing from a primary screening plate culture medium, transferring the single colony to a fresh liquid re-screening culture medium, and carrying out shaking culture at 37 ℃ and 200rpm for 48 hours to obtain a fermentation liquid; the liquid re-screening culture medium comprises the following components: 20g/L glucose, 10g/L peptone, 3g/L beef extract, 5g/L NaCl and the balance of distilled water, wherein the initial pH of the liquid re-screening culture medium is 7.0, the initial pH is 121 ℃, and the sterilization is carried out for 30min.
1.4 extraction and detection of gamma-polyglutamic acid: and (3) removing thalli in the fermentation liquor obtained in the step 1.3 by centrifugation, taking supernatant, adding 4 times of volume of precooled absolute ethyl alcohol, putting the mixture into a refrigerator at 4 ℃ for precipitation for 12 hours, taking out the mixture, centrifuging the mixture for 10min at 4 ℃, removing the supernatant, washing the precipitate for 3 times by using 75% ethanol, drying the precipitate at 105 ℃ to constant weight, taking out the precipitate, cooling the precipitate to room temperature to obtain a gamma-polyglutamic acid crude product, and weighing the gamma-polyglutamic acid crude product to determine the yield of the polyglutamic acid.
1.5 identification of Gamma-polyglutamic acid: weighing 2g of the gamma-polyglutamic acid product obtained in the step 1.4, adding ultrapure water for redissolution, fixing the volume to 100ml, filtering with a 0.22 mu m filter membrane to remove impurities, taking 5ml of the filtered sample, adding 6mol/L hydrochloric acid solution with the same volume, carrying out oil bath hydrolysis at 110 ℃ for 12 hours, adding 6mol/LNaOH solution for neutralization after hydrolysis until the pH value is 7.0, analyzing the diluted hydrolysate by using a high performance liquid chromatography, analyzing the glutamic acid standard by using the high performance liquid chromatography, comparing the liquid chromatography peak time of the hydrolysate and the glutamic acid standard, and identifying the prepared gamma-polyglutamic acid.
Screening results of the gamma-polyglutamic acid-producing strains:
single colonies which are in an emulsion shape and can be drawn into wires are obtained through primary screening, the single colonies are respectively transferred to a fresh liquid re-screening culture medium, after 12 hours of culture, gamma-polyglutamic acid is extracted and detected according to the step 1.4 and the step 1.5, the strain producing the gamma-polyglutamic acid is obtained, the yield is high and reaches 32.72g/L, and the number is GXU-1.
2. Identification of Bacillus licheniformis
2.1 morphological characterisation
A single colony of the strain with the number GXU-1 is picked by an inoculating needle, inoculated on an LB solid culture medium (which consists of peptone 10g/L, yeast powder 5g/L, naCl 10g/L, agar powder 20g/L and the balance of distilled water, the initial pH of the culture medium is 7.0, and the culture medium is sterilized at 121 ℃ for 30 min), inversely cultured at 37 ℃, and the colony morphology is observed, and the result is shown in figure 1. As can be seen from FIG. 1, the GXU-1 strain has irregular shape of colony on LB solid medium, regular edge, moist lawn, rough surface, white color and opaque surface.
2.2 physiological and biochemical identification:
the GXU-1 strain somatic cell is in a short rod shape, is gram-positive, can decompose and utilize glucose, sucrose, lactose, maltose, fructose and mannitol, can hydrolyze starch, can generate acid by utilizing glucose, has negative oxidase reaction, has positive nitrate reduction reaction, has negative citrate utilization test and has positive gelatin liquefaction test.
2.3 molecular biological identification
Extracting genome DNA of GXU-1 strain, adopting bacteria universal primer (27F, 1492R), PCR amplifying its 16S rDNA sequence, and sequencing to obtain its 16S rDNA sequence shown in SEQ ID NO.1, with length of 1448bp. The homology of the sequence is compared and analyzed in a GeneBank database on NCBI by using BLAST software, and the result shows that the homology of the 16S rDNA sequence of the GXU-1 strain and Bacillus paralicheniformis reaches 99 percent. Combining the morphological and physiological biochemical identification results, GXU-1 is identified as Bacillus licheniformis, and a phylogenetic tree is shown in figure 2.
Example 2
A method for preparing gamma-polyglutamic acid by utilizing fermentation of Bacillus licheniformis comprises the following steps:
1. seed culture
The Bacillus paracasei (Bacillus paraccheniformis) GXU-1 separated in the example 1 is streaked and inoculated on an LB solid medium (which consists of 10g/L of peptone, 5g/L of yeast powder, 10g/L of NaCl, 20g/L of agar powder and the balance of distilled water, the initial pH of the medium is 7.0, the medium is sterilized at 121 ℃ for 30 min), inverted culture is carried out at 37 ℃, a single colony is selected and inoculated in a liquid seed medium (which consists of 20g/L of glucose, 10g/L of peptone, 3g/L of beef extract, 5g/L of NaCl and the balance of distilled water, the initial pH of the medium is 7.0, the medium is sterilized at 115 ℃ for 20 min), and shaking culture is carried out at 37 ℃ and 220rpm for 18h to the middle logarithmic growth phase, so as to obtain a seed solution.
2. Fermentation culture
Culturing step 1 according to an inoculum size of 8% (v/v)The resulting seed solution was inoculated into a fermentation medium (composed of 50g/L of glucose, (NH) 4 ) 2 SO 4 7g/L, 8g/L corn steep liquor, 50g/L sodium glutamate and K 2 HPO 4 2.5g/L and the balance of distilled water, the initial pH of the culture medium is 7.0, the culture medium is sterilized for 30min at 121 ℃), the liquid loading of 500ml shake flask fermentation is 100ml, and shaking culture is carried out for 24h at 37 ℃ and 220rpm.
Example 3
A method for preparing gamma-polyglutamic acid by fermentation of Bacillus licheniformis similar to example 2, except that the temperature is 30 deg.C during fermentation and culture in step 2.
Example 4
A method for preparing gamma-polyglutamic acid by fermentation of Bacillus licheniformis similar to example 2, except that the temperature is 45 deg.C during fermentation culture in step 2.
Comparative example 1
A method for preparing gamma-polyglutamic acid by fermentation of Bacillus licheniformis similar to example 2, except that the temperature is 25 deg.C during fermentation culture in step 2.
Comparative example 2
A method for preparing gamma-polyglutamic acid by fermentation of Bacillus licheniformis similar to example 2, except that the temperature is 50 deg.C during fermentation culture in step 2.
The contents of gamma-polyglutamic acid in the fermentation broths after the completion of the cultivation in examples 2 to 4, comparative example 1 and comparative example 2 were measured in the manner of step 1.4 in example 1, and the results are shown in Table 1.
TABLE 1 content (g/L) of gamma-polyglutamic acid in fermentation broth after completion of cultivation by different methods
As can be seen from Table 1, the yield decreases below 30 ℃ and the yield is below 45 ℃ when the temperature is above 45 ℃ under the condition of increasing energy consumption, and the optimum fermentation temperature is 30-45 ℃ when shaking flask fermentation is carried out.
Example 5
A method for preparing gamma-polyglutamic acid by fermentation of Bacillus licheniformis similar to the embodiment 2, except that in the step 2 fermentation culture process, shaking table cultivation at 150rpm is carried out for 24h.
Example 6
A method for preparing gamma-polyglutamic acid by fermentation of Bacillus licheniformis similar to the embodiment 2, except that in the step 2 fermentation culture process, shaking table shaking culture at 200rpm is carried out for 24h.
Comparative example 3
A method for preparing gamma-polyglutamic acid by fermentation of Bacillus licheniformis similar to example 2, except that shaking culture at 120rpm for 24h is performed during the fermentation culture in step 2.
Comparative example 4
A method for preparing gamma-polyglutamic acid by fermentation of Bacillus licheniformis similar to the embodiment 2, except that in the step 2 fermentation culture process, shaking table at 250rpm is used for 24h.
The contents of gamma-polyglutamic acid in the fermentation broths after the completion of the cultivation in examples 2, 5, 6, comparative example 3 and comparative example 4 were measured in the manner of step 1.4 in example 1, and the results are shown in Table 2.
TABLE 2 content (g/L) of gamma-polyglutamic acid in fermentation broth after completion of cultivation by different methods
| Group of | Example 2 | Example 5 | Example 6 | Comparative example 3 | Comparative example 4 |
| Rotation speed of fermentation | 220rpm | 150rpm | 200rpm | 120rpm | 250rpm |
| Content of gamma-polyglutamic acid | 36.86 | 35.38 | 36.24 | 34.26 | 36.85 |
As shown in Table 2, the optimum rotation speed for the shake flask fermentation was 150 to 220rpm.
Example 7
A method for preparing gamma-polyglutamic acid by fermenting bacillus licheniformis similar to the example 2, wherein the difference is that the fermentation time is 12 hours in the fermentation culture process of the step 2.
Example 8
A method for preparing gamma-polyglutamic acid by fermenting bacillus licheniformis similar to the embodiment 2, wherein the difference is that the fermentation time is 36h in the fermentation culture process in the step 2.
Comparative example 5
A method for preparing gamma-polyglutamic acid by fermenting bacillus licheniformis similar to the embodiment 2, wherein the difference is that the fermentation time is 10 hours in the fermentation culture process of the step 2.
Comparative example 6
A method for preparing gamma-polyglutamic acid by fermenting bacillus licheniformis similar to the embodiment 2, wherein the difference is that the fermentation time is 40h in the fermentation culture process in the step 2.
The contents of gamma-polyglutamic acid in the fermentation broths after the completion of the cultivation in examples 2, 7, 8, comparative example 5 and comparative example 6 were measured in the manner of step 1.4 in example 1, and the results are shown in Table 3.
TABLE 3 content (g/L) of gamma-polyglutamic acid in fermentation broth after completion of cultivation by different methods
| Group of | Example 2 | Example 7 | Example 8 | Comparative example 5 | Comparative example 6 |
| Time of fermentation | 24h | 12h | 36h | 10h | 40h |
| Content of gamma-polyglutamic acid | 36.86 | 35.36 | 34.82 | 23.13 | 35.87 |
As shown in Table 3, the optimum fermentation time was 12 to 36 hours in the case of shake flask fermentation.
Example 9
A method for preparing gamma-polyglutamic acid by fermentation of Bacillus licheniformis similar to the embodiment 2, except that the concentration of glucose in the fermentation medium during the fermentation culture of the step 2 is 20g/L.
Example 10
A method for preparing gamma-polyglutamic acid by fermentation of Bacillus licheniformis similar to example 2, except that during the fermentation culture in step 2, the concentration of glucose in the fermentation medium is 100g/L, (NH) 4 ) 2 SO 4 The concentration of (b) is 11g/L, and the corn steep liquor concentration is 0g/L.
Example 11
A method for producing gamma-polyglutamic acid by fermentation of Bacillus licheniformis similar to that in example 2, except that the concentration of glucose in the fermentation medium during the fermentation culture in step 2 is 150g/L.
Comparative example 7
A method for preparing gamma-polyglutamic acid by fermentation of Bacillus licheniformis similar to example 2, except that the concentration of glucose in the fermentation medium during the fermentation culture of step 2 is 15g/L.
Comparative example 8
A method for preparing gamma-polyglutamic acid by fermentation of Bacillus licheniformis similar to the embodiment 2, except that the concentration of glucose in the fermentation medium during the fermentation culture of the step 2 is 180g/L.
The contents of gamma-polyglutamic acid in the fermentation broths after the completion of the cultivation in examples 2, 9, 10, 11, comparative example 7 and comparative example 8 were measured in the same manner as in step 1.4 of example 1, and the results are shown in Table 4.
TABLE 4 content (g/L) of gamma-polyglutamic acid in fermentation broth after completion of cultivation by different methods
As is clear from Table 4, the optimum glucose concentration (carbon source) of the fermentation medium during the fermentation culture was 20 to 150g/L.
Example 12
A method for preparing gamma-polyglutamic acid by fermentation of Bacillus paracasei similar to example 2, except that (NH) is added to the fermentation medium during the fermentation culture in step 2 4 ) 2 SO 4 And the concentration of the corn steep liquor is 1g/L and 2g/L in sequence.
Example 13
A method for preparing gamma-polyglutamic acid by fermentation of Bacillus paracasei similar to example 2, except that (NH) is added to the fermentation medium during the fermentation culture in step 2 4 ) 2 SO 4 And the concentration of the corn steep liquor is 4g/L and 6g/L in sequence.
Example 14
A method for preparing gamma-polyglutamic acid by fermentation of Bacillus licheniformis similar to example 2, except that in the step 2 fermentation culture process, the fermentation medium contains (NH) 4 ) 2 SO 4 And the concentration of the corn steep liquor is 5g/L and 3g/L in sequence.
Comparative example 9
A method for preparing gamma-polyglutamic acid by fermentation of Bacillus paracasei similar to example 2, except that (NH) is added to the fermentation medium during the fermentation culture in step 2 4 ) 2 SO 4 And the concentration of the corn steep liquor is 1g/L and 1g/L in sequence.
Comparative example 10
A method for preparing gamma-polyglutamic acid by fermentation of Bacillus paracasei similar to example 2, except that (NH) is added to the fermentation medium during the fermentation culture in step 2 4 ) 2 SO 4 And the concentration of the corn steep liquor is 8g/L and 9g/L in sequence.
The contents of gamma-polyglutamic acid in the fermentation broths after the completion of the cultivation in examples 2, 12 to 14, comparative example 9 and comparative example 10 were measured in the manner of step 1.4 in example 1, and the results are shown in Table 5.
TABLE 5 content (g/L) of gamma-polyglutamic acid in fermentation broth after completion of cultivation by different methods
As is clear from Table 5, the optimum nitrogen source concentration in the fermentation medium during the fermentation culture was 3 to 15g/L.
Example 15
A method for preparing gamma-polyglutamic acid by fermentation of Bacillus licheniformis similar to example 2, except that during the fermentation culture in step 2, the concentration of glucose is 20g/L, and the concentration of sodium glutamate in the fermentation medium is 0g/L.
Example 16
A method for preparing gamma-polyglutamic acid by fermentation of Bacillus licheniformis similar to the embodiment 2, except that during the fermentation culture in the step 2, the concentration of sodium glutamate in the fermentation medium is 40g/L.
Example 17
A method for preparing gamma-polyglutamic acid by fermentation of Bacillus licheniformis similar to example 2, except that during the fermentation culture in step 2, the concentration of sodium glutamate in the fermentation medium is 90g/L.
Comparative example 11
A method for preparing gamma-polyglutamic acid by fermentation of Bacillus licheniformis similar to example 2, except that during the fermentation culture in step 2, the concentration of sodium glutamate in the fermentation medium is 100g/L.
The contents of gamma-polyglutamic acid in the fermentation broths after the completion of the cultivation in examples 2, 15 to 18 and comparative example 11 were measured in the same manner as in step 1.4 of example 1, and the results are shown in Table 6.
TABLE 6 content (g/L) of gamma-polyglutamic acid in fermentation broth after completion of cultivation by different methods
| Group of | Example 2 | Example 15 | Example 16 | Example 17 | Comparative example 11 |
| Glucose concentration (g/L) | 50 | 20 | 50 | 50 | 50 |
| Concentration of sodium glutamate (g/L) | 50 | 0 | 40 | 90 | 100 |
| Content of gamma-polyglutamic acid | 36.86 | 14.32 | 25.60 | 52.36 | 58.64 |
As can be seen from Table 6, the Bacillus licheniformis GXU-1 provided by the invention is a glutamic acid independent strain, but can also rely on glutamic acid to produce gamma-polyglutamic acid, namely, the gamma-polyglutamic acid can be produced in high yield in both a fermentation medium containing sodium glutamate and a fermentation medium without sodium glutamate.
Example 18
A method for preparing gamma-polyglutamic acid by fermenting bacillus licheniformis similar to the example 2, wherein the difference is that during the fermentation culture process in the step 2, glucose in the fermentation culture medium is replaced by cane sugar, and the content of the gamma-polyglutamic acid is detected to be 34.53g/L.
Example 19
A method for preparing gamma-polyglutamic acid by fermenting bacillus licheniformis similar to the example 2, wherein the difference is that in the fermentation culture process of the step 2, glucose in a fermentation culture medium is replaced by fructose, and the content of the gamma-polyglutamic acid is detected to be 33.32g/L.
Example 20
A method for preparing gamma-polyglutamic acid by fermenting bacillus licheniformis similar to the example 2, except that during the fermentation culture in the step 2, glucose in the fermentation culture medium is replaced by lactose, and the content of gamma-polyglutamic acid is detected to be 35.24g/L.
Example 21
A method for preparing gamma-polyglutamic acid by fermentation of Bacillus licheniformis similar to example 2, except that in the step 2 fermentation culture process, (NH) in the fermentation medium 4 ) 2 SO 4 Replacing the ammonium chloride, and detecting the content of the gamma-polyglutamic acid to be 32.39g/L.
Example 22
A method for preparing gamma-polyglutamic acid by fermentation of Bacillus licheniformis similar to example 2, except that in the step 2 fermentation culture process, (NH) in the fermentation medium 4 ) 2 SO 4 Replacing with peptone, and detecting the content of the gamma-polyglutamic acid to be 31.91g/L.
Example 23
A method for preparing gamma-polyglutamic acid by fermentation of bacillus paracasei, which is similar to that of example 2, except that in the fermentation culture process of step 2, ammonium sulfate and corn steep liquor in the fermentation culture medium are replaced by soybean cake powder, and the content of gamma-polyglutamic acid is detected to be 21.97g/L.
Example 24
A method for preparing gamma-polyglutamic acid by fermentation of Bacillus licheniformis similar to example 2, except that in the step 2 fermentation culture process, (NH) in the fermentation medium 4 ) 2 SO 4 7g/L of corn steep liquor and 8g/L of corn steep liquor are replaced by 7g/L of yeast powder and 8g/L of beef extract, and the content of the gamma-polyglutamic acid is 34.18g/L by detection.
Example 25
A method for preparing gamma-polyglutamic acid by fermentation of Bacillus paracasei similar to example 2, except that in the step 2 fermentation culture process, (NH) is added to the fermentation medium 4 ) 2 SO 4 7g/L and 8g/L of corn steep liquor are replaced by 5g/L of ammonium chloride, 5g/L of peptone and 5g/L of soybean cake powder, and the content of the gamma-polyglutamic acid is detected to be 36.59g/L.
Example 26
A method for preparing gamma-polyglutamic acid by fermenting bacillus paraci similar to example 2, which is characterized in that in the fermentation culture process of step 2, a shake flask is replaced by a 10L fermentation tank, the liquid filling amount is 8L, the ventilation amount is 0.8vvm, the culture is carried out for 24h at 37 ℃ and 220rpm stirring speed, and finally the content of the gamma-polyglutamic acid is measured to be 38.33g/L.
Example 27
A method for producing gamma-polyglutamic acid by fermentation of bacillus paracasei similar to example 26, except that the ventilation amount during the fermentation culture of step 2 is 1.2vvm, and the content of gamma-polyglutamic acid is finally measured to be 38.67g/L.
Comparative example 12
A method for producing gamma-polyglutamic acid by fermentation of bacillus paracasei similar to that of example 26, except that the ventilation amount during the fermentation culture of step 2 is 0.5vvm, and the content of gamma-polyglutamic acid was finally determined to be 32.58g/L.
Comparative example 13
A method for preparing gamma-polyglutamic acid by fermentation of Bacillus licheniformis similar to example 26, except that in the fermentation culture of step 2, the ventilation amount is 1.5vvm, and the content of gamma-polyglutamic acid is 35.37g/L.
From the results of example 5, example 6, comparative example 3 and comparative example 4, it was found that the optimum aeration amount during fermentation culture in the fermentation tank was 0.8 to 1.2vvm.
From the results of example 26, example 27, comparative example 12 and comparative example 13, it is understood that the optimum aeration amount during the fermentation tank culture is 0.8 to 1.2vvm.
Example 28
A method for preparing gamma-polyglutamic acid by fermenting bacillus paracasei similar to example 26, except that during the fermentation culture of step 2, the stirring speed is 250rpm, and finally the content of gamma-polyglutamic acid is determined to be 36.72g/L.
Example 29
A method for preparing gamma-polyglutamic acid by fermentation of Bacillus licheniformis similar to example 26, except that during the fermentation culture in step 2, the stirring speed is 150rpm, and the content of gamma-polyglutamic acid is 34.46g/L.
Example 30
A method for preparing gamma-polyglutamic acid by fermenting bacillus paracasei similar to example 26, except that during the fermentation culture in step 2, the fermentation time is 36 hours, and the content of gamma-polyglutamic acid is finally determined to be 38.52g/L.
Example 31
A method for preparing gamma-polyglutamic acid by fermenting bacillus paracasei similar to example 26, except that during the fermentation culture in step 2, the fermentation time is 48 hours, and the content of gamma-polyglutamic acid is finally determined to be 39.73g/L.
According to the embodiment, the bacillus licheniformis GXU-1 is cultured, the obtained fermentation culture is rich in gamma-polyglutamic acid, the yield can reach more than 50g/L at most, the fermentation process is simple and convenient to operate, the components of the culture medium are simple, the cost is low (the bacillus licheniformis GXU-1 can use various different carbon sources and nitrogen sources, and can utilize various cheap carbon and nitrogen sources such as starch, corn flour and the like), the fermentation period is short, and industrial large-scale production can be realized.
Although the present invention has been described in detail with reference to the above embodiments, it is only a part of the embodiments of the present invention, not all of the embodiments, and other embodiments can be obtained without inventive step according to the embodiments, and the embodiments are within the scope of the present invention.
Claims (9)
1. The bacillus licheniformis (Bacillus parachuieniformis) GXU-1 is characterized in that the preservation number of the bacillus parachuieniformis GXU-1 is CGMCC No.24578.
2. The use of the Bacillus licheniformis GXU-1 of claim 1 for the preparation of gamma-polyglutamic acid.
3. A method for preparing gamma-polyglutamic acid, comprising the steps of:
inoculating a seed solution containing the bacillus licheniformis GXU-1 of claim 1 into a fermentation culture medium, and performing aerobic fermentation to obtain gamma-polyglutamic acid;
the fermentation medium comprises 20-150 g/L of carbon source, 3-15 g/L of nitrogen source, 0-90 g/L of sodium glutamate and K 2 HPO 4 2.5g/L and the balance of distilled water.
4. The method of claim 3, wherein the aerobic fermentation comprises a shake flask fermentation or a fermentor fermentation;
when the aerobic fermentation mode is shake flask fermentation, the fermentation conditions comprise: the temperature is 30-45 ℃, the rotating speed is 150-220 rpm, and the time is 12-36 h;
when the aerobic fermentation mode is fermentation in a fermentation tank, the fermentation conditions comprise: the temperature is 30-45 ℃, the stirring speed is 150-250 rpm, the time is 24-48 h, and the ventilation volume is 0.8-1.2 vvm.
5. The method according to claim 3, wherein the volume ratio of the seed solution to the fermentation medium at the time of inoculation is (1-15): 100.
6. the method of claim 3 or 5, wherein the initial pH of the fermentation medium is between 6.0 and 7.5.
7. The method of claim 3 or 5, wherein the seed liquid is prepared by a method comprising: inoculating a single colony of the Bacillus licheniformis GXU-1 of claim 1 into a seed culture medium, and carrying out shaking culture at the temperature of 30-37 ℃ and the rpm of 150-20 for 12-18 h to the middle logarithmic phase of growth.
8. The method of claim 7, wherein the seed medium comprises glucose 20g/L, peptone 10g/L, beef extract 3g/L, naCl5g/L and balance distilled water, initial ph7.0.
9. The method of claim 3, wherein the carbon source comprises one or more of glucose, sucrose, maltose, fructose, lactose, and starch;
the nitrogen source comprises one or more of peptone, beef extract, yeast extract, ammonium chloride, ammonium sulfate, soybean cake powder and corn steep liquor.
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