阅读说明：本技术 烟酰胺高效率转化为烟酸的菌种的方法 (Method for efficiently converting nicotinamide into nicotinic acid strains ) 是由 周媛 胡昊 齐勇 夏伟 李玉山 于 2021-08-12 设计创作，主要内容包括：本发明公开了烟酰胺高效率转化为烟酸的菌种的方法,包括如下步骤：S100：烟酰胺菌的筛选与鉴定；S200：生物催化反应条件探究；S300：催化反应条件确定及小试放大实验验证。筛选出可以将烟酰胺中的酰胺转化为羧酸的菌种,通过生物催化的方法,将烟酰胺转化为烟酸,告别了传统化学方法的高能耗和高污染,具有成本低、污染小、反应温和,适合工业化生产。(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.)

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.

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.