阅读说明：本技术 一种β-烟酰胺核糖二核苷酸的制备方法 (Preparation method of beta-nicotinamide ribodinucleotide ) 是由 王杰 颜祥飞 彭文静 李卓 王姝昕 宋辉 冯晓燕 于 2020-06-11 设计创作，主要内容包括：本发明涉及生物酶催化制备领域,具体涉及一种β-烟酰胺核糖二核苷酸的制备方法。该方法首先通过高密度发酵法制备烟酰胺核糖激酶和烟酰胺单核苷酸转移酶,然后烟酰胺核糖和ATP在烟酰胺核糖激酶和烟酰胺单核苷酸转移酶作用下生产β-烟酰胺核糖二核苷酸。本发明提供制备烟酰胺核糖激酶和烟酰胺单核苷酸转移酶的高密度发酵方法,方法中使用发酵培养基中含有适合大肠杆菌生长的必需元素,在诱导剂IPTG的作用下,使得大肠杆菌在发酵培养基中良好生长并且能够高产烟酰胺核糖激酶和烟酰胺单核苷酸转移酶,酶活比较高。本发明提供β-烟酰胺核糖二核苷酸的制备方法效率高,操作简单,收率高,使用的烟酰胺核糖和ATP是常见的原料,成本低,适合产业化生产。(The invention relates to the field of bio-enzyme catalytic preparation, and in particular relates to a preparation method of beta-nicotinamide ribodinucleotide. The method comprises the steps of firstly preparing nicotinamide ribokinase and nicotinamide mononucleotide transferase by a high-density fermentation method, and then producing beta-nicotinamide ribodinucleotide by nicotinamide ribose and ATP under the action of the nicotinamide ribokinase and the nicotinamide mononucleotide transferase. The invention provides a high-density fermentation method for preparing nicotinamide ribokinase and nicotinamide mononucleotide transferase, which uses a fermentation culture medium containing essential elements suitable for growth of escherichia coli, enables the escherichia coli to grow well in the fermentation culture medium under the action of an inducer IPTG, can produce high-yield nicotinamide ribokinase and nicotinamide mononucleotide transferase, and has high enzyme activity. The preparation method of the beta-nicotinamide riboside provided by the invention has the advantages of high efficiency, simple operation and high yield, and the used nicotinamide riboside and ATP are common raw materials, so that the cost is low, and the preparation method is suitable for industrial production.)

1. A preparation method of beta-nicotinamide ribodinucleotide is characterized by comprising the following steps:

step one, inoculating escherichia coli to a solid culture medium of LB, and culturing for 12-18 h at 37 ℃ to obtain a solid inclined plane containing the escherichia coli;

step two, inoculating the slant inoculation amount containing the escherichia coli into the sterilized seed liquid, and culturing at the temperature of 37 ℃ and the rotating speed of a shaking table of 180-200 r/min until the OD600 is 3.2, wherein the seed liquid in the shaking bottle is good in length;

step three, preparing nicotinamide ribokinase and nicotinamide mononucleotide transferase by a high-density fermentation method, wherein a fermentation medium consists of the following raw materials in percentage by mass: peptone 1.0-2.0%, yeast extract 0.2-1.0%, potassium dihydrogen phosphate 0.05-0.15%, dipotassium hydrogen phosphate 0.03-0.10%, glycerol 0.5-1.0%, magnesium sulfate 0.05-0.2%, defoaming agent 0.05-0.10%, and the balance of drinking water; sterilizing the culture medium by using steam, respectively inoculating escherichia coli containing nicotinamide ribokinase and nicotinamide mononucleotide transferase genes, culturing at the temperature of 33-37 ℃, the ventilation volume of 3-10L/min and the stirring speed of 300-600rpm in a fermentation tank for 5-6 hours, adding 0.5g of isopropyl thiogalactoside IPTG for induction, and continuously culturing for 15-22 hours to obtain a culture solution;

step four, respectively centrifuging the two culture solutions in the step three to obtain thalli, homogenizing and crushing the thalli by using the pressure of 0.2-0.6MPa, filtering to obtain filtrate, and filtering and concentrating the filtrate by using a hollow membrane to obtain an enzyme concentrate of the amide ribokinase and the nicotinamide mononucleotide transferase;

step five, dissolving 34g of nicotinamide riboside, 100g of ATP and 8-16g of magnesium chloride into 2L of purified water, respectively adding 20-80g of enzyme concentrated solution of nicotinamide riboside kinase and nicotinamide mononucleotide transferase in the step four, controlling the temperature at 30-37 ℃, controlling the pH value to be 6.40-6.60 by using sodium hydroxide solution in the process, detecting the residue of nicotinamide riboside and the generated beta-nicotinamide riboside, and under the action of enzyme, the nicotinamide riboside is remained below 0.02g/L within 4 hours, so that the generated beta-nicotinamide riboside reaches the highest concentration.

2. The method of claim 1, wherein the fermentation medium of step three comprises the following raw materials by weight: peptone 1.0-1.5%, yeast extract 0.5-0.8%, potassium dihydrogen phosphate 0.05-0.10%, dipotassium hydrogen phosphate 0.05-0.08%, glycerol 0.5-0.8%, magnesium sulfate 0.10-0.15%, defoaming agent 0.05-0.08%, and water in balance.

3. The method of claim 1, wherein the amount of magnesium chloride added in step five is 10-15 g.

4. The method of claim 1, wherein 30-60g of the enzyme concentrate of nicotinamide ribokinase and nicotinamide mononucleotide transferase is added in step five.

5. The method of claim 1, wherein the temperature in step five is controlled to 35-37 ℃.

6. The method of claim 1, wherein the pH is controlled to 6.45-6.55 in step five.

Technical Field

The invention relates to the field of bio-enzyme catalytic preparation, and in particular relates to a preparation method of beta-nicotinamide ribodinucleotide.

Background

Of all the enzymes currently found, about 30-35% are oxidoreductases. In industrial biotechnology, especially in biopharmaceutical and fine chemical industries, oxidoreductases are important enzymes. In the case of the reaction catalyzed by the oxidoreductase, the production of the product is accompanied by the consumption of a certain coenzyme for the transfer of electrons and protons. According to statistics, about 80% of reactions need nicotinamide ribodinucleotide, namely oxidized coenzyme I (NAD +) as coenzyme, and the phosphorylation derivative of 2' -position of a ribose ring system connected with adenine in the NAD + is nicotinamide adenine dinucleotide phosphate, also called oxidized coenzyme II, (NADP +) is an important coenzyme. The oxidized coenzyme II transfers protons, electrons and energy in a redox reaction mode, participates in a plurality of metabolic reactions of cells, synthesizes lipids, fatty acid and nucleotide, can activate a multienzyme system, promotes the synthesis and metabolism of nucleic acid, protein and polysaccharide, improves the material transfer and regulation control, and improves the metabolic function. Research shows that the oxidized coenzyme II can promote metabolism, energy metabolism, resist cell senility and resist oxidation.

NAD + is widely present in living organisms, but is present in very low amounts, with NAD + extracted from living organisms being at a market price of tens of thousands of dollars per kilogram. NAD + is used in industrial production, but the production method is less. Current chemical and biological enzymatic methods of NAD +. The chemical method uses nicotinamide ribose as a raw material, and also uses expensive reagents and explosive reagents, so that accidents can occur due to high cost. Biological enzyme catalysis is gradually the mainstream method for industrially preparing nicotinamide ribodinucleotide (NAD +) because of the inherent advantages of high efficiency, environmental protection and the like, but the existing biological enzyme method has complex operation and low yield.

Disclosure of Invention

Aiming at the problems of complex operation and low yield of the preparation method of beta-nicotinamide ribodinucleotide in the prior art, the invention aims to provide a preparation method of beta-nicotinamide ribodinucleotide.

In order to achieve the purpose, the invention adopts the following technical scheme.

A preparation method of beta-nicotinamide ribodinucleotide comprises the following steps.

Step one, inoculating escherichia coli to a solid culture medium of LB, and culturing for 12-18 h at 37 ℃ to obtain a solid inclined plane containing the escherichia coli.

And step two, inoculating the slant inoculation amount containing the escherichia coli into the sterilized seed liquid, and culturing at the temperature of 37 ℃ and the rotation speed of a shaking table of 180-200 r/min until the OD600 is 3.2, wherein the seed liquid in the shaking bottle grows well.

Step three, preparing nicotinamide ribokinase and nicotinamide mononucleotide transferase by a high-density fermentation method, wherein a fermentation medium consists of the following raw materials in percentage by mass: peptone 1.0-2.0%, yeast extract 0.2-1.0%, potassium dihydrogen phosphate 0.05-0.15%, dipotassium hydrogen phosphate 0.03-0.10%, glycerol 0.5-1.0%, magnesium sulfate 0.05-0.2%, defoaming agent 0.05-0.10%, and drinking water in balance; sterilizing the culture medium by using steam, respectively inoculating escherichia coli containing nicotinamide ribokinase and nicotinamide mononucleotide transferase genes, culturing at the temperature of 33-37 ℃, the ventilation volume of 3-10L/min and the stirring speed of 300-600rpm in a fermentation tank for 5-6 hours, adding 0.5g of isopropyl thiogalactoside IPTG for induction, and continuously culturing for 15-22 hours to obtain a culture solution.

Step four, respectively centrifuging the two culture solutions in the step three to obtain thalli, homogenizing and crushing the thalli by using the pressure of 0.2-0.6MPa, filtering to obtain filtrate, and filtering and concentrating the filtrate by using a hollow membrane to obtain an enzyme concentrate of the amide ribokinase and the nicotinamide mononucleotide transferase.

Step five, dissolving 34g of nicotinamide riboside, 100g of ATP and 8-16g of magnesium chloride into 2L of purified water, respectively adding 20-80g of enzyme concentrated solution of nicotinamide riboside kinase and nicotinamide mononucleotide transferase in the step four, controlling the temperature at 30-37 ℃, controlling the pH value to be 6.40-6.60 by using sodium hydroxide solution in the process, detecting the residue of nicotinamide riboside and the generated beta-nicotinamide riboside, and under the action of enzyme, the nicotinamide riboside is remained below 0.02g/L within 4 hours, so that the generated beta-nicotinamide riboside reaches the highest concentration.

And step six, carrying out post-treatment ultrafiltration, nanofiltration, resin adsorption elution and crystallization on the reaction liquid to obtain a product.

Further, the fermentation medium in the third step is composed of the following raw materials by mass percent: peptone 1.0-1.5%, yeast extract 0.5-0.8%, potassium dihydrogen phosphate 0.05-0.10%, dipotassium hydrogen phosphate 0.05-0.08%, glycerol 0.5-0.8%, magnesium sulfate 0.10-0.15%, defoaming agent 0.05-0.08%, and water in balance.

Further, the amount of magnesium chloride added in step five is 10-15 g.

Further, the amount of the enzyme concentrate of nicotinamide ribokinase and nicotinamide mononucleotide transferase added in step five is 30-60 g.

Further, the temperature in the fifth step is controlled to be 35-37 ℃.

Further, the pH value is controlled to be 6.45-6.55 in the fifth step.

Compared with the prior art, the invention has the following beneficial effects.

The enzymatic activities of nicotinamide ribokinase and nicotinamide mononucleotide transferase in the preparation method for preparing beta-nicotinamide ribodinucleotide provided by the invention are high, nicotinamide ribokinase and ATP can be rapidly converted into beta-nicotinamide ribodinucleotide, the conversion rate is high, the purity of the produced NAD + is high, the fermentation process is simple, the extraction is easy, the cost is low, and the industrial production is easy.

The fermentation medium contains essential elements suitable for growth of escherichia coli, the escherichia coli can grow well in the fermentation medium under the action of an inducer IPTG, high yield of nicotinamide ribokinase and nicotinamide mononucleotide transferase can be achieved, the enzyme activity is high, the medium is simple in component, low in cost and wide in application prospect, and the fermentation method is simple, practical and easy to operate.

The invention provides an enzymatic reaction method of nicotinamide riboside, which has high efficiency, simple operation and high yield, and the used nicotinamide riboside and ATP are common raw materials, so the cost is low and the method can be industrialized.

Detailed Description

For better understanding of the present invention, the following examples are further illustrated, but the following examples do not limit the scope of the present invention. In the following examples, experimental procedures and methods not described in detail are conventional means well known in the art.