CN107384827B - Escherichia coli JL-GlcN and application thereof - Google Patents
Escherichia coli JL-GlcN and application thereof Download PDFInfo
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Abstract
The invention relates to Escherichia coli JL-GlcN and application thereof, wherein the Escherichia coli JL-GlcN has a strain preservation number of CGMCC NO. 13924; activating the Escherichia coli JL-GlcN by a slant, culturing for 20h at 37 ℃ in a biochemical incubator, performing gradient dilution, coating on an LB culture medium, selecting single colonies, respectively inoculating to the LB slant culture medium, and culturing for 12h at 30-36 ℃ in the biochemical incubator; inoculating the seed solution into a fermentation tank filled with a fermentation culture medium for culture, adding IPTG (isopropyl-beta-D-thiogalactoside) for induction when the OD (OD) of the thallus is 10-15, and culturing for 10-20h to obtain a fermentation liquor containing N-acetylglucosamine; extracting N-acetylglucosamine from the fermentation broth containing N-acetylglucosamine. The fermentation of the Escherichia coli JL-GlcN strain is aerobic fermentation, and the growth of the strain is fast; the fermentation process is simple, easy to control and easy to enlarge for industrial production.
Description
Technical Field
The invention relates to escherichia coli, in particular to escherichia coli JL-GlcN and application thereof.
Background
N-acetylglucosamine is a monomer of chitin, and the chitin is very large in stock in nature, and is second to cellulose. N-acetylglucosamine has important physiological functions in medicine, has the functions of diminishing inflammation, resisting tumor and resisting oxidation, is an effective medicine for treating osteoarthritis and rheumatic arthritis, and has important application in the industries of food, chemical industry and cosmetics.
The existing preparation method of N-acetylglucosamine comprises a sodium methoxide method, a direct chitin enzymolysis method and the like. The process of the sodium methoxide method comprises the following steps: adding sodium methoxide into glucosamine raw materials for replacement reaction, removing chloride ions in the glucosamine, adding acetic anhydride for acetylation, removing harmful substances such as bacteria, rust, colloid and the like in the glucosamine raw materials through ultrafiltration, then carrying out fractional precipitation and filtration, then carrying out re-dissolution crystallization and solid-liquid separation, and obtaining the finished product after vacuum drying. The direct enzymolysis method means that chitin can be directly produced by the enzymolysis of chitin with chitinase, the enzymolysis process of the chitin is longer due to the particularity of the chemical property of the chitin, the hydrolysis is incomplete, the hydrolysis rate is below 75 percent, the efficiency is lower, the actual large-scale industrial production cost is higher, and the benefit is poor.
Escherichia coli (Bacillus subtilis) is a production host widely used as food enzyme preparation and important nutrition and chemicals, and the product is certified as "general regulated as safe" (GRAS) level by FDA. Therefore, the construction of recombinant Escherichia coli by using metabolic engineering means is an effective way for producing food safety-grade N-acetylglucosamine. However, N-acetylglucosamine is a carbon source substance preferentially used by Escherichia coli, and when glucose is consumed in a culture medium, extracellular N-acetylglucosamine is transported to the intracellular space, enters a cell glycolysis pathway in the form of fructose-6-phosphate through deamination and deacetylation, and participates in cell metabolism. Therefore, it is necessary to knock out the N-acetylglucosamine deacetylase coding gene (nagA) and the N-acetylglucosamine deaminase coding gene (nagB) to block the degradation of N-acetylglucosamine and increase the yield of N-acetylglucosamine. The N-acetylglucosamine deacetylase coding gene (nagA) and the N-acetylglucosamine deaminase coding gene (nagB) are knocked out through homologous recombination, and the way of degrading N-acetylglucosamine by host bacteria is blocked.
Disclosure of Invention
The invention aims to solve the defects of the technical problems and provide the Escherichia coli JL-GlcN and the application thereof, wherein the strain improves the yield of N-acetylglucosamine, shortens the production period and reduces the production cost.
The Escherichia coli JL-GlcN is classified and named as Escherichia coli (Escherichia coli), is preserved in China general microbiological culture Collection center (CGMCC), has a strain preservation number of CGMCC No.13924, and has a preservation date of 2017, 3 months and 23 days.
After activation and amplification culture of Escherichia coli JL-GlcN, the strain can be inoculated into a fermentation tank for fermentation to prepare N-acetylglucosamine.
The application of Escherichia coli JL-GlcN in preparing N-acetylglucosamine includes the following steps:
step one, activating a colon bacillus JL-GlcN by a slant, culturing for 20h in a biochemical incubator at 37 ℃, then performing gradient dilution, coating on an LB culture medium, selecting single colonies, respectively inoculating to the LB slant culture medium, and culturing for 12h in the biochemical incubator at 30-36 ℃; inoculating into a seed bottle filled with a seed culture medium, and culturing under the following conditions: the temperature is 30-36 ℃, the rotating speed of a shaking table is 200rmp, and the culture time is 6-8 h; when the OD value is 1.0-2.0, obtaining a seed culture solution;
the seed culture medium contains 20-30g/L of glucose and K2HPO410-20g/L, MgSO4·7H2O 0.5-3.0g/L，KH2PO410-20g/L, citric acid 0.5-3.0g/L, (NH)4)2SO45-10.0g/L, 5-10.0mL/L of microelement stock solution, the balance of water and pH 6.7;
the microelement stock solution contains FeSO in each liter of microelement stock solution41.0-3.0g，MnSO40.2-1.0g，ZnSO40.2-1.0g，CuSO40.2-1.0g and CoCl20.2-1.0g。
Inoculating the seed solution into a fermentation tank filled with a fermentation culture medium for culture, adding IPTG (isopropyl-beta-D-thiogalactoside) for induction when the OD (origin-degree of gravity) of the thallus is 10-15, and culturing for 10-20h to obtain a fermentation liquor containing N-acetylglucosamine; inoculation amount: 5 to 15 percent; the culture conditions are as follows: the initial fermentation temperature is 32-38 ℃, the dissolved oxygen is maintained at 10-30%, and the fermentation pH value is controlled between 7.0-7.2;
the fermentation medium contains 20-30g/L of glucose and K2HPO410-20g/L, MgSO4·7H2O0.5-3.0 g/L, citric acid 0.5-3.0g/L, (NH)4)2SO45-10.0g/L, 5-10.0mL/L of microelement stock solution and 7.0 pH; the microelement stock solution contains FeSO in each liter of microelement stock solution41.0-3.0g，MnSO40.2-1.0g，ZnSO40.2-1.0g，CuSO40.2-1.0g，CoCl20.2-1.0g。
And step three, extracting the N-acetylglucosamine from the fermentation liquor containing the N-acetylglucosamine.
The method for extracting the N-acetylglucosamine from the fermentation liquor containing the N-acetylglucosamine comprises the following steps: adjusting pH of the fermentation liquid containing acetylglucosamine to 2-5 with 2mo1/L hydrochloric acid, microfiltering with 30nm ceramic membrane, washing with purified water for 2-3 times, passing the supernatant through strong acid cation exchange column, and performing static adsorption: adding cationic resin according to the volume ratio of 20%, stirring and adsorbing for 1 h; dynamic adsorption: filling column resin according to 15% volume, feeding at the flow rate of 2BV/h, and washing the column with 0.5-1BV water at the final stage; collecting the supernatant; adding activated carbon into the supernatant according to the volume ratio of 0.5-1% for decolorization for 0.5-2h at 60 ℃; filtering to remove carbon, collecting the obtained N-acetylglucosamine supernatant, and vacuum concentrating to obtain N-acetylglucosamine; the vacuum concentration conditions are as follows: vacuum degree of-0.1 MPa, rotation speed of 100-.
The beneficial effects are that:
1. the fermentation of the Escherichia coli JL-GlcN strain is aerobic fermentation, and the growth of the strain is fast; the fermentation process is simple, easy to control and easy to enlarge for industrial production.
2. The invention has short fermentation period (10-20 h) and large cell density (OD = 110-150). High yield and yield of N-acetylglucosamine, short cell culture time and production period, high production level of N-acetylglucosamine per cell, no consumption of N-acetylglucosamine as carbon source, simple extraction process, easy control, high recovery rate, and high purity.
Preservation of biological materials
Escherichia coli JL-GlcN, classified and named as Escherichia coli (Escherichia coli), with a date of 3/23 in 2017, and a collection unit and its abbreviation: china general microbiological culture Collection center (CGMCC), the preservation address is as follows: xilu No.1 Hospital No. 3, Beijing, Chaoyang, North; the preservation number is CGMCC No. 13924.
Detailed Description
The Escherichia coli JL-GlcN is classified and named as Escherichia coli (Escherichia coli), is preserved in China general microbiological culture Collection center (CGMCC), has a strain preservation number of CGMCC No.13924, and has a preservation date of 2017, 3 months and 23 days.
After activation and amplification culture of Escherichia coli JL-GlcN, the strain can be inoculated into a fermentation tank for fermentation to prepare N-acetylglucosamine.
The application of Escherichia coli JL-GlcN in preparing N-acetylglucosamine includes the following steps:
step one, activating a colon bacillus JL-GlcN by a slant, culturing for 20h in a biochemical incubator at 37 ℃, then performing gradient dilution, coating on an LB culture medium, selecting single colonies, respectively inoculating to the LB slant culture medium, and culturing for 12h in the biochemical incubator at 30-36 ℃; inoculating into a seed bottle filled with a seed culture medium, and culturing under the following conditions: the temperature is 30-36 ℃, the rotating speed of a shaking table is 200rmp, and the culture time is 6-8 h; when the OD value is 1.0-2.0, obtaining a seed culture solution;
the seed culture medium: the seed culture medium contains glucose 20-30g/L, K2HPO410-20g/L,MgSO4·7H2O 0.5-3.0g/L，KH2PO410-20g/L, citric acid 0.5-3.0g/L, (NH)4)2SO45-10.0g/L, 5-10.0mL/L of microelement stock solution, the balance of water and pH 6.7;
inoculating the seed solution into a fermentation tank filled with a fermentation culture medium for culture, adding IPTG (isopropyl-beta-D-thiogalactoside) for induction when the OD (origin-degree of gravity) of the thallus is 10-15, and culturing for 10-20h to obtain a fermentation liquor containing N-acetylglucosamine; inoculation amount: 5 to 15 percent; the culture conditions are as follows: the initial fermentation temperature is 32-38 ℃, the dissolved oxygen is maintained at 10-30%, and the fermentation pH value is controlled between 7.0-7.2;
the fermentation medium comprises: each liter of fermentation medium contains 20-30g/L of glucose, K2HPO410-20g/L,MgSO4·7H2O0.5-3.0 g/L, citric acid 0.5-3.0g/L, (NH)4)2SO45-10.0g/L, 5-10.0mL/L of microelement stock solution and 7.0 pH;
and step three, extracting the N-acetylglucosamine from the fermentation liquor containing the N-acetylglucosamine.
The microelement stock solution contains FeSO in each liter of microelement stock solution41.0-3.0g，MnSO40.2-1.0g，ZnSO40.2-1.0g，CuSO40.2-1.0g，CoCl20.2-1.0g。
The method for extracting the N-acetylglucosamine from the fermentation liquor containing the N-acetylglucosamine comprises the following steps: adjusting pH of the fermentation liquid containing N-acetylglucosamine to 2-5 with 2mo1/L hydrochloric acid, microfiltering with 30nm ceramic membrane, washing with purified water for 2-3 times, passing the supernatant through strong acid cation exchange column under static adsorption conditions: adding cationic resin according to the volume ratio of 20%, stirring and adsorbing for 1 h; dynamic adsorption: filling column resin according to 15% volume, feeding at the flow rate of 2BV/h, and washing the column with 0.5-1BV water at the final stage; collecting the supernatant; adding activated carbon into the supernatant according to the volume ratio of 0.5-1% for decolorization for 0.5-2h at 60 ℃; filtering to remove carbon, collecting the obtained N-acetylglucosamine supernatant, and vacuum concentrating to obtain N-acetylglucosamine; the vacuum concentration conditions are as follows: vacuum degree of-0.1 MPa, rotation speed of 100-.
In the following examples, the yield of N-acetylglucosamine was calculated by: n-acetylglucosamine yield = total mass of N-acetylglucosamine in the fermentation broth/volume of fermentation broth.
Example 1
Respectively taking Escherichia coli JL-GlcN and common Escherichia coli producing N-acetylglucosamine to inoculate to the inclined plane, culturing for 20h at 37 ℃ in a biochemical incubator, then performing gradient dilution, coating on a complete culture medium (LB), respectively selecting single bacterial colonies to inoculate to the inclined plane, and culturing for about 12h at 30-36 ℃ in the biochemical incubator. Inoculated into a 500mL seed flask, culture conditions: the temperature is 30-36 ℃, the rotating speed of a shaking table is 200rmp, and the culture time is 6-8 h; when the OD value is about 1.0-2.0, inoculating into a 500ml fermentation bottle, wherein the inoculation amount is as follows: about 7%, culture conditions: when the temperature is 30-36 ℃, the rotating speed of a shaking table is 200rmp and the OD of the thalli is about 10-15, IPTG is added for induction, and the cultivation is continued for 10-20 h. After the fermentation is finished, the content of N-acetylglucosamine is detected to be 60.26g/L and 52.38 g/L. The yield of the N-acetylglucosamine is improved by 15.04 percent.
Example 2
Separately, Escherichia coli JL-GlcN and common N-acetylglucosamine-producing Escherichia coli were selected from a fresh slant and inoculated into 500mL seed flasks under the following culture conditions: the temperature is 30-36 ℃, the rotating speed of a shaking table is 200rmp, and the culture time is 6-8 h; when the OD value is about 1.0-4.0, inoculating the shake flask seeds with 10% of inoculum size into a 5L full-automatic fermentation tank filled with 3L fermentation medium for secondary seed culture. The culture temperature is 30-36 ℃, the tank pressure is 0.01-0.05MPa, the air flow is 1-8L/min, the stirring speed is 200-. Inoculating the second-level seeds into a 30L full-automatic fermentation tank filled with 18L fermentation medium at an inoculation amount of 10% to perform enzyme-producing fermentation. The fermentation temperature is 30-36 ℃, the tank pressure is 0.01-0.15MPa, the air flow is 5-50L/min, the stirring speed is 200-. The N-acetylglucosamine content was determined to be 180.74g/L and 140.63 g/L. The yield of N-acetylglucosamine is improved by 28.52%.
Example 3
Separately, Escherichia coli JL-GlcN and common N-acetylglucosamine-producing Escherichia coli were selected from a fresh slant and inoculated into 500mL seed flasks under the following culture conditions: the temperature is 30-36 ℃, the rotating speed of a shaking table is 200rmp, and the culture time is 6-8 h; when the OD value is about 1.0-4.0, inoculating the shake flask with 0.6m seed32m of fermentation Medium3And performing secondary seed culture in a fermentation tank. The culture temperature is 30-36 ℃, the tank pressure is 0.01-0.05MPa, and the air flow is 0.1-0.5m3The dissolved oxygen is maintained at 10-50% at a stirring speed of 100-300r/min, the pH value is controlled between 7.0-7.2, and the culture time is 5-8 h. Inoculating the second-level seeds with a seed size of 6m at 10%310m of fermentation Medium3Carrying out enzyme-producing fermentation in a fermentation tank. The fermentation temperature is 30-36 ℃, the tank pressure is 0.01-0.15MPa, and the air flow is 0.5-6m3At 300r/min and stirring speed of 100-. The N-acetylglucosamine content was determined to be 179.31g/L and 137.63 g/L. The yield of the N-acetylglucosamine is improved by 30.28 percent.
Example 4
Adjusting pH of the fermentation liquid to 2-5 with 2mo1/L hydrochloric acid, microfiltering with 30nm ceramic membrane, washing with purified water for 2-3 times to obtain supernatant, and passing the supernatant through 10000 molecular weight hollow fiber bundle membrane, wherein the recovery rate of N-acetylglucosamine is 95.3-98.4%. Passing the supernatant through a strong acid cation exchange column under the conditions of static adsorption: adding cationic resin according to the volume ratio of 20%, stirring and adsorbing for 1 h; dynamic adsorption: filling column resin according to 15% volume, feeding at the flow rate of 2BV/h, and washing the column with 0.5-1BV water at the final stage; the supernatant was collected. Adding activated carbon into the supernatant according to the volume ratio of 0.5-1% for decolorization for 0.5-2h at 60 ℃, and filtering to remove carbon. Collecting the obtained N-acetylglucosamine supernatant, and concentrating under vacuum at the rotation speed of 100-. And (3) cooling and crystallizing the N-acetylglucosamine concentrated solution. The extraction yield is 85.0-92.5%.
The screening method of the Escherichia coli JL-GlcN comprises the following steps:
1. directional breeding of escherichia coli: the N-acetylglucosamine high-yield strain is obtained by performing protoplast ultraviolet mutagenesis, ultraviolet mutagenesis and diethyl sulfate (DES) chemical mutagenesis on an original Escherichia coli strain for producing N-acetylglucosamine, and then separating and purifying (after gradient dilution, plate coating and culture, and then picking out single colony) a genetic marker endowed with N-acetylglucosamine resistance (which cannot be used as a C source). The protoplast ultraviolet mutagenesis method comprises the following steps: adding 3-5ml of the prepared protoplast into a dish with the diameter of 5cm, placing under an ultraviolet lamp with the power of 20w, vertically irradiating for 30-120s, sucking 0.2ml by using a pipette, coating the protoplast on a culture dish, and culturing for 36-60h at 28-35 ℃ in the dark. Chemical mutagenesis method of diethyl sulfate (DES): after the glutathione high-yield strain slant strain is subjected to primary seed culture, centrifugal sedimentation is carried out for 10min (3000 + 7000r \ min), thalli are collected, the thalli are washed for 2-3 times by using sterile water, the thalli are collected by centrifugation again, phosphate buffer solution with ph =7.0 is added to the original volume, treatment is carried out for 20-60min by using diethyl sulfate with the concentration of 1% (v \ v), and primary culture is carried out after dilution by using sterile water.
2. Genetic stability test: and (3) carrying out single colony separation on the N-acetylglucosamine high-yield strain obtained in the step (1), carrying out continuous shake flask subculture for 10 generations, carrying out a genetic marker experiment and a shake flask fermentation acid production experiment, and selecting a strain with stable genetic marker and acid production rate for further research. Shake flask passaging method: transferring the N-acetylglucosamine high-producing strain from the inclined plane into a flask, and transferring to a second generation flask after the strain is cultured to the logarithmic phase.
3. The Escherichia coli JL-GlcN was examined by the institute of microbiology of Chinese academy of sciences in morphology, physiological morphology, 16RS gene, and Antibiotics sensitivity, and the results showed that:
1) the complete 16srDNA sequence (the gene sequence is shown as SEQ NO. 1) of the JL-GlcN of the Escherichia coli and the recN sequence (the gene sequence is shown as SEQ NO. 2) are subjected to NCBI blast analysis, and the comparison result has 100 percent of similarity with the Escherichia coli. The results of the sequence alignment, combined with morphological and physiological biochemical features, indicate that they belong to the strain Escherichia coli (Escherichia coli).
2) The current cell state and the results of physicochemical experiments are as follows:
table 1:
table 2:
table 3:
comparative experiments related to the invention:
the fermentation experiments were divided into two groups, each group was run several times with 500ml shake flasks, 30L fermentors and 10m3Culturing in a fermentation tank, and taking an average value; the group A adopts the process of the invention, and the Escherichia coli JL-GlcN is used as a strain to ferment and produce the N-acetylglucosamine; group B adopts conventional process and common production of N-BEscherichia coli with glucosamine amido;
after fermentation is finished, respectively calculating the N-acetylglucosamine yield and the saccharic acid conversion rate of the two experiments; the specific calculation method comprises the following steps: n-acetylglucosamine yield = total mass of N-acetylglucosamine in the fermentation broth/volume of fermentation broth; saccharic acid conversion rate = (fermentation broth volume N-acetylglucosamine yield g/L)/total sugar consumption g of fermentation; the results are shown in Table 4:
table 4:
the experimental result shows that compared with the common process, the process method provided by the invention has the advantages that the yield of N-acetylglucosamine and the conversion rate of saccharic acid are both greatly improved, the process method is remarkably improved, and the process method is more suitable for process production.
SEQUENCE LISTING
<110> Henan Julong bioengineering GmbH
<120> Escherichia coli JL-GlcN and application thereof
<130>1
<160>2
<170>PatentIn version 3.3
<210>1
<211>1451
<212>DNA
<213> Escherichia coli
<400>1
acacatgcaa gtcgaacggt aacaggaagc aagcttgctt ctttgctgac gagtggcgga 60
cgggtgagta atgtctggga aactgcctga tggaggggga taactactgg aaacggtagc 120
taataccgca taacgtcgca agaccaaaga gggggacctt cgggcctctt gccatcggat 180
gtgcccagat gggattagct tgtaggtggg gtaacggctc acctaggcga cgatccctag 240
ctggtctgag aggatgacca gccacactgg aactgagaca cggtcagact cctacgggag 300
gcagcagtgg ggaatattgc acaatgggcg caagcctgat gcagccatgc cgcgtgtatg 360
aagaaggcct tcgggttgta aagtactttc agcggggagg aagggagtaa agttaatacc 420
tttgctcatt gacgttaccc gcagaagaag caccggctaa ctccgtgcca gcagccgcgg 480
taatacggag ggtgcaagcg ttaatcggaa ttactgggcg taaagcgcac gcaggcggtt 540
tgttaagtca gatgtgaaat ccccgggctc aacctgggaa ctgcatctga tactggcaag 600
cttgagtctc gtagaggggg gtagaattcc aggtgtagcg gtgaaatgcg tagagatctg 660
gaggaatacc ggtggcgaag gcggccccct ggacgaagac tgacgctcag gtgcgaaagc 720
gtggggagca aacaggatta gataccctgg tagtccacgc cgtaaacgat gtgacttgga 780
ggttgtgccc ttgaggcgtg gcttccggag ctaacgcgtt aagtgaccgc ctggggagta 840
cggccgcaag gttaaaactc aaatgaattg acgggggccc gcacaagcgg tggagcatgt 900
ggtttaattc gatgcaacgc gaagaacctt acctggtctt gaatccacgg aagttttcag 960
agatgagaat gtgccttcgg gaaccgtgag acaggtgctg catggctgtc gtcagctcgt 1020
gttgtgaaat gttgggttaa gtcccgcaac gagcgcaacc cttatccttt gttgccagcg 1080
gtccggccgg gaactcaaag gagactgcca gtgataaact ggaggaaggt ggggatgacg 1140
tcaagtcatc atggccctta cgaccagggc tacacacgtg ctacaatggc gcatacaaag 1200
agaagcgacc tcgcgagagc aagcggacct cataaagtgc gtcgtagtcc ggattggagt 1260
ctgcaactcg actccatgaa gtcggaatcg ctagtaatcg tggatcagaa tgccacggtg 1320
aatacgttcc cgggccttgt acacaccgcc cgtcacacca tgggagtggg ttgcaaaaga 1380
agtaggtagc ttaaccttcg ggagggcgct taccactttg tgattcatga ctggggtgaa 1440
gtcgtaacaa g 1451
<210>2
<211>959
<212>DNA
<213> Escherichia coli
<400>2
cgtgagcttg agattgattt tcatagcggc atgaccgtaa taactggcga gaccggcgcg 60
ggtaaatcta ttgcaataga tgccctcggt ctttgtctcg gtggtcgcgc tgaagccgac 120
atggtgcgta ccggcgctgc tcgcgctgac ctgtgcgccc gtttttctct gaaagatacg 180
ccagcggctc tgcgctggct ggaagaaaac cagcttgaag acgggcatga atgtttgctt 240
cgtcgcgtga tcagcagcga tggtcgctcc cgtggtttca tcaacggtac agctgttcct 300
ctgtcacaac tgcgcgaact gggtcagttg ctgattcaga tccatggtca gcacgctcat 360
caattactca ccaaacctga gcaccaaaaa ttcctgcttg atggctatgc caatgaaacc 420
tctctactgc aggaaatgac cgcacgttat cagttgtggc atcaaagctg ccgtgacctc 480
gcgcatcatc aacagttaag tcaggaacgc gccgcccgtg cggaactgct gcaataccaa 540
ttaaaagaac ttaacgaatt taatccgcag cccggagagt ttgaacaaat cgacgaagag 600
tacaaacgtc tggcgaacag cggtcaattg ctgaccacag ccagaatgca ttggcattaa 660
tggccgacgg tgaagacgca aacctgcaaa gtcagcttta cacggctaaa caactggtga 720
gcgaattgat tggcatggac agcaaactgt ccggcgtact tgatatgctg gaagaagcta 780
ccatccagat tgctgaagcc agcgatgaac tgcgccacta ctgcgatcgt ctggatctcg 840
atcccaaccg actatttgaa cttgaacagc gcatctcaaa acagatttcg ctggcacgta 900
aacatcacgt cagccctgag gcattgccac agtattacca gtcgctactg gaagaacag 959
Claims (3)
1. Escherichia coli JL-GlcN, characterized in that: classified and named as Escherichia coli (Escherichia coli), and has been preserved in China general microbiological culture Collection center (CGMCC), the strain preservation number is CGMCC NO.13924, and the preservation date is 2017, 3 months and 23 days.
2. Use of Escherichia coli JL-GlcN of claim 1 for the production of N-acetylglucosamine, wherein: the method for preparing N-acetylglucosamine by fermenting Escherichia coli as a strain comprises the following steps:
step one, activating a colon bacillus JL-GlcN by a slant, culturing for 20h in a biochemical incubator at 37 ℃, then performing gradient dilution, coating on an LB culture medium, selecting single colonies, respectively inoculating to the LB slant culture medium, and culturing for 12h in the biochemical incubator at 30-36 ℃; inoculating into a seed bottle filled with a seed culture medium, and culturing under the following conditions: the temperature is 30-36 ℃, the rotating speed of a shaking table is 200rmp, and the culture time is 6-8 h; when the OD value is 1.0-2.0, obtaining a seed culture solution;
the seed culture medium contains 20-30g/L of glucose and K2HPO410-20g/L, MgSO4·7H2O 0.5-3.0g/L，KH2PO410-20g/L, citric acid 0.5-3.0g/L, (NH)4)2SO45-10.0g/L, 5-10.0mL/L of microelement stock solution, the balance of water and pH 6.7;
the microelement stock solution contains FeSO in each liter of microelement stock solution41.0-3.0g，MnSO40.2-1.0g，ZnSO40.2-1.0g，CuSO40.2-1.0g and CoCl20.2-1.0g；
Inoculating the seed solution into a fermentation tank filled with a fermentation culture medium for culture, adding IPTG (isopropyl-beta-D-thiogalactoside) for induction when the OD (origin-degree of gravity) of the thallus is 10-15, and culturing for 10-20h to obtain a fermentation liquor containing N-acetylglucosamine; inoculation amount: 5 to 15 percent; the culture conditions are as follows: the initial fermentation temperature is 32-38 ℃, the dissolved oxygen is maintained at 10-30%, and the fermentation pH value is controlled between 7.0-7.2;
the fermentation medium contains 20-30g/L of glucose and K2HPO410-20g/L, MgSO4·7H2O0.5-3.0 g/L, citric acid 0.5-3.0g/L, (NH)4)2SO45-10.0g/L, 5-10.0mL/L of microelement stock solution and 7.0 pH; the microelement stock solution contains FeSO in each liter of microelement stock solution41.0-3.0g，MnSO40.2-1.0g，ZnSO40.2-1.0g，CuSO40.2-1.0g，CoCl20.2-1.0g；
And step three, extracting the N-acetylglucosamine from the fermentation liquor containing the N-acetylglucosamine.
3. Use of Escherichia coli JL-GlcN in the production of N-acetylglucosamine according to claim 2, wherein: the method for extracting the N-acetylglucosamine from the fermentation liquor containing the N-acetylglucosamine comprises the following steps: adjusting pH of the fermentation liquid containing N-acetylglucosamine to 2-5 with 2mo1/L hydrochloric acid, microfiltering with 30nm ceramic membrane, washing with purified water for 2-3 times, passing the supernatant through strong acid cation exchange column under static adsorption conditions: adding cationic resin according to the volume ratio of 20%, stirring and adsorbing for 1 h; dynamic adsorption: filling column resin according to 15% volume, feeding at the flow rate of 2BV/h, and washing the column with 0.5-1BV water at the final stage; collecting the supernatant; adding activated carbon into the supernatant according to the volume ratio of 0.5-1% for decolorization for 0.5-2h at 60 ℃; filtering to remove carbon, collecting the obtained N-acetylglucosamine supernatant, and vacuum concentrating to obtain N-acetylglucosamine; the vacuum concentration conditions are as follows: vacuum degree of-0.1 MPa, rotation speed of 100-.
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