CN113637715A - Method for efficiently converting nicotinamide into nicotinic acid strains - Google Patents
Method for efficiently converting nicotinamide into nicotinic acid strains Download PDFInfo
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- DFPAKSUCGFBDDF-UHFFFAOYSA-N Nicotinamide Chemical compound NC(=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-UHFFFAOYSA-N 0.000 title claims abstract description 126
- PVNIIMVLHYAWGP-UHFFFAOYSA-N Niacin Chemical compound OC(=O)C1=CC=CN=C1 PVNIIMVLHYAWGP-UHFFFAOYSA-N 0.000 title claims abstract description 81
- 229960003966 nicotinamide Drugs 0.000 title claims abstract description 62
- 235000005152 nicotinamide Nutrition 0.000 title claims abstract description 62
- 239000011570 nicotinamide Substances 0.000 title claims abstract description 62
- 229960003512 nicotinic acid Drugs 0.000 title claims abstract description 44
- 239000011664 nicotinic acid Substances 0.000 title claims abstract description 43
- 235000001968 nicotinic acid Nutrition 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 title claims abstract description 20
- 238000006243 chemical reaction Methods 0.000 claims abstract description 41
- 241000894006 Bacteria Species 0.000 claims abstract description 8
- 238000006555 catalytic reaction Methods 0.000 claims abstract description 8
- 238000012216 screening Methods 0.000 claims abstract description 8
- 230000002210 biocatalytic effect Effects 0.000 claims abstract description 4
- 238000011172 small scale experimental method Methods 0.000 claims abstract description 4
- 108090000790 Enzymes Proteins 0.000 claims description 46
- 102000004190 Enzymes Human genes 0.000 claims description 46
- 239000000243 solution Substances 0.000 claims description 28
- 239000000758 substrate Substances 0.000 claims description 28
- 230000000694 effects Effects 0.000 claims description 19
- 239000007788 liquid Substances 0.000 claims description 17
- 238000012258 culturing Methods 0.000 claims description 15
- 238000006911 enzymatic reaction Methods 0.000 claims description 13
- QAOWNCQODCNURD-UHFFFAOYSA-N Sulfuric acid Chemical compound OS(O)(=O)=O QAOWNCQODCNURD-UHFFFAOYSA-N 0.000 claims description 10
- DFPAKSUCGFBDDF-ZQBYOMGUSA-N [14c]-nicotinamide Chemical compound N[14C](=O)C1=CC=CN=C1 DFPAKSUCGFBDDF-ZQBYOMGUSA-N 0.000 claims description 10
- 238000000855 fermentation Methods 0.000 claims description 10
- 230000004151 fermentation Effects 0.000 claims description 10
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 claims description 9
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 claims description 9
- 230000001580 bacterial effect Effects 0.000 claims description 9
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 claims description 6
- 238000001514 detection method Methods 0.000 claims description 6
- 238000002474 experimental method Methods 0.000 claims description 6
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 6
- 238000012544 monitoring process Methods 0.000 claims description 6
- 239000002351 wastewater Substances 0.000 claims description 6
- BPHPUYQFMNQIOC-NXRLNHOXSA-N isopropyl beta-D-thiogalactopyranoside Chemical compound CC(C)S[C@@H]1O[C@H](CO)[C@H](O)[C@H](O)[C@H]1O BPHPUYQFMNQIOC-NXRLNHOXSA-N 0.000 claims description 5
- 239000003795 chemical substances by application Substances 0.000 claims description 4
- 238000010790 dilution Methods 0.000 claims description 4
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- 240000004808 Saccharomyces cerevisiae Species 0.000 claims description 3
- 229960000583 acetic acid Drugs 0.000 claims description 3
- 230000003213 activating effect Effects 0.000 claims description 3
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- 230000003197 catalytic effect Effects 0.000 claims description 3
- 239000012295 chemical reaction liquid Substances 0.000 claims description 3
- 239000011248 coating agent Substances 0.000 claims description 3
- 238000000576 coating method Methods 0.000 claims description 3
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- 239000012362 glacial acetic acid Substances 0.000 claims description 3
- 239000000411 inducer Substances 0.000 claims description 3
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- 230000010355 oscillation Effects 0.000 claims description 3
- 239000008055 phosphate buffer solution Substances 0.000 claims description 3
- 238000013341 scale-up Methods 0.000 claims description 3
- REFMEZARFCPESH-UHFFFAOYSA-M sodium;heptane-1-sulfonate Chemical compound [Na+].CCCCCCCS([O-])(=O)=O REFMEZARFCPESH-UHFFFAOYSA-M 0.000 claims description 3
- 239000006228 supernatant Substances 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
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- 238000004811 liquid chromatography Methods 0.000 claims description 2
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- 238000009776 industrial production Methods 0.000 abstract description 3
- 239000000126 substance Substances 0.000 abstract description 3
- 150000001408 amides Chemical class 0.000 abstract description 2
- 150000001732 carboxylic acid derivatives Chemical class 0.000 abstract description 2
- 238000005265 energy consumption Methods 0.000 abstract 1
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 description 3
- 239000003814 drug Substances 0.000 description 3
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 description 2
- JUJWROOIHBZHMG-UHFFFAOYSA-N Pyridine Chemical compound C1=CC=NC=C1 JUJWROOIHBZHMG-UHFFFAOYSA-N 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 239000006227 byproduct Substances 0.000 description 2
- HVYWMOMLDIMFJA-DPAQBDIFSA-N cholesterol Chemical compound C1C=C2C[C@@H](O)CC[C@]2(C)[C@@H]2[C@@H]1[C@@H]1CC[C@H]([C@H](C)CCCC(C)C)[C@@]1(C)CC2 HVYWMOMLDIMFJA-DPAQBDIFSA-N 0.000 description 2
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- 108090000623 proteins and genes Proteins 0.000 description 2
- 102000004169 proteins and genes Human genes 0.000 description 2
- JBYSVQKTMBXLKJ-UHFFFAOYSA-N pyridine-3-carboxamide;pyridine-3-carboxylic acid Chemical compound NC(=O)C1=CC=CN=C1.OC(=O)C1=CC=CN=C1 JBYSVQKTMBXLKJ-UHFFFAOYSA-N 0.000 description 2
- 235000019160 vitamin B3 Nutrition 0.000 description 2
- 239000011708 vitamin B3 Substances 0.000 description 2
- GZPHSAQLYPIAIN-UHFFFAOYSA-N 3-pyridinecarbonitrile Chemical compound N#CC1=CC=CN=C1 GZPHSAQLYPIAIN-UHFFFAOYSA-N 0.000 description 1
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 1
- 208000024172 Cardiovascular disease Diseases 0.000 description 1
- 108020005199 Dehydrogenases Proteins 0.000 description 1
- 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 description 1
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 description 1
- 239000012880 LB liquid culture medium Substances 0.000 description 1
- BAWFJGJZGIEFAR-NNYOXOHSSA-N NAD zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 BAWFJGJZGIEFAR-NNYOXOHSSA-N 0.000 description 1
- XJLXINKUBYWONI-NNYOXOHSSA-N NADP zwitterion Chemical compound NC(=O)C1=CC=C[N+]([C@H]2[C@@H]([C@H](O)[C@@H](COP([O-])(=O)OP(O)(=O)OC[C@@H]3[C@H]([C@@H](OP(O)(O)=O)[C@@H](O3)N3C4=NC=NC(N)=C4N=C3)O)O2)O)=C1 XJLXINKUBYWONI-NNYOXOHSSA-N 0.000 description 1
- 206010029400 Nicotinic acid deficiency Diseases 0.000 description 1
- 208000002141 Pellagra Diseases 0.000 description 1
- LCTONWCANYUPML-UHFFFAOYSA-M Pyruvate Chemical compound CC(=O)C([O-])=O LCTONWCANYUPML-UHFFFAOYSA-M 0.000 description 1
- 208000007536 Thrombosis Diseases 0.000 description 1
- 229930003270 Vitamin B Natural products 0.000 description 1
- 229930003537 Vitamin B3 Natural products 0.000 description 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 description 1
- 150000003855 acyl compounds Chemical class 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000000996 additive effect Effects 0.000 description 1
- 230000037354 amino acid metabolism Effects 0.000 description 1
- 150000001413 amino acids Chemical class 0.000 description 1
- 239000003674 animal food additive Substances 0.000 description 1
- 230000002180 anti-stress Effects 0.000 description 1
- 239000000987 azo dye Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000010364 biochemical engineering Methods 0.000 description 1
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- 230000008468 bone growth Effects 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 235000012000 cholesterol Nutrition 0.000 description 1
- 239000005515 coenzyme Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 239000012847 fine chemical Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000002778 food additive Substances 0.000 description 1
- 210000000232 gallbladder Anatomy 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000034659 glycolysis Effects 0.000 description 1
- 230000012447 hatching Effects 0.000 description 1
- 239000012760 heat stabilizer Substances 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- JRFKIOFLCXKVOT-UHFFFAOYSA-N hydroxymethylnicotinamide Chemical compound OCNC(=O)C1=CC=CN=C1 JRFKIOFLCXKVOT-UHFFFAOYSA-N 0.000 description 1
- 229910052742 iron Inorganic materials 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- JIAOUYONZMRJJD-UHFFFAOYSA-N n-benzylpyridine-3-carboxamide Chemical compound C=1C=CN=CC=1C(=O)NCC1=CC=CC=C1 JIAOUYONZMRJJD-UHFFFAOYSA-N 0.000 description 1
- 229950006238 nadide Drugs 0.000 description 1
- 230000003647 oxidation Effects 0.000 description 1
- 238000007254 oxidation reaction Methods 0.000 description 1
- 150000002972 pentoses Chemical class 0.000 description 1
- 229940083256 peripheral vasodilators nicotinic acid and derivative Drugs 0.000 description 1
- 239000004033 plastic Substances 0.000 description 1
- 229920003023 plastic Polymers 0.000 description 1
- 238000006116 polymerization reaction Methods 0.000 description 1
- 230000008092 positive effect Effects 0.000 description 1
- 244000144977 poultry Species 0.000 description 1
- 230000022558 protein metabolic process Effects 0.000 description 1
- 230000004144 purine metabolism Effects 0.000 description 1
- UMJSCPRVCHMLSP-UHFFFAOYSA-N pyridine Natural products COC1=CC=CN=C1 UMJSCPRVCHMLSP-UHFFFAOYSA-N 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 238000006479 redox reaction Methods 0.000 description 1
- 238000007670 refining Methods 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- UFTFJSFQGQCHQW-UHFFFAOYSA-N triformin Chemical compound O=COCC(OC=O)COC=O UFTFJSFQGQCHQW-UHFFFAOYSA-N 0.000 description 1
- 229940088594 vitamin Drugs 0.000 description 1
- 229930003231 vitamin Natural products 0.000 description 1
- 235000013343 vitamin Nutrition 0.000 description 1
- 239000011782 vitamin Substances 0.000 description 1
- 235000019156 vitamin B Nutrition 0.000 description 1
- 239000011720 vitamin B Substances 0.000 description 1
- 239000011701 zinc Substances 0.000 description 1
- 229910052725 zinc Inorganic materials 0.000 description 1
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/10—Nitrogen as only ring hetero atom
- C12P17/12—Nitrogen as only ring hetero atom containing a six-membered hetero ring
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/978—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5)
- G01N2333/98—Hydrolases (3) acting on carbon to nitrogen bonds other than peptide bonds (3.5) acting on amide bonds in linear amides (3.5.1)
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Abstract
The invention discloses a method for efficiently converting nicotinamide into nicotinic acid strains, which comprises the following steps: s100: screening and identifying nicotinamide bacteria; s200: researching biocatalytic reaction conditions; s300: and (3) determining catalytic reaction conditions and verifying by a small-scale experiment. The strain capable of converting amide in nicotinamide into carboxylic acid is screened out, and nicotinamide is converted into nicotinic acid by a biocatalysis method, so that the method is distinguished from the high energy consumption and high pollution of the traditional chemical method, and has the advantages of low cost, small pollution, mild reaction and suitability for industrial production.
Description
Technical Field
The invention relates to the technical field of biochemical engineering, in particular to a method for efficiently converting nicotinamide into nicotinic acid strains.
Background
Nicotinic acid, also known as nicotinic acid, is a generic name for all pyridine-3-carboxylic acids and derivatives thereof having biochemical activity. Nicotinic acid belongs to vitamin B group, also called vitamin B3 or vitamin PP, is one of 13 vitamins essential to human body, is widely applied to food and feed additives at present, and is also an important medical raw material and chemical intermediate with certain economic value.
In medicine, nicotinic acid is an important participant in redox reactions in organisms, and mainly takes the forms of coenzyme I and coenzyme II as coenzymes of dehydrogenases, which play a role as hydrogen carriers in biological oxidation and participate in pyruvate metabolism, glucose glycolysis, pentose biosynthesis and fat, amino acid, protein and purine metabolism. Nicotinic acid plays an important role in increasing zinc and iron availability in humans. Nicotinic acid is an important factor in combating pellagra in humans. Many acyl compounds of nicotinic acid also have the effects of reducing blood fat, enhancing metabolism among cells, inhibiting the formation of cholesterol and plasma triglyceride and have good effect of preventing and treating cardiovascular diseases. In addition, nicotinic acid derivative nicotinoylbenzylamine can be used as a high-efficiency medicament for treating thrombus; the nicotinoylhydroxymethylamine can be used as a good medicine for protecting the liver, benefiting the gallbladder and inhibiting bacteria; nicotinic acid has positive effects on the bone growth, anti-stress response, egg production and hatching rate of eggs of poultry. Is an important additive in feed. In the dye industry. Nicotinic acid can synthesize various intermediates of the active dye combined with azo dye. Nicotinic acid is also a heat stabilizer for chain transfer agents in the polymerization of PVC plastics and acrylamide. In conclusion, with the continuous and intensive research on the effects of nicotinic acid and derivatives thereof in the fine chemical industry, the application of nicotinic acid is increasingly wide, and the importance of nicotinic acid is gradually known.
At present, the main product nicotinamide is produced by a chemical-enzyme cascade technology, 3-cyanogen wastewater is generated in the industrial production process of 3-cyanopyridine, wherein the 3-cyanogen wastewater contains nicotinic acid, nicotinamide, pyridine, benzene and other substances, and after concentrated solution of part of process routes is recycled, the content of the byproduct nicotinic acid exceeds the standard, and certain influence is caused on a finished product. Meanwhile, the waste water discharged from the working section contains a large amount of nicotinamide and nicotinic acid, so that the problem of the byproduct nicotinic acid in the production process is solved and the nicotinic acid is recycled.
Disclosure of Invention
The invention aims to provide a method for efficiently converting nicotinamide into nicotinic acid strains, which is used for screening out strains capable of converting amide in nicotinamide into carboxylic acid and converting nicotinamide into nicotinic acid by a biocatalysis method.
In order to achieve the purpose, the invention provides the following technical scheme:
the method for efficiently converting nicotinamide into nicotinic acid strains comprises the following steps:
s100: screening and identifying nicotinamide bacteria;
s200: researching biocatalytic reaction conditions;
s300: and (3) determining catalytic reaction conditions and verifying by a small-scale experiment.
Further, the step of S100 is as follows:
s101: collecting 3-cyanogen workshop wastewater, and carrying out enrichment screening to obtain a strain capable of converting nicotinamide into nicotinic acid;
s102: inoculating the screened dominant strains on a slant of a enrichment medium according to the requirement of aseptic operation, culturing at 30 ℃ for 3 days, taking out, growing strains, growing colonies, performing amplification culture on the grown colonies for 3 days at 30 ℃, observing, and storing in a refrigerator at 4 ℃ with good effect;
s103: activating a flat plate: diluting and coating the preserved nicotinamide bacterium on a K + resistant LB flat plate, and culturing for 16h in a constant-temperature incubator at 37 ℃;
s104: seed liquid transfer: taking out the plate, selecting 50 monoclonal colonies, transferring to 50K + resistant LB single tubes, and culturing for 16h at 37 ℃ by a shaking table at 220 rpm;
s105: transferring the yeast liquid: taking out the single tube after culture, respectively absorbing 1.5ml of bacterial liquid, inoculating the bacterial liquid into 50K + resistant shake flasks, culturing for about 1.5h at 220rpm of a shaking table at 37 ℃, taking out when the cultured bacterial liquid is flocculent, adding an inducer IPTG (isopropyl-beta-thiogalactoside) and culturing for 22h at 220rpm of a shaking table at 18 ℃;
s106: and (3) enzyme activity detection: taking out the fermentation liquor, measuring the nicotinamide enzyme activity in each bottle by an HPLC method, and simultaneously measuring an OD600 value, wherein HPLC detection parameters are as follows: a chromatographic column: agent C18(4.6mm 250mm,5ul), flow rate 0.8ml/min, column temperature 30 ℃, mobile phase a: water + sodium heptanesulfonate + triethylamine + glacial acetic acid 80mL +0.1g +0.02mL +0.1 mL; mobile phase: 90% mobile phase a + 10% acetonitrile, substrate solution: 100ul of 70g/L nicotinamide solution +690ul of pH 7.0, 0.05mol/L phosphate buffer solution;
s107: placing the substrate solution on a uniform mixing oscillator with the temperature controlled at 30 ℃ for oscillation, adding 10ul of the treated fermentation liquor, reacting for 10min, adding 200ul of 0.5mol/L sulfuric acid solution to terminate the reaction, and placing the reacted solution at 12000; centrifuging for 3min on a centrifuge with rpm;
s108: taking the centrifuged reaction liquid supernatant to dilute by 6 times and loading;
s109: and (3) calculating: and (5) calculating and obtaining a result.
Further, the calculation formula of S109:
crude enzyme liquid enzyme activity (U/mL) ═ CxV xX/123.1/T/V
c-Niacin concentration by liquid chromatography,. mu.g/mL;
v-reaction volume, here 800. mu.L reaction solution + 200. mu.L sulfuric acid test solution;
x-dilution multiple (6) before reaction sample injection;
t-reaction time, 10 min;
v-volume of crude enzyme solution added, 10. mu.L
123.1-nicotinic acid molecular weight, g/moL;
the enzyme activity of the fermentation broth (U/mL) is equal to the enzyme activity of the crude enzyme and the dilution factor (N) of the fermentation broth.
Further, the step of S200 is as follows:
s201: optimum substrate concentration: setting the concentration gradients of 10%, 20%, 30% and 40% nicotinamide for the substrate, reacting at the temperature of 25 ℃, the pH value of 7.55 and the enzyme addition amount of 4.8U/ml, counting the relation between the substrate and the enzymatic reaction rate after a certain time (10h), and determining the optimal substrate concentration;
s202: optimal enzyme amount: setting concentration gradients of enzyme dosage of 6.0U/ml, 7.2U/ml, 8.4U/ml, 9.6U/ml and 10.8U/ml, reacting under the conditions that the substrate concentration is 20% nicotinamide, the temperature is 25 ℃ and the pH value is 7.55, counting the relation between the enzyme dosage and the nicotinamide conversion rate after a certain time, and determining the optimal enzyme dosage;
s203: optimum temperature: setting temperature gradients of 25 ℃, 30 ℃, 35 ℃, 40 ℃ and 45 ℃, reacting under the conditions that the substrate concentration is 20 percent of nicotinamide, the pH value is 7.55 and the enzyme addition amount is 8.4U/ml, counting the relation between the temperature and the enzymatic reaction rate after a certain time (6h), and determining the optimal temperature;
s204: optimum pH: setting pH at 5.0-9.0, reacting every 1.0 gradient under the conditions that the substrate concentration is 20% nicotinamide, the temperature is 36-38 ℃, and the enzyme addition amount is 8.4U/ml, and counting the relation between the pH and the enzymatic reaction rate after a certain time (6h) to determine the optimum pH.
Further, the step of S300 is as follows:
s301: determination of nicotinamide catalysis reaction bench (2L) protocol: adding nicotinamide enzyme 6.6U/ml into 20% nicotinamide solution, reacting at 36-38 deg.C, controlling pH at 7.4 + -0.2, and monitoring nicotinamide and nicotinic acid content change every half an hour during reaction;
s302: catalytic experiment bench scale-up experiment verifies: adding nicotinamide enzyme 6.6U/ml into 20% nicotinamide solution, reacting at 36-38 deg.C, controlling pH at 7.4 + -0.2, and monitoring nicotinamide and nicotinic acid content change every half an hour during reaction process.
Compared with the prior art, the invention has the beneficial effects that:
1. the technical scheme adopted by the invention has the advantages of low cost, convenient operation, mild reaction, little pollution and certain industrial application potential.
2. The technical scheme adopted by the invention screens out the strain with high protein expression, can be used as a catalyst for converting nicotinamide into nicotinic acid in industrial production, and can ensure that the conversion rate of nicotinamide reaches more than 99.99 percent, the conversion rate is high and the performance is stable.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
The method for efficiently converting nicotinamide into nicotinic acid strains comprises the following steps:
s100: screening and identification of nicotinamide bacteria
S101: collecting 3-cyanogen workshop wastewater, and carrying out enrichment screening to obtain a strain capable of converting nicotinamide into nicotinic acid;
s102: inoculating the screened dominant strains on a slant of a enrichment medium according to the requirement of aseptic operation, culturing at 30 ℃ for 3 days, taking out, growing strains, growing colonies, performing amplification culture on the grown colonies for 3 days at 30 ℃, observing, and storing in a refrigerator at 4 ℃ with good effect;
s103: activating a flat plate: diluting and coating the preserved nicotinamide bacterium on a K + resistant LB flat plate, and culturing for 16h in a constant-temperature incubator at 37 ℃;
s104: seed liquid transfer: taking out the plate, selecting 50 monoclonal colonies, transferring to 50K + resistant LB single tubes, and culturing for 16h at 37 ℃ by a shaking table at 220 rpm;
s105: transferring the yeast liquid: taking out the single tube after culture, respectively absorbing 1.5ml of bacterial liquid, inoculating the bacterial liquid into 50K + resistant shake flasks (50 ml of LB liquid culture medium in each shake flask), culturing for about 1.5h at 220rpm of a shaking table at 37 ℃, taking out when the cultured bacterial liquid is flocculent, adding an inducer IPTG (final concentration of 100uM), and culturing for 22h at 220rpm in the shaking table at 18 ℃;
s106: and (3) enzyme activity detection: taking out the fermentation liquor, measuring the nicotinamide enzyme activity in each bottle by an HPLC method, and simultaneously measuring an OD600 value, wherein HPLC detection parameters are as follows: a chromatographic column: agent C18(4.6mm 250mm,5ul), flow rate 0.8ml/min, column temperature 30 ℃, mobile phase a: water + sodium heptanesulfonate + triethylamine + glacial acetic acid 80mL +0.1g +0.02mL +0.1 mL; mobile phase: 90% mobile phase a + 10% acetonitrile, substrate solution: 100ul of 70g/L nicotinamide solution +690ul of pH 7.0, 0.05mol/L phosphate buffer solution;
s107: placing the substrate solution on a uniform mixing oscillator with the temperature controlled at 30 ℃ for oscillation, adding 10ul of the treated fermentation liquor, reacting for 10min, adding 200ul of 0.5mol/L sulfuric acid solution to terminate the reaction, and placing the reacted solution at 12000; centrifuging for 3min on a centrifuge with rpm;
s108: taking the centrifuged reaction liquid supernatant to dilute by 6 times and loading;
s109: and (3) calculating: and (5) calculating and obtaining a result.
S200: exploration of biocatalytic reaction conditions
S201: optimum substrate concentration: setting the concentration gradients of 10%, 20%, 30% and 40% nicotinamide for the substrate, reacting at the temperature of 25 ℃, the pH value of 7.55 and the enzyme addition amount of 4.8U/ml, counting the relation between the substrate and the enzymatic reaction rate after a certain time (10h), and determining the optimal substrate concentration;
relationship between substrate and enzymatic reaction rate
S202: optimal enzyme amount: setting concentration gradients of enzyme dosage of 6.0U/ml, 7.2U/ml, 8.4U/ml, 9.6U/ml and 10.8U/ml, reacting under the conditions that the substrate concentration is 20% nicotinamide, the temperature is 25 ℃ and the pH value is 7.55, counting the relation between the enzyme dosage and the nicotinamide conversion rate after a certain time, and determining the optimal enzyme dosage;
correlation between Nicotinamide enzyme dosage and conversion
S203: optimum temperature: setting temperature gradients of 25 ℃, 30 ℃, 35 ℃, 40 ℃ and 45 ℃, reacting under the conditions that the substrate concentration is 20 percent of nicotinamide, the pH value is 7.55 and the enzyme addition amount is 8.4U/ml, counting the relation between the temperature and the enzymatic reaction rate after a certain time (6h), and determining the optimal temperature;
relationship between reaction temperature and enzymatic reaction rate
S204: optimum pH: setting pH at 5.0-9.0, reacting every 1.0 gradient under the conditions that the substrate concentration is 20% nicotinamide, the temperature is 36-38 ℃, and the enzyme addition amount is 8.4U/ml, and counting the relation between the pH and the enzymatic reaction rate after a certain time (6h) to determine the optimum pH.
Relationship between reaction pH and enzymatic reaction Rate
Refining the relationship between catalytic reaction pH and enzymatic reaction rate
S300: and (3) determining catalytic reaction conditions and verifying by a small-scale experiment.
S301: determination of nicotinamide catalysis reaction bench (2L) protocol: adding nicotinamide enzyme 6.6U/ml into 20% nicotinamide solution, reacting at 36-38 deg.C, controlling pH at 7.4 + -0.2, monitoring nicotinamide and nicotinic acid content change every half an hour, finishing reaction for 4 hr, wherein nicotinamide conversion rate is above 99.99%, and nicotinic acid content is about 20%.
Nicotinamide nicotinic acid content change and conversion rate statistics at each stage of reaction
S302: catalytic experiment bench scale-up experiment verifies: adding nicotinamide enzyme 6.6U/ml into 20% nicotinamide solution, reacting at 36-38 deg.C, controlling pH at 7.4 + -0.2, monitoring nicotinamide and nicotinic acid content change every half an hour during reaction process, finishing reaction for 4 hr, wherein nicotinamide conversion rate is above 99.99%, and nicotinic acid content is about 20%.
Nicotinamide nicotinic acid content change and conversion rate statistics at each stage of reaction
The above description is only for the preferred embodiment of the present invention, but the scope of the present invention is not limited thereto, and any person skilled in the art should be able to cover the technical solutions and the inventive concepts of the present invention within the technical scope of the present invention.
Claims (5)
1. The method for efficiently converting nicotinamide into nicotinic acid strains is characterized by comprising the following steps:
s100: screening and identifying nicotinamide bacteria;
s200: researching biocatalytic reaction conditions;
s300: and (3) determining catalytic reaction conditions and verifying by a small-scale experiment.
2. The method of claim 1 wherein the step of S100 is as follows:
s101: collecting 3-cyanogen workshop wastewater, and carrying out enrichment screening to obtain a strain capable of converting nicotinamide into nicotinic acid;
s102: inoculating the screened dominant strains on a slant of a enrichment medium according to the requirement of aseptic operation, culturing at 30 ℃ for 3 days, taking out, growing strains, growing colonies, performing amplification culture on the grown colonies for 3 days at 30 ℃, observing, and storing in a refrigerator at 4 ℃ with good effect;
s103: activating a flat plate: diluting and coating the preserved nicotinamide bacterium on a K + resistant LB flat plate, and culturing for 16h in a constant-temperature incubator at 37 ℃;
s104: seed liquid transfer: taking out the plate, selecting 50 monoclonal colonies, transferring to 50K + resistant LB single tubes, and culturing for 16h at 37 ℃ by a shaking table at 220 rpm;
s105: transferring the yeast liquid: taking out the single tube after culture, respectively absorbing 1.5ml of bacterial liquid, inoculating the bacterial liquid into 50K + resistant shake flasks, culturing for about 1.5h at 220rpm of a shaking table at 37 ℃, taking out when the cultured bacterial liquid is flocculent, adding an inducer IPTG (isopropyl-beta-thiogalactoside) and culturing for 22h at 220rpm of a shaking table at 18 ℃;
s106: and (3) enzyme activity detection: taking out the fermentation liquor, measuring the nicotinamide enzyme activity in each bottle by an HPLC method, and simultaneously measuring an OD600 value, wherein HPLC detection parameters are as follows: a chromatographic column: agent C18(4.6mm 250mm,5ul), flow rate 0.8ml/min, column temperature 30 ℃, mobile phase a: water + sodium heptanesulfonate + triethylamine + glacial acetic acid 80mL +0.1g +0.02mL +0.1 mL; mobile phase: 90% mobile phase a + 10% acetonitrile, substrate solution: 100ul of 70g/L nicotinamide solution +690ul of pH 7.0, 0.05mol/L phosphate buffer solution;
s107: placing the substrate solution on a uniform mixing oscillator with the temperature controlled at 30 ℃ for oscillation, adding 10ul of the treated fermentation liquor, reacting for 10min, adding 200ul of 0.5mol/L sulfuric acid solution to terminate the reaction, and placing the reacted solution at 12000; centrifuging for 3min on a centrifuge with rpm;
s108: taking the centrifuged reaction liquid supernatant to dilute by 6 times and loading;
s109: and (3) calculating: and (5) calculating and obtaining a result.
3. The method of claim 2, wherein the formula for S109 is:
crude enzyme liquid enzyme activity (U/mL) ═ CxV xX/123.1/T/V
c-Niacin concentration by liquid chromatography,. mu.g/mL;
v-reaction volume, here 800. mu.L reaction solution + 200. mu.L sulfuric acid test solution;
x-dilution multiple (6) before reaction sample injection;
t-reaction time, 10 min;
v-volume of crude enzyme solution added, 10. mu.L
123.1-nicotinic acid molecular weight, g/moL;
the enzyme activity of the fermentation broth (U/mL) is equal to the enzyme activity of the crude enzyme and the dilution factor (N) of the fermentation broth.
4. The method of claim 1 wherein step S200 is as follows:
s201: optimum substrate concentration: setting the concentration gradients of 10%, 20%, 30% and 40% nicotinamide for the substrate, reacting at the temperature of 25 ℃, the pH value of 7.55 and the enzyme addition amount of 4.8U/ml, counting the relation between the substrate and the enzymatic reaction rate after a certain time (10h), and determining the optimal substrate concentration;
s202: optimal enzyme amount: setting concentration gradients of enzyme dosage of 6.0U/ml, 7.2U/ml, 8.4U/ml, 9.6U/ml and 10.8U/ml, reacting under the conditions that the substrate concentration is 20% nicotinamide, the temperature is 25 ℃ and the pH value is 7.55, counting the relation between the enzyme dosage and the nicotinamide conversion rate after a certain time, and determining the optimal enzyme dosage;
s203: optimum temperature: setting temperature gradients of 25 ℃, 30 ℃, 35 ℃, 40 ℃ and 45 ℃, reacting under the conditions that the substrate concentration is 20 percent of nicotinamide, the pH value is 7.55 and the enzyme addition amount is 8.4U/ml, counting the relation between the temperature and the enzymatic reaction rate after a certain time (6h), and determining the optimal temperature;
s204: optimum pH: setting pH at 5.0-9.0, reacting every 1.0 gradient under the conditions that the substrate concentration is 20% nicotinamide, the temperature is 36-38 ℃, and the enzyme addition amount is 8.4U/ml, and counting the relation between the pH and the enzymatic reaction rate after a certain time (6h) to determine the optimum pH.
5. The method of claim 1, wherein the step of S300 is as follows:
s301: determination of nicotinamide catalysis reaction bench (2L) protocol: adding nicotinamide enzyme 6.6U/ml into 20% nicotinamide solution, reacting at 36-38 deg.C, controlling pH at 7.4 + -0.2, and monitoring nicotinamide and nicotinic acid content change every half an hour during reaction;
s302: catalytic experiment bench scale-up experiment verifies: adding nicotinamide enzyme 6.6U/ml into 20% nicotinamide solution, reacting at 36-38 deg.C, controlling pH at 7.4 + -0.2, and monitoring nicotinamide and nicotinic acid content change every half an hour during reaction process.
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