CN118064334A - Paenibacillus polymyxa T9 and application thereof - Google Patents
Paenibacillus polymyxa T9 and application thereof Download PDFInfo
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- CN118064334A CN118064334A CN202410478723.6A CN202410478723A CN118064334A CN 118064334 A CN118064334 A CN 118064334A CN 202410478723 A CN202410478723 A CN 202410478723A CN 118064334 A CN118064334 A CN 118064334A
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- paenibacillus polymyxa
- pyrrolidone
- gamma
- aminobutyric acid
- conversion
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- 241000194105 Paenibacillus polymyxa Species 0.000 title claims abstract description 44
- BTCSSZJGUNDROE-UHFFFAOYSA-N gamma-aminobutyric acid Chemical compound NCCCC(O)=O BTCSSZJGUNDROE-UHFFFAOYSA-N 0.000 claims abstract description 86
- 238000006243 chemical reaction Methods 0.000 claims abstract description 52
- HNJBEVLQSNELDL-UHFFFAOYSA-N pyrrolidin-2-one Chemical compound O=C1CCCN1 HNJBEVLQSNELDL-UHFFFAOYSA-N 0.000 claims abstract description 52
- 229960003692 gamma aminobutyric acid Drugs 0.000 claims abstract description 50
- OGNSCSPNOLGXSM-UHFFFAOYSA-N (+/-)-DABA Natural products NCCC(N)C(O)=O OGNSCSPNOLGXSM-UHFFFAOYSA-N 0.000 claims abstract description 36
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 32
- 229910052799 carbon Inorganic materials 0.000 claims description 18
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 17
- 229910052757 nitrogen Inorganic materials 0.000 claims description 16
- 239000001963 growth medium Substances 0.000 claims description 15
- 108090000790 Enzymes Proteins 0.000 claims description 13
- 102000004190 Enzymes Human genes 0.000 claims description 13
- 238000000034 method Methods 0.000 claims description 13
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 claims description 12
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 claims description 11
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims description 10
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- NLXLAEXVIDQMFP-UHFFFAOYSA-N Ammonia chloride Chemical compound [NH4+].[Cl-] NLXLAEXVIDQMFP-UHFFFAOYSA-N 0.000 claims description 7
- 239000001888 Peptone Substances 0.000 claims description 7
- 108010080698 Peptones Proteins 0.000 claims description 7
- 235000019319 peptone Nutrition 0.000 claims description 7
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 6
- 239000007836 KH2PO4 Substances 0.000 claims description 6
- 239000001110 calcium chloride Substances 0.000 claims description 6
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 6
- BNIILDVGGAEEIG-UHFFFAOYSA-L disodium hydrogen phosphate Chemical compound [Na+].[Na+].OP([O-])([O-])=O BNIILDVGGAEEIG-UHFFFAOYSA-L 0.000 claims description 6
- 229910000397 disodium phosphate Inorganic materials 0.000 claims description 6
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- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 claims description 6
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 claims description 5
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- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 claims description 4
- VBIXEXWLHSRNKB-UHFFFAOYSA-N ammonium oxalate Chemical compound [NH4+].[NH4+].[O-]C(=O)C([O-])=O VBIXEXWLHSRNKB-UHFFFAOYSA-N 0.000 claims description 4
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 claims description 4
- 229910052921 ammonium sulfate Inorganic materials 0.000 claims description 4
- 235000011130 ammonium sulphate Nutrition 0.000 claims description 4
- 239000004202 carbamide Substances 0.000 claims description 4
- 235000013877 carbamide Nutrition 0.000 claims description 4
- MNNHAPBLZZVQHP-UHFFFAOYSA-N diammonium hydrogen phosphate Chemical compound [NH4+].[NH4+].OP([O-])([O-])=O MNNHAPBLZZVQHP-UHFFFAOYSA-N 0.000 claims description 4
- 229910000388 diammonium phosphate Inorganic materials 0.000 claims description 4
- 235000019838 diammonium phosphate Nutrition 0.000 claims description 4
- 239000008101 lactose Substances 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 108090000623 proteins and genes Proteins 0.000 claims description 4
- 108020004465 16S ribosomal RNA Proteins 0.000 claims description 2
- 235000019270 ammonium chloride Nutrition 0.000 claims description 2
- KLOIYEQEVSIOOO-UHFFFAOYSA-N carbocromen Chemical compound CC1=C(CCN(CC)CC)C(=O)OC2=CC(OCC(=O)OCC)=CC=C21 KLOIYEQEVSIOOO-UHFFFAOYSA-N 0.000 claims description 2
- 235000011187 glycerol Nutrition 0.000 claims 1
- 244000005700 microbiome Species 0.000 abstract description 2
- 230000000694 effects Effects 0.000 description 20
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- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 3
- KGBXLFKZBHKPEV-UHFFFAOYSA-N boric acid Chemical compound OB(O)O KGBXLFKZBHKPEV-UHFFFAOYSA-N 0.000 description 3
- 239000004327 boric acid Substances 0.000 description 3
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- 241000894006 Bacteria Species 0.000 description 2
- 239000005696 Diammonium phosphate Substances 0.000 description 2
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- 238000001212 derivatisation Methods 0.000 description 2
- 238000001514 detection method Methods 0.000 description 2
- YXVFQADLFFNVDS-UHFFFAOYSA-N diammonium citrate Chemical compound [NH4+].[NH4+].[O-]C(=O)CC(O)(C(=O)O)CC([O-])=O YXVFQADLFFNVDS-UHFFFAOYSA-N 0.000 description 2
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 2
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- WHUUTDBJXJRKMK-UHFFFAOYSA-N Glutamic acid Natural products OC(=O)C(N)CCC(O)=O WHUUTDBJXJRKMK-UHFFFAOYSA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
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- ZWLUXSQADUDCSB-UHFFFAOYSA-N phthalaldehyde Chemical compound O=CC1=CC=CC=C1C=O ZWLUXSQADUDCSB-UHFFFAOYSA-N 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention belongs to the technical field of microorganisms, and particularly relates to paenibacillus polymyxa T9 and application thereof. The Paenibacillus polymyxa T9 (Paenibacillus polymyxa T) is preserved in China center for type culture Collection (CCTCC NO) at the year 2022, month 05 and 24: m2022706. The invention provides a Paenibacillus polymyxa T9, wherein the Paenibacillus polymyxa T9 can convert 2-pyrrolidone into gamma-aminobutyric acid, and according to the experimental result, the conversion rate is highest and reaches 86.0% when the concentration of the 2-pyrrolidone is 5.0 g/L, and the strain can tolerate the 2-pyrrolidone with the concentration of 20 g/L, so that the Paenibacillus polymyxa T9 has good industrial application prospect.
Description
Technical Field
The invention belongs to the technical field of microorganisms, and particularly relates to paenibacillus polymyxa T9 and application thereof.
Background
Gamma-aminobutyric acid (Gamma-aminobutyric acid, GABA) is a four-carbon, non-protein natural amino acid widely distributed in nature, has a molecular formula of C 4H9NO2 and a relative molecular weight of 103.12, has a plurality of important physiological activities, is an important inhibitory neurotransmitter of the central nervous system in a human body, participates in the regulation of various vital activities, and has various functions of resisting diabetes, reducing blood pressure, resisting arrhythmia, resisting anxiety, treating epilepsy, regulating hormone secretion, improving liver and kidney functions, promoting sleep, regulating intestinal microbiota and the like; the GABA can improve nutrient metabolism of weaned pigs and improve immunity and oxidation resistance of weaned pigs; GABA can promote the growth of chicken and fish and improve the immunity; GABA is a main metabolite of plants resisting adversity stress, is accumulated in a large amount in the plants under adversity stress, is likely to improve the adaptability of the plants to adversity stress, and has the research that GABA treatment can enhance the cold resistance of the post-harvest refrigerated guava fruits and maintain higher storage quality; GABA has been added to many foods such as green tea, wheat, distiller's yeast and the like and processed materials thereof, and has been used as a bioactive substance in various medicines and foods. Therefore, GABA has wide application in the fields of food and pharmaceutical chemicals.
Since GABA is naturally present in a low amount, it is difficult to separate and extract a large amount from some natural animal and plant tissues, and the methods for obtaining GABA at present mainly include chemical synthesis methods, plant enrichment methods and microbial transformation methods. The chemical synthesis method has serious environmental pollution and poor safety. The plant enrichment method is not suitable for mass production due to the low GABA content. The microbial conversion method for producing GABA has the advantages of short period, low production cost, high safety and high yield, and is an ideal way for industrially producing GABA in large scale. However, the current microbial transformation method uses glutamic acid as a substrate, and a large amount of grains are consumed.
Therefore, the invention provides a method for converting gamma-aminobutyric acid to solve the problems in the prior art, which is very significant.
Disclosure of Invention
The invention aims to solve the defects in the prior art, provides a Paenibacillus polymyxa T9 and application thereof, and adopts the following technical scheme:
The invention utilizes 2-pyrrolidone to screen a strain of Paenibacillus polymyxa T9, wherein the name of Paenibacillus polymyxa T9 (Paenibacillus polymyxa T) is preserved in China Center for Type Culture Collection (CCTCC) in 2022, 05 and 24 days, and the preservation number is CCTCC NO: m2022706. The gene sequence of the 16S rDNA of the Paenibacillus polymyxa T9 is shown as SEQ ID No: 1.
The invention also provides application of the Paenibacillus polymyxa T9 in preparing gamma-aminobutyric acid. Preferably, paenibacillus polymyxa T9 is prepared by converting 2-pyrrolidone to gamma-aminobutyric acid.
The invention also provides a preparation method of the gamma-aminobutyric acid, which comprises the following steps:
Inoculating the Paenibacillus polymyxa T9 into a liquid enzyme-producing culture medium for fermentation culture, then adding a 2-pyrrolidone solution, and carrying out conversion at 20-60 ℃ for 12 h-48 h to obtain gamma-aminobutyric acid;
The liquid enzyme-producing culture medium comprises the following components: 10 g/L of carbon source, 10g of nitrogen source 2 g/L,Na2HPO4·12H2O 5 g,KH2PO42.0 g,MgSO4·7H2O 1.0 g, CaCl2·2H2O 10 mg,FeSO4·7H2O 8 mg,2- pyrrolidone and pH value of 3-10.
As a further preferred embodiment, the carbon source comprises any one of glucose, lactose, soluble starch, citric acid, sucrose, glycerol. More preferably, the carbon source is glucose.
As a further preferred embodiment, the nitrogen source comprises any one of ammonium chloride, peptone, urea, ammonium sulfate, diammonium hydrogen phosphate, ammonium oxalate, diammonium hydrogen citrate. More preferably, the nitrogen source is peptone.
As a further preferred embodiment, the concentration of 2-pyrrolidone is from 5 g/L to 10.0 g/L. More preferably, the concentration of 2-pyrrolidone is 5 g/L.
As a further preferable embodiment, the fermentation culture temperature is 20℃to 40 ℃. More preferably, the temperature of the fermentation culture is 30 ℃.
As a further preferred embodiment, the above-mentioned conversion temperature is 30 ℃.
The beneficial effects of the invention are as follows:
The invention provides a Paenibacillus polymyxa T9, wherein the Paenibacillus polymyxa T9 can convert 2-pyrrolidone into gamma-aminobutyric acid, and according to the experimental result, the conversion rate is highest and reaches 86.0% when the concentration of the 2-pyrrolidone is 5.0 g/L, and the strain can tolerate the 2-pyrrolidone with the concentration of 20 g/L, so that the Paenibacillus polymyxa T9 has good industrial application prospect.
Biological preservation information
Paenibacillus polymyxa (Paenibacillus polymyxa) T9, deposited at the China center for type culture Collection, at a location of: eight paths of 299 of Wuchang district of Wuhan, hubei province are provided, the preservation number is CCTCC NO: m2022706.
Drawings
FIG. 1 shows a colony morphology of Paenibacillus polymyxa Paenibacillus polymyxaT.
Detailed Description
The conception, specific structure, and technical effects produced by the present application will be clearly and completely described below with reference to the embodiments and the drawings to fully understand the objects, aspects, and effects of the present application. It should be noted that, without conflict, the embodiments of the present application and features of the embodiments may be combined with each other.
Example 1
Screening and related experiments of Paenibacillus polymyxa (Paenibacillus polymyxa) T9
(1) Screening the culture medium:
Flat-plate screening medium (LB)：Na2HPO4·12H2O 2.0g,KH2PO42.0 g,MgSO4·7H2O 0.5 g,CaCl2·2H2O 10 mg,FeSO4·7H2O 8 mg,ZnSO4·7H2O 0.1 mg,NH4Cl 3 g,2－ pyrrolidone 5g, agar powder 15 g, pH 7.0, and sterilization at 121℃20 min.
Strain preservation medium (slant): the following ingredients were added per liter except for the unchanged ingredients in the screening medium: yeast extract 0.2g, soluble starch 0.2g, tryptone 0.2 g;
Liquid enzyme-producing medium (LB)：Na2HPO4·12H2O 5 g,KH2PO42.0 g,MgSO4·7H2O 1.0 g,CaCl2·2H2O 10 mg,FeSO4·7H2O 8 mg,NH4Cl 2 g,2- pyrrolidone 10 g, glucose 10 g, pH 7.0, and sterilization at 121℃20min.
Carbon source optimization culture medium: different carbon sources 10 g/L,Na2HPO4·12H2O 5 g,KH2PO42.0 g,MgSO4·7H2O 1.0 g,CaCl2·2H2O 10 mg,FeSO4·7H2O 8 mg,NH4Cl 2 g,2- pyrrolidone 10 g, pH 7.0, sterilization 20. 20 min at 121 ℃; different carbon sources include glucose, lactose, soluble starch, citric acid, sucrose and glycerol.
Nitrogen source optimization culture medium: different nitrogen sources 2 g/L,Na2HPO4·12H2O 5 g,KH2PO42.0 g,MgSO4·7H2O 1.0 g, CaCl2·2H2O 10 mg,FeSO4·7H2O 8 mg,2- pyrrolidone 10g, glucose 10 g/L, pH 7.0, sterilization at 121 ℃ 20 min; different nitrogen sources include peptone, urea, ammonium sulfate, diammonium phosphate, ammonium oxalate and diammonium citrate.
(2) Screening of Paenibacillus polymyxa T9:
a: screening culture of strain in soil
Taking underwater silt of a Nanchang Aixi lake wetland park, carrying out gradient dilution on the underwater silt by using sterile water to obtain 10 -1、10-2 and 10 -3, respectively coating the diluted underwater silt on a plate screening culture medium, and culturing the diluted underwater silt at 30 ℃; after 2d, the strain growth was observed on the plate, the single colonies were kept on the medium slope, the strain was grown successively after 2d, and the strain was taken out and kept in a refrigerator at 4 ℃.
B: liquid transformation culture and pretreatment of strain
Inoculating bacteria on the inclined plane into a liquid enzyme-producing culture medium, culturing at 30 ℃ at 220 rpm/min, diluting 0.1 mL with water for 10 times after culturing at 48: 48 h, and measuring the absorbance (OD value) of the bacteria at 660: 660 nm by taking a blank culture medium as a control; and centrifuging the residual bacterial liquid to collect bacterial cells.
C: conversion of 2-pyrrolidone by Paenibacillus polymyxa T9
Preparing 2-pyrrolidone solution into 10 g/L with 0.05 mol/L sodium phosphate buffer (pH 7.0), adding 5mL to wet thallus, oscillating, pouring into a large test tube, 30 ℃,220 rpm/min, transforming for 24h, centrifuging at 6000 r.min -1 for 10min, collecting supernatant, and identifying transformation result by HPLC method.
(3) Determination of gamma-aminobutyric acid by HPLC method
1. Formulation of HPLC reagents:
20 mM sodium acetate buffer (pH 7.3): adding 2.72 g NaAc 3H2 O and 200 mu L of triethylamine into pure water, fixing the volume to 1L, and regulating the pH to 7.3 by using glacial acetic acid;
0.4 M boric acid buffer (pH 10.4): 24.7 Dissolving g boric acid in 500 mL ultrapure water, dissolving to 1L, and regulating the pH to 10.4 by using 1M NaOH solution;
amino acid derivatizing reagent: dissolving 10 mg o-phthalaldehyde in 2.5 mL acetonitrile, and then adding 10 mu L beta-mercaptoethanol for preparation;
HPLC mobile phase: 20mM sodium acetate buffer with acetonitrile: (4:1, v/v) mixing, preparing, ultrasonically degassing 10 min, and preparing for use;
2. sample derivatization: sucking 100 mu L of boric acid buffer solution, 10 mu L of amino acid derivative reagent, 10 mu L of sample to be tested, and uniformly mixing and then reacting at room temperature for 5 min;
hplc conditions:
The chromatographic column model is C18, the sample injection amount of all samples is 10 mu L, the flow rate of the mobile phase is 0.8 mL/min, the detection wavelength is 338 nm, and the column temperature is 30 ℃. And (3) manufacturing a standard curve: GABA standard solutions of 0.05 g/L,0.1 g/L,0.5 g/L,1.0 g/L and 1.5 g/L were prepared and peak times and peak areas were recorded by the derivatization and HPLC detection described above. And drawing a standard curve by taking the peak area as an abscissa and the concentration of the GABA standard solution as an ordinate. A standard curve with a linear fit correlation coefficient R 2 >0.999 was used.
4. Calculation of conversion
Conversion = concentration of gamma-aminobutyric acid (mol/L) converted/initial concentration of 2-pyrrolidone (mol/L). Times.100%
5. Optimizing the enzyme production conditions of the liquid fermentation of the strain: the carbon source, nitrogen source, culture temperature (15-40 ℃) and pH value (3-10) are optimized under different culture conditions, and the optimized result is detected by liquid chromatography.
6. And (3) optimizing conversion conditions: and (3) optimizing conversion conditions at different temperatures (20-60 ℃) and different pH values (3-10), and detecting an optimized result by using liquid chromatography.
7. The influence of different concentrations of 2-pyrrolidone on the conversion of 2-pyrrolidone by Paenibacillus polymyxa T9 to obtain gamma-aminobutyric acid is detected and optimized by liquid chromatography.
Screening results and related experimental results of Paenibacillus polymyxa (Paenibacillus polymyxa) T9
(1) The invention uses 2-pyrrolidone as the only carbon source to screen a strain of Paenibacillus polymyxa T9 which can convert 2-pyrrolidone to obtain gamma-aminobutyric acid, and the strain is circular in colony on LB culture medium, slightly convex in bacterial surface, neat in edge, smooth and moist in surface, semitransparent, and the colony morphology is shown in figure 1;
Extracting a T9 genome by using a bacterial genome DNA extraction kit, carrying out PCR by using 27F and 1492R primers, and carrying out Blast comparison on the sequencing result of a PCR product, wherein the result is identified as Paenibacillus Polymyxa, so the kit is named as Paenibacillus polymyxa Paenibacillus polymyxaT9, and the gene sequence SEQ ID No:1 is shown as follows:
AAATCTCCACCTTCGGCGGCTGGCTCCTTGCGGTTACCTCACCGACTTCGGGTGTTGTAAACTCTCGTGGTGTGACGGGCGGTGTGTACAAGACCCGGGAACGTATTCACCGCGGCATGCTGATCCGCGATTACTAGCAATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGACCGGCTTTTCTAGGATTGGCTCCACCTCGCGGCTTCGCTTCCCGTTGTACCGGCCATTGTAGTACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTGTCACCGGCAGTCTGCTTAGAGTGCCCAGCTTGACCTGCTGGCAACTAAGCATAAGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTCTCCTCTGTCCCGAAGGAAAGGCCTATCTCTAGACCGGTCAGAGGGATGTCAAGACCtGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATACTCCACTGCTTGTGCGGGTCCCCGTCAATTCCTTTGAGTTTCAGTCTTGCGACCGTACTCCCCAGGCGGAATGCTTAATGTGTTAACTTCGGCACCAAGGGTATCGAAACCCCTAACACCTAGCATTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGCGCCTCAGCGTCAGTTACAGCCCAGAGAGTCGCCTTCGCCACTGGTGTTCCTCCACATCTCTACGCATTTCACCGCTACACGTGGAATTCCACTCTCCTCTTCTGCACTCAAGCTCYCCAGTTTCCAGTGCGACCCGAAGTTGAGCCTCGGGATTAAACACCAGACTTAAAGAGCCGCCTGCGCGCGCTTTACGCCCAATAATTCCcGGACAACGCTTGCCCCCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCcGGGGCTTTCTTCTCAGgTACCGTCACTCTTGTAGCAGTTACTCTACAAGACGTTCTTCCCTGGCAACAGAGCTTTACGATCCGAAAACCTTCATCACTCACGCGGCGTTGCTCCGTCAGGCTTTCGCCCATTGCGGAAGATTCCCTACTGCTGCCTCCCGTAGGAGTCTGGGCCGTGTCTCAGTCCCAGTGTGGCCGATCACCCTCTCAGGTCGGCTACGCATCGTCGCCTTGGTAGGCCTTTACCCCACCAACTAGCTAATGCGCCGCAGGCCCATCCACAAGTGACAGATTGCTCCGTCTTTCCTCCTTCTCCCATGCAGGAAAAGGATGTATCGGGTATTAGCTACCGTTTCCGGTAGTTATCCCTGTCTTGTGGGCAGGTTGCCTACGTGTTACTCACCCGTCCGCCGCTAGGTTATTTAGAAGCAAGCTTCTAAATAATCCCGCTCGACTTGCATGTATAGCACCCGCCGCCGG.
(2) Effect of different carbon sources on Paenibacillus polymyxa T biomass and conversion of 2-pyrrolidone to gamma-aminobutyric acid
The enzyme-producing culture medium takes 2 g/L NH 4 Cl as a nitrogen source, and the following carbon sources are respectively added according to the concentration of 10 g/L: glucose, lactose, soluble starch, citric acid, sucrose and glycerol. The results are shown in Table 1, and it is clear from Table 1 that the carbon source has a significant effect on biomass of the cells, and has a large effect on the conversion rate, and when glucose and sucrose are used as the carbon sources, the cells grow vigorously, the biomass is relatively high, and the conversion rate reaches 72.5% and 68.5%, respectively, wherein the optimal carbon source is glucose.
TABLE 1 influence of different carbon sources on Paenibacillus polymyxaT biomass and conversion of 2-pyrrolidone to gamma-aminobutyric acid
(3) Effect of different Nitrogen sources on Paenibacillus polymyxaT biomass and conversion of 2-pyrrolidone to gamma-aminobutyric acid
The enzyme-producing culture medium takes 10g/L glucose as a carbon source, and the following nitrogen sources are respectively added according to 2 g/L: peptone, urea, ammonium sulfate, diammonium phosphate, ammonium oxalate and diammonium citrate. 27. 2d cells were cultured at 220 rpm/min and subjected to transformation experiments. As a result, as shown in Table 2, it was found from Table 2 that the nitrogen source had a large influence on both biomass and conversion rate of the cells, and that the cell growth was vigorous when peptone was used as the nitrogen source, the biomass was relatively high, the conversion rate was 81.5%, and the optimum nitrogen source was peptone.
TABLE 2 Effect of different Nitrogen sources on Paenibacillus polymyxaT biomass and conversion of 2-pyrrolidone to gamma-aminobutyric acid
(4) Effect of culture temperature on Paenibacillus polymyxa T biomass and conversion of 2-pyrrolidone to gamma-aminobutyric acid
The temperature is an important factor influencing cell growth and fermentation enzyme production, and the influence of different fermentation enzyme production temperatures on the cell yield and conversion rate of Paenibacillus polymyxaT strains is examined. As a result, as shown in Table 3, it was found from Table 3 that the temperature had a certain effect on both biomass and conversion rate, and that the biomass and conversion rate were the highest and the conversion rate reached 81.8% when the cell culture temperature was 30℃and therefore the optimum fermentation temperature was 30 ℃.
TABLE 3 influence of the culture temperature on Paenibacillus polymyxaT biomass and conversion of 2-pyrrolidone to gamma-aminobutyric acid
(5) Effect of the initial pH of the Medium on Paenibacillus polymyxaT biomass and conversion of 2-pyrrolidone to gamma-aminobutyric acid
Cell growth and the activity of the various enzymes of the cell are affected by the initial pH of the medium. To examine the effect of initial pH on Paenibacillus polymyxaT biomass and conversion of 2-pyrrolidone to gamma-aminobutyric acid. And (3) adjusting the enzyme-producing culture medium to different pH values, and carrying out an experiment of converting 2-pyrrolidone into gamma-aminobutyric acid on the cultured thalli. The results are shown in Table 4, and it is found from Table 4 that the culture medium cells having pH=4.0 to pH=10.0 were grown, and that the biomass and the conversion rate were the highest at pH=7, and the conversion rate was 82.0%.
TABLE 4 influence of initial pH of the Medium on Paenibacillus polymyxaT biomass and conversion of 2-pyrrolidone to gamma-aminobutyric acid
(6) Effect of the conversion temperature on Paenibacillus polymyxaT conversion of 2-pyrrolidone to gamma-aminobutyric acid
The effect of the conversion temperature on the effect of Paenibacillus polymyxaT on the conversion of 2-pyrrolidone to gamma-aminobutyric acid is shown in Table 5, with 30℃being the most suitable conversion temperature and the conversion rate decreasing with increasing temperature exceeding 30 ℃.
TABLE 5 influence of the conversion temperature on the conversion of Paenibacillus polymyxa T to 2-pyrrolidone to gamma-aminobutyric acid
(7) Effect of the conversion pH on Paenibacillus polymyxaT on conversion of 2-pyrrolidone to gamma-aminobutyric acid
The effect of pH on Paenibacillus polymyxaT on conversion of 2-pyrrolidone to gamma-aminobutyric acid is shown in Table 6. It can be seen from table 6 that the catalytic effect is best at the optimum conversion ph=7, with either too high or too low a pH being detrimental to the conversion.
TABLE 6 influence of the conversion pH on Paenibacillus polymyxaT conversion of 2-pyrrolidone to gamma-aminobutyric acid
(8) Effect of different concentrations of 2-pyrrolidone on Paenibacillus polymyxaT conversion of 2-pyrrolidone to gamma-aminobutyric acid
The effect of varying concentrations of 2-pyrrolidone on Paenibacillus polymyxaT conversion of 2-pyrrolidone to gamma-aminobutyric acid is shown in Table 7. As is clear from Table 7, the conversion was highest at a concentration of 5.0 g/L and reached 86.0%, and when the concentration of 2-pyrrolidone exceeded 5.0 g/L, the conversion gradually decreased with increasing 2-pyrrolidone concentration, but 20 g/L of 2-pyrrolidone was tolerated.
TABLE 7 influence of different concentrations of 2-pyrrolidone on Paenibacillus polymyxaT conversion of 2-pyrrolidone to gamma-aminobutyric acid
While the present invention has been described in considerable detail and with particularity with respect to several described embodiments, it is not intended to be limited to any such detail or embodiments or any particular embodiment, but is to be construed as providing broad interpretation of such claims by reference to the appended claims in view of the prior art so as to effectively encompass the intended scope of the invention. Furthermore, the foregoing description of the invention has been presented in its embodiments contemplated by the inventors for the purpose of providing a useful description, and for the purposes of providing a non-essential modification of the invention that may not be presently contemplated, may represent an equivalent modification of the invention.
Claims (10)
1. The Paenibacillus polymyxa T9 is characterized in that the Paenibacillus polymyxa T9 (Paenibacillus polymyxa T) is preserved in China Center for Type Culture Collection (CCTCC) at 2022, month 05 and 24, and the preservation number is CCTCC NO: m2022706.
2. The paenibacillus polymyxa T9 according to claim 1, wherein the gene sequence of the 16S rDNA of paenibacillus polymyxa T9 is as shown in SEQ ID No: 1.
3. Use of a strain of paenibacillus polymyxa T9 according to any one of claims 1-2 for the preparation of gamma-aminobutyric acid.
4. Use according to claim 3, characterized in that paenibacillus polymyxa T9 is obtained by converting 2-pyrrolidone into gamma-aminobutyric acid.
5. The preparation method of the gamma-aminobutyric acid is characterized by comprising the following steps:
Inoculating the Paenibacillus polymyxa T9 according to claim 1 or 2 into a liquid enzyme-producing culture medium for fermentation culture, adding a 2-pyrrolidone solution, and performing conversion at 20-60 ℃ for 12 h-48 h to obtain gamma-aminobutyric acid;
the liquid enzyme-producing culture medium comprises the following components: 10 g/L of carbon source, 10 g of nitrogen source 2 g/L,Na2HPO4·12H2O 5 g,KH2PO42.0 g,MgSO4·7H2O 1.0 g, CaCl2·2H2O 10 mg,FeSO4·7H2O 8 mg,2- pyrrolidone and pH value of 3-10.
6. The method according to claim 5, wherein the carbon source comprises any one of glucose, lactose, soluble starch, citric acid, sucrose, and glycerin.
7. The method according to claim 5, wherein the nitrogen source comprises any one of ammonium chloride, peptone, urea, ammonium sulfate, diammonium hydrogen phosphate, ammonium oxalate, and diammonium hydrogen citrate.
8. The method according to claim 5, wherein the concentration of 2-pyrrolidone is 5 g/L to 10.0 g/L.
9. The process according to claim 5, wherein the fermentation culture is carried out at a temperature of 20℃to 40 ℃.
10. The process of claim 5, wherein the conversion temperature is 30 ℃.
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559564A (en) * | 2012-03-06 | 2012-07-11 | 福州大学 | Method for culturing paenibacillus polymyxa at high density |
| WO2019222253A1 (en) * | 2018-05-17 | 2019-11-21 | Bayer Cropscience Lp | Stabilized fungicidal composition |
| CN112501071A (en) * | 2020-12-15 | 2021-03-16 | 河北省科学院生物研究所 | Paenibacillus polymyxa SWGC4112 and culture method and application thereof |
| CN114717154A (en) * | 2022-05-06 | 2022-07-08 | 兰州交通大学 | Application of paenibacillus polymyxa YF in preventing and treating plant blight |
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102559564A (en) * | 2012-03-06 | 2012-07-11 | 福州大学 | Method for culturing paenibacillus polymyxa at high density |
| WO2019222253A1 (en) * | 2018-05-17 | 2019-11-21 | Bayer Cropscience Lp | Stabilized fungicidal composition |
| CN112423593A (en) * | 2018-05-17 | 2021-02-26 | 拜耳作物科学有限合伙公司 | Stable fungicidal compositions |
| CN112501071A (en) * | 2020-12-15 | 2021-03-16 | 河北省科学院生物研究所 | Paenibacillus polymyxa SWGC4112 and culture method and application thereof |
| CN114717154A (en) * | 2022-05-06 | 2022-07-08 | 兰州交通大学 | Application of paenibacillus polymyxa YF in preventing and treating plant blight |
Non-Patent Citations (2)
| Title |
|---|
| RUI LI 等: "Dietary L-theanine alleviated lipopolysaccharide-induced immunological stress in yellow-feathered broilers", ANIMAL NUTRITION, vol. 4, 23 May 2018 (2018-05-23), pages 265 - 272 * |
| 杨少波 等: "多粘类芽孢杆菌及其产生的生物活性 物质研究进展", 微生物学通报, vol. 35, no. 10, 20 October 2008 (2008-10-20), pages 1621 - 1625 * |
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