CN108330094B - Free recombinant mycobacterium smegmatis producing nicotinic acid and construction method thereof - Google Patents
Free recombinant mycobacterium smegmatis producing nicotinic acid and construction method thereof Download PDFInfo
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Abstract
The invention discloses free recombinant mycobacterium smegmatis for producing nicotinic acid and a construction method thereof. The invention claims a recombinant mycobacterium smegmatis for producing nicotinic acid, which contains recombinant episomal plasmids containingnudCThe gene(s) is (are),nudCthe nucleotide sequence of the gene is shown as SEQ ID NO: 1 is shown. The engineering bacteria prepared by the method can directly obtain nicotinic acid in one step without depending on the addition of 3-cyanopyridine, so that various defects in the existing chemical synthesis process of the nicotinic acid are avoided, the production condition is simple, pollution is avoided, the method is simple and easy to implement, easy to amplify and low in cost, and the method is suitable for large-scale industrial production and application and has great popularization and application values.
Description
Technical Field
The invention relates to the technical field of production of nicotinic acid, and more particularly relates to free recombinant mycobacterium smegmatis for producing nicotinic acid and a construction method thereof.
Background
Nicotinic acid, 3-picolinic acid, also known as vitamin B3, belongs to the B vitamin group, is a water-soluble vitamin, belongs to the B vitamin group, and is one of the 13 vitamins essential to the human body. Nicotinic acid is an important factor for resisting a hypecopathy and a hydrogen carrier in body tissues, and has the effects of maintaining the health of skin and nerves and promoting digestion. In the absence of it, pellagra may occur, manifested as dermatitis, glossitis, oropharynx, diarrhea, dysphoria, insomnia, and paresthesia. Nicotinamide, collectively known as vitamin PP, is used for combating pellagra and also as a vasodilator. As a medical intermediate, is used for producing isoniazid, nicotinamide, nicousamide, inositol nicotinate and the like. Niacin is also used in food products, meat additives and feed additives to prevent pellagra. Especially, the feed added with nicotinic acid can improve the disease resistance of livestock and poultry, accelerate the growth, improve the utilization rate of the feed, save a large amount of feed and reduce the feeding cost. Compared with the control group, the milk yield of the dairy cows fed with the feed added with the nicotinic acid in the United states can be improved by 15 to 20 percent. In addition, nicotinic acid can also be used as a biochemical hormone for forming activated sludge, and a deodorant for air and exhaust gas. Nicotinic acid also has certain application in the dye industry, the photosensitive material industry, hair dyeing auxiliaries, detergents and the like.
At present, the chemical method is mainly used in the industry for producing nicotinic acid by a chemical synthesis method, the synthesis method mainly comprises a liquid phase method (a potassium permanganate oxidation method and a nitric acid oxidation method) and a gas phase method (an ozone oxidation method, an ammonia oxidation method and an air oxidation method), and the used raw materials mainly comprise 3-methylpyridine, 2-methyl-5-ethylpyridine, 3-cyanopyridine, alein and the like. The most common method at present is to synthesize nicotinic acid by air oxidation method by using 3-methylpyridine as a raw material. Introducing 3-methylpyridine, air and ammonia gas into a fluidized bed reactor in proportion, and reacting at 290-360 ℃ under the catalysis of V2O5 to generate nicotinonitrile; hydrolyzing in sodium hydroxide water solution at 160 deg.C under high pressure to obtain sodium nicotinate, and acidifying with hydrochloric acid to obtain nicotinic acid. In the synthesis process, the intermediate product of nicotinonitrile is needed, the nicotinonitrile is hydrolyzed into nicotinic acid through chemical reaction under the conditions of high temperature, high pressure, strong acid and strong alkali, so that the method has certain requirements on equipment, can corrode the equipment, and is not environment-friendly. Patent CN114288A of qinghua violet corporation reports that 3-methylpyridine is used to generate nicotinic acid in one step, the process is to react 3-methylpyridine with sulfuric acid to generate methylpyridine sulfate, then to generate nicotinic acid sulfate by oxidation under the action of nitric acid as oxidant, the nicotinic acid sulfate is added into alkali solution to prepare nicotinic acid, the yield of raw material is about 90%. In the process, strong acid and strong alkali substances such as sulfuric acid, nitric acid, alkali solution and the like are needed, and the pollution is serious.
In the chemical synthesis process of the nicotinic acid, a specific high-temperature and high-pressure environment is required or strong acid, strong base or a chemical catalyst is adopted for treatment, the reaction selectivity is not high, a large number of byproducts are generated, the product yield is not high, and the environmental pollution is large. In contrast, the biological method for preparing nicotinic acid has the characteristics of high substrate selectivity, high catalytic efficiency, mild reaction conditions, small environmental pollution and the like. In addition, the biological method is easy to amplify, has low cost and is suitable for large-scale industrial production and application. The biocatalytic production of nicotinic acid from 3-cyanopyridine has been reported using the microorganisms Bacillus subtilis, Rhodococcus rhodochrous, Nocardia, Fusarium solani and Pseudomonas putida for fermentation. However, the biocatalytic method for preparing nicotinic acid mainly relies on microbial fermentation to produce nitrilase, and the nitrilase catalyzes the conversion of 3-cyanopyridine into nicotinic acid by the nitrilase, and the raw material 3-cyanopyridine still needs to be added.
Disclosure of Invention
The technical problem to be solved by the invention is to overcome the defects of the existing nicotinic acid preparation technology and provide an engineering bacterium capable of directly producing nicotinic acid, wherein the engineering bacterium can expressnudCEngineering bacteria of genes. The engineering bacteria arenudCThe gene ORF sequence is connected into episomal plasmid and transformed into Mycobacterium smegmatis, and is overexpressednudCThe nicotinic acid can be obtained in a large amount from the bacterial liquid of the gene mycobacterium smegmatis.
The first purpose of the invention is to provide a recombinant mycobacterium smegmatis for producing nicotinic acid.
The second object of the present invention is to provide a method for preparing recombinant Mycobacterium smegmatis that produces nicotinic acid.
The third purpose of the invention is to provide the recombinant mycobacterium smegmatis for producing the nicotinic acid constructed by the method.
The fourth purpose of the invention is to provide the application of the recombinant mycobacterium smegmatis in the production of nicotinic acid.
A fifth object of the present invention is to provide a method for producing nicotinic acid.
In order to achieve the purpose, the invention is realized by the following technical scheme:
produce nicotinic acidThe recombinant Mycobacterium smegmatis of (1), which contains a recombinant episomal plasmid, wherein the recombinant episomal plasmid containsnudCThe gene(s) is (are),nudCthe nucleotide sequence of the gene is shown as SEQ ID NO: 1 is shown.
The recombinant Mycobacterium smegmatis is constructed bynudCRecombinant episomal plasmids of the gene sequences were transformed into M.smegmatis.
Preferably, the episomal plasmid is the pMV261 plasmid.
Preferably, the Mycobacterium smegmatis is Mycobacterium smegmatis mc2155。
Preferably, the transformation is performed by electric shock method, and the electric shock parameters are as follows: the voltage is 2.5kV, the resistance is 1000 Ω, and the capacitance is 25 μ F.
Preferably, the construction method of the recombinant mycobacterium smegmatis is divided into the following steps:
s1 preparation of Mycobacterium smegmatis mc2155 competent cells;
s2 transformation of Mycobacterium smegmatis mc2155；
S3, screening positive recombinant bacteria.
More preferably, the construction method of the recombinant Mycobacterium smegmatis is divided into the following steps:
s1, preparing the recombinant Mycobacterium smegmatis:
mixing Mycobacterium smegmatis mc2155 Single colony in 7H9 liquid medium, 37 degrees C200 rpm vibration culture to logarithmic growth phase (OD)6000.5 to 1.0); the culture is inoculated in a fresh 7H9 liquid culture medium according to the proportion of 1 (80-120), and cultured overnight at 37 ℃ to OD600Centrifuging at 4 deg.C and 5000rpm for 10 min to collect thallus, washing thallus with pre-cooled 10% sterile glycerol, adding 10 ml of pre-cooled 10% glycerol, resuspending thallus, and freezing at-80 deg.C.
S2, culturing the recombinant Mycobacterium smegmatis:
adding 200 mu l of Mycobacterium smegmatis mc into positive plasmid2155 electric transfer competent cells, incubating for 8-12 min on ice, transferring into a 2 mm BTX electric transfer cup, and wiping off the outer wall of the electric transfer cupThen shocked using a BTX ECM630 electrotransformer, the shock parameters being: the voltage is 2.5kV, the resistance is 1000 Ω, and the capacitance is 25 μ F. After electric shock, 1 ml of 7H9 liquid culture medium is immediately added, the culture medium is incubated overnight in an incubator at 37 ℃, then a proper amount of culture medium is taken out to coat a 7H10 solid plate (containing 40-60 mu g/ml kanamycin sulfate), and the plate is placed in the incubator at 37 ℃ for culture for 3-5 days.
S3, collecting and purifying nicotinic acid in the culture solution:
selecting monoclonals growing on a plate, inoculating the monoclonals into a 7H9 liquid culture medium, culturing for 2-3 days until the logarithmic phase, centrifugally collecting thalli, washing the thalli with sterile water, re-suspending the thalli with sterile water, boiling the thalli in boiling water for 10-20 minutes, centrifuging the thalli at 14000-16000 rpm for 15 minutes, taking supernate as a DNA template of PCR, wherein the primer pair used in the PCR is as follows: 5'-GTGGCAGCGAGGACAACTTG-3', 5'-GATGCCTGGCAGTCGATCGTAC-3', PCR verification of plasmid transfer into M.smegmatis.
The recombinant mycobacterium smegmatis for producing nicotinic acid constructed by the method also belongs to the protection scope of the invention.
The application of the recombinant mycobacterium smegmatis for producing the nicotinic acid in the production of the nicotinic acid also belongs to the protection scope of the invention.
A method for producing niacin, comprising the steps of:
s1, preparing the recombinant mycobacterium smegmatis;
s2, culturing the recombinant mycobacterium smegmatis;
s3, collecting and purifying the nicotinic acid in the culture solution.
Preferably, in the step S2, the liquid medium 7H9 is used for the cultivation.
Preferably, in step S3, the collecting nicotinic acid in the culture solution specifically comprises: filtering the culture solution with sterile filter, collecting filtrate, removing protein and other macromolecular substances in the filtrate with ultrafiltration concentration tube, collecting filtrate, performing high performance liquid chromatography, and separating nicotinic acid.
Preferably, the parameters of the high performance liquid chromatography are, column: thermo Fisher, Hypersil Goldaq, 150X 4.6mm, 3 μm; column temperature: 30 ℃; mobile phase: the water phase is deionized water containing 0.1% formic acid, and the organic phase is methanol; gradient elution: at 0min, 95% water phase +5% organic phase, and at 30min, 5% water phase +95% organic phase; flow rate: 0.3 mL/min; sample introduction amount: 10 mu L of the solution; wavelength: 284 nm.
Compared with the prior art, the invention has the following beneficial effects:
the engineering bacteria prepared by the invention can directly obtain nicotinic acid in one step without depending on the addition of 3-cyanopyridine, thereby not only avoiding various defects in the existing chemical synthesis process of the nicotinic acid, but also having simple production conditions, no pollution, simplicity, convenience, practicability, easy amplification and low cost, and being suitable for large-scale industrial production and application.
Drawings
FIG. 1 shows pMV261-NudCmsThe plasmid was digested with BamHI and HindIII restriction enzymes followed by nucleic acid gel electrophoresis, M: DNA marker; 1-3: and (3) positive plasmids.
FIG. 2 shows pMV261-NudCmsSchematic representation of the plasmid.
FIG. 3 shows the nucleic acid gel electrophoresis of the PCR amplification product; m: DNA marker; 1-3: positive clones.
FIG. 4 is a HPLC separation chromatogram of control strain, over-expression strain culture solution filtrate and nicotinic acid standard; a is control strain Mycobacterium smegmatis mc2155 culture solution filtrate; b: mycobacterium smegmatis mc overexpressing the NudC gene2155-pMV261-NudCmsA culture solution filtrate; c: and (4) a nicotinic acid standard substance.
Detailed Description
The invention is described in further detail below with reference to the drawings and specific examples, which are provided for illustration only and are not intended to limit the scope of the invention. The test methods used in the following examples are all conventional methods unless otherwise specified; the materials, reagents and the like used are, unless otherwise specified, commercially available reagents and materials.
Example 1nudCGene amplification and construction of plasmid pMV261-NudCms
1、nudCGene amplification
Using Mycobacterium smegmatis mc2155 genomic DNA as template, amplified using primer pair 5'-ATCGGGATCCATGAGCGAACACCGCACGT-3'/5'-TGCAAAGCTTTCAGTCGAGTGCGGCCCAGG-3', PCRnudCGene ORF sequence. Separating target DNA fragment from PCR product by nucleic acid gel electrophoresis, recovering target DNA fragment with Omega gel recovery kit, and sequencing to obtainnudCThe gene ORF sequence is shown as SEQ ID NO: 1, and the amino acid sequence of the encoded protein is shown as SEQ ID NO: 2. the recovered DNAs were digested with BamHI and HindIII restriction enzymes and recovered by Omega Cycle-pure recovery kit for use.
2. Enzyme digestion
The Escherichia coli DH5 α strain containing pMV261 plasmid was cultured in LB, plasmid was extracted using Omega plasmid extraction kit, the pMV261 plasmid obtained was digested with BamHI and Hind III restriction enzymes, nucleic acid gel electrophoresis was performed, and linearized plasmid was recovered using Omega gel recovery kit.
3. Connection of
The recovered DNA fragment was digested with the linearized plasmid pMV261 recovered by the same digestion and reacted with T4 DNA ligase at 16 ℃ overnight, the ligation product transformed E.coli DH5 α competent cells and plated on LB solid plate (containing 100. mu.g/ml kanamycin sulfate), which was incubated overnight in a 37 ℃ incubator.
4. Screening for Positive plasmids
The monoclonal colonies growing on the plates were picked and inoculated into LB liquid medium and cultured overnight with shaking in a shaker at 200rpm at 37 ℃. Then extracting the plasmid by using a plasmid extraction kit of Omega company, carrying out nucleic acid gel electrophoresis detection and verification on the obtained plasmid after the obtained plasmid is cut by using BamHI and Hind III restriction enzymes,nudCthe gene ORF sequence was ligated into the pMV261 vector (see FIG. 1). Verification of the correct plasmid pMV261-NudCmsThe sequencing of the Oncorhynchus company verifies that the sequence has no mutation, and successfully constructs the pMV261-NudCmsThe plasmid map is shown in FIG. 2.
Example 2 construction of NudC overexpressing Mycobacterium smegmatis strains
1. Preparation of Mycobacterium smegmatis mc2155 competent cell
Picking fresh M.smegmatis mc2155 single colony is inoculated in 5ml of 7H9 liquid culture medium, and is subjected to shaking culture at the temperature of 37 ℃ and the rpm of 200 until the logarithmic phase (OD 0.5-1.0) is reached; the culture was inoculated into 100 ml of fresh 7H9 liquid medium at a ratio of 1:100, and cultured overnight at 37 ℃ to OD600Centrifuging at 4 deg.C and 5000rpm for 10 min to collect thallus, washing thallus with pre-cooled 10% sterile glycerol at least twice, adding 10 ml (appropriate amount) of pre-cooled 10% glycerol, resuspending thallus, and freezing at-80 deg.C.
2. Transformation of Mycobacterium smegmatis mc2155
Taking a positive plasmid pMV261-NudC with correct constructionmsAdding 200 mu l of Mycobacterium smegmatis mc into the DNA2155 in the electric transfer competent cells, incubating for 10 min on ice, then transferring into a 2 mm BTX electric transfer cup, wiping off water on the outer wall of the electric transfer cup, and then using a BTX ECM630 electric converter to shock, wherein the shock parameters are as follows: the voltage is 2.5kV, the resistance is 1000 Ω, and the capacitance is 25 μ F. After electric shock, 1 ml of 7H9 liquid medium was immediately added, incubated overnight in an incubator at 37 ℃ and then an appropriate amount of the culture was spread on a 7H10 solid plate (containing 50. mu.g/ml kanamycin sulfate), and the plate was incubated in an incubator at 37 ℃ for 3 to 5 days.
3. Screening positive recombinant bacteria
Selecting monoclonals grown on a plate, inoculating the monoclonals into 5mL of 7H9 liquid culture medium, culturing for 2-3 days until logarithmic phase, centrifugally collecting thalli, washing the thalli for 2 times by using sterile water, boiling the thalli for 15 minutes after re-suspending the thalli by using sterile water, centrifuging the thalli for 15 minutes at 15000rpm, taking supernatant as a DNA template of PCR (polymerase chain reaction), using a primer pair 5'-GTGGCAGCGAGGACAACTTG-3', 5'-GATGCCTGGCAGTCGATCGTAC-3' for PCR, taking extracted genomic DNA as a template, and verifying pMV261-NudC by PCRmsThe plasmid was transferred into M.smegmatis (PCR results are shown in FIG. 3). Will verify the correct NudCmsThe overexpressed strain was named Mycobacterium smegmatis mc2155-pMV261-NudCms。
Example 3 recombinant Mycobacterium smegmatis mc2155-pMV261-NudCmsSecretion of nicotinic acid
1. M. smegmatisc2155-pMV261-NudCmsAfter inoculating 7H9 liquid medium and culturing at 37 ℃ to a logarithmic growth phase, the culture solution was filtered using a Millipore 0.22 μm sterile filter and the filtered filtrate was collected, and then macromolecules such as proteins in the filtrate were removed using a Millipore3-kDa ultrafiltration concentration tube and the filtrate after filtration was collected. The obtained filtrate was separated and analyzed for nicotinic acid using High Performance Liquid Chromatography (HPLC).
Specific conditions of HPLC:
the equipment model is as follows: thermo Fisher Scientific UltiMate 3000 ultra-high-performance chromatography
A chromatographic column: thermo Fisher, Hypersil Gold aQ, 150X 4.6mm, 3 μm, column temperature: 30 ℃;
mobile phase: water phase: deionized water (containing 0.1% formic acid); organic phase: methanol;
gradient elution: at 0min, 95% aqueous phase +5% organic phase;
at 30min, 5% aqueous phase +95% organic phase;
flow rate: 0.3 mL/min;
sample introduction amount: 10 mu L of the solution;
wavelength: 284 nm.
2. HPLC results showed M.smegmatis mc2155-pMV261-NudCmsThe culture medium contains nicotinic acid (see FIG. 4).
Sequence listing
<110> Shanghai Jingnuo Biotech Co., Ltd
<120> free recombinant mycobacterium smegmatis for producing nicotinic acid and construction method thereof
<160>2
<170>SIPOSequenceListing 1.0
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<211>936
<212>DNA
<213> Mycobacterium smegmatis (Mycobacterium smegmatis)
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atgagcgaac accgcacgtt cgggctccgt aacgtcccgc tgctgtcccg ggtcggcgcc 60
gatcgcgccg ataccttgcg caccgacgtc gacgccgccc tggcgggctg gcccgacgcg 120
ctggtgctac gcgtggaccg ccgcaaccag gtgctcatcg ccaacggtca ggtggtgctc 180
ggtgaggccg gcgcactcgg agaccggccg cccgagcacg cggtgttcct gggacgtctg 240
caggacggca ggcacgtatg gggtatccgg gcggatctgg aggcgcccga ggatgccgac 300
ctggggaccg aggtgctcga cctgcgccgg gccgggcaga tcttcgacga caccagcgcc 360
cagttggtgg cgaccgccac ggcgctgctc aactggcatg acaacgcgcg gcacagcgcg 420
atcgacgggg cgccgacccg gcccgccaag ggcggctggt cgcgcgtcaa cccgctgacc 480
ggccacgagg agttcccgcg gatcgacccc gccatcatct gcctggtgca cgacgggcat 540
gaccgggcgg tgctggcccg tcagacgctg tggccggagc ggttgttctc gatcctggcc 600
ggcttcgtcg aggccggcga gtcgttcgag acatgcgtgc agcgcgagat cgccgaggag 660
atcgggctca cggtcaccga cgtgcagtac ctcggcagtc agccgtggcc gttcccgcgc 720
tcgctcatgg tgggattcca cgcgatcggc gacccggagc agccgttctc ctacaacgac 780
ggcgagatcg ccgaggccgc gtggttcacc cgcgatgaga tccgcgcggc actcgatcag 840
ggggactgga acagcgactc gccgtcacgg ctcctgctgc caggctccat ctcgatcgcc 900
cgcgagatca tcgaatcctg ggccgcactc gactga 936
<210>2
<211>311
<212>PRT
<213> Mycobacterium smegmatis (Mycobacterium smegmatis)
<400>2
Met Ser Glu His Arg Thr Phe Gly Leu Arg Asn Val Pro Leu Leu Ser
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Arg Val Gly Ala Asp Arg Ala Asp Thr Leu Arg Thr Asp Val Asp Ala
20 25 30
Ala Leu Ala Gly Trp Pro Asp Ala Leu Val Leu Arg Val Asp Arg Arg
35 40 45
Asn Gln Val Leu Ile Ala Asn Gly Gln Val Val Leu Gly Glu Ala Gly
50 55 60
Ala Leu Gly Asp Arg Pro Pro Glu His Ala Val Phe Leu Gly Arg Leu
65 70 75 80
Gln Asp Gly Arg His Val Trp Gly Ile Arg Ala Asp Leu Glu Ala Pro
85 90 95
Glu Asp Ala Asp Leu Gly Thr Glu Val Leu Asp Leu Arg Arg Ala Gly
100 105 110
Gln Ile Phe Asp Asp Thr Ser Ala Gln Leu Val Ala Thr Ala Thr Ala
115 120 125
Leu Leu Asn Trp His Asp Asn Ala Arg His Ser Ala Ile Asp Gly Ala
130 135 140
Pro Thr Arg Pro Ala Lys Gly Gly Trp Ser Arg Val Asn Pro Leu Thr
145 150 155 160
Gly His Glu Glu Phe Pro Arg Ile Asp Pro Ala Ile Ile Cys Leu Val
165 170 175
His Asp Gly His Asp Arg Ala Val Leu Ala Arg Gln Thr Leu Trp Pro
180 185 190
Glu Arg Leu Phe Ser Ile Leu Ala Gly Phe Val Glu Ala Gly Glu Ser
195 200 205
Phe Glu Thr Cys Val Gln Arg Glu Ile Ala GluGlu Ile Gly Leu Thr
210 215 220
Val Thr Asp Val Gln Tyr Leu Gly Ser Gln Pro Trp Pro Phe Pro Arg
225 230 235 240
Ser Leu Met Val Gly Phe His Ala Ile Gly Asp Pro Glu Gln Pro Phe
245 250 255
Ser Tyr Asn Asp Gly Glu Ile Ala Glu Ala Ala Trp Phe Thr Arg Asp
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Glu Ile Arg Ala Ala Leu Asp Gln Gly Asp Trp Asn Ser Asp Ser Pro
275 280 285
Ser Arg Leu Leu Leu Pro Gly Ser Ile Ser Ile Ala Arg Glu Ile Ile
290 295 300
Glu Ser Trp Ala Ala Leu Asp
305 310
Claims (9)
1. The recombinant mycobacterium smegmatis for producing nicotinic acid is characterized by comprising a recombinant episomal plasmid, wherein the recombinant episomal plasmid contains a nucc gene, and the nucleotide sequence of the nucc gene is shown as SEQ ID NO: 1 is shown.
2. The method of constructing recombinant Mycobacterium smegmatis of claim 1, wherein the recombinant episomal plasmid containing a nucC gene sequence is transformed into Mycobacterium smegmatis.
3. The method of claim 2, wherein the episomal plasmid is the pMV261 plasmid.
4. The method of claim 2, wherein the transformation is performed by electrical shock, and the shock parameters are: voltage 2.5kV, resistance 1000 Ω, capacitance 25 μ F.
5. Use of the recombinant Mycobacterium smegmatis of claim 1 for the production of niacin.
6. A method for producing nicotinic acid is characterized by comprising the following steps,
s1, preparing the recombinant Mycobacterium smegmatis of claim 1;
s2, culturing the recombinant mycobacterium smegmatis;
and S3, collecting and purifying nicotinic acid in the culture solution.
7. The method according to claim 6, wherein in step S2, the culture is performed using a 7H9 liquid medium.
8. The method according to claim 6, wherein the step S3 of collecting nicotinic acid from the culture solution comprises: filtering the culture solution with sterile filter, collecting filtrate, removing protein and other macromolecular substances in the filtrate with ultrafiltration concentration tube, collecting filtrate, performing high performance liquid chromatography, and separating nicotinic acid.
9. The method of claim 8, wherein the parameters of the high performance liquid chromatography are, column: thermo Fisher, Hypersil Gold aQ, 150X 4.6mm, 3 μm; column temperature: 30 ℃; mobile phase: the water phase is deionized water containing 0.1% formic acid, and the organic phase is methanol; gradient elution: at 0min, 95% water phase +5% organic phase, and at 30min, 5% water phase +95% organic phase; flow rate: 0.3 mL/min; sample introduction amount: 10 mu L of the solution; wavelength: 284 nm.
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|---|---|---|---|---|
| CN101326292A (en) * | 2005-12-08 | 2008-12-17 | 诺瓦提斯公司 | Effects of inhibitors of FGFR3 on gene transcription |
| WO2009045116A1 (en) * | 2007-10-05 | 2009-04-09 | University Of Otago | Detection of volatile compounds as markers for mycobacteria tuberculosis |
| CN105283554A (en) * | 2013-06-05 | 2016-01-27 | 朗泽科技新西兰有限公司 | Recombinant microorganisms exhibiting increased flux through a fermentation pathway |
| CN107217056A (en) * | 2017-08-04 | 2017-09-29 | 遵义医学院 | The structure and authentication method of taeniasis suis Recombinant Lactococcus lactis secretion type expression system |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101326292A (en) * | 2005-12-08 | 2008-12-17 | 诺瓦提斯公司 | Effects of inhibitors of FGFR3 on gene transcription |
| WO2009045116A1 (en) * | 2007-10-05 | 2009-04-09 | University Of Otago | Detection of volatile compounds as markers for mycobacteria tuberculosis |
| CN105283554A (en) * | 2013-06-05 | 2016-01-27 | 朗泽科技新西兰有限公司 | Recombinant microorganisms exhibiting increased flux through a fermentation pathway |
| CN107217056A (en) * | 2017-08-04 | 2017-09-29 | 遵义医学院 | The structure and authentication method of taeniasis suis Recombinant Lactococcus lactis secretion type expression system |
Non-Patent Citations (4)
| Title |
|---|
| Investigations on the inducible nicotinic acid hydroxylase formation in Mycobacterium smegmatis;Bonicke,R et al.;《Pneumonologie》;19701231;第142卷(第2期);第173-181页 * |
| Mycobacterium smegmatis str. MC2 155, complete genome,GenBank: CP009494.1;Mohan,A.et al.;《GenBank》;20150226;第1-2页 * |
| NADH pyrophosphatase NudC，UniProtKB-I7G5D5;EMBL;《EMBL》;20170510;第1-2页 * |
| 基于挥发性代谢产物质谱快速鉴定结核分枝杆菌复合群;刘衍伶等;《临床检验杂志》;20160831;第34卷(第8期);第571-574页 * |
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