CN105695429A - Fermentation medium and method for fermenting nicotinamide monoucleotide (NMN) transferase by same - Google Patents
Fermentation medium and method for fermenting nicotinamide monoucleotide (NMN) transferase by same Download PDFInfo
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Abstract
The invention provides a fermentation medium and a method for fermenting nicotinamide monoucleotide (NMN) transferase by the same.The method includes the following steps that 1, a solid medium containing Escherichia coli is prepared; 2, activation liquor is prepared; 3, Escherichia coli is inoculated into the activation liquor, and kanamycin sulfate is added to obtain liquor containing activated Escherichia coli thalli; 4, the liquor containing thalli is inoculated into a sterilized fermentation medium for fermentation cultivation, an inductive agent IPTG is added for inductive cultivation, and fermentation liquor containing NMN transferase is collected; 5, the fermentation liquor is centrifuged, sediment is taken, and then Escherichia coli wet thalli containing NMN transferase are obtained.The fermentation medium has simple components, is low in cost and has broad application prospects, and the fermentation method is simple, practical and easy to implement.
Description
Technical field
The present invention relates to microbial fermentation engineering field, be specifically related to a kind of fermentation medium and the method for the NMN transferring enzyme that ferments with this fermentation medium。
Background technology
Research finds, NMN transferring enzyme is widely present in animal, plant, prokaryote and Eukaryotic cell。The NMN transferring enzyme already known at present existence form in cell mainly has three kinds of hypotypes, is Nmnat1, Nmnat2 and Nmnat3 respectively, and its molecular size range, character and feature difference occur along with its distributing position difference in histiocyte。Nicotinamide mononucleotide. (NMN) plays key player in human body cell energy generates, and it participates in the synthesis of nicotinamide adenine dinucleotide in cell (NAD, the important coenzyme that cellular energy converts)。In the biological tissue of all work, NMN transferring enzyme is the indispensable enzyme of synthesis NAD, research proves, it is particularly significant to the survival of prokaryote, be uniquely a kind of can the enzyme of NAD synthesis in activated cell core, and, NMN transferring enzyme can also as the target of biological agent, aging and the death of biological cell can be reduced to a certain extent, reduce the incidence rate of some diseases。Such as: the degeneration of the aixs cylinder caused by gene can be reduced, the biological activity of antitumor drug is improved。
The method synthesizing NMN transferring enzyme at present is mainly biological synthesis process, can be obtained some with traditional screening and induced-mutation technique and there are the high enzyme strain such as staphylococcus aureus of NMN transferring enzyme alive, Ai Xishi bacillus, extreme thermophilic archeobacteria etc., but, these strain enzymatic productivities and catalytic capability are relatively low, it is impossible to meet the demand of industrial development。
Escherichia coli are all higher than these antibacterial enzymatic productivities above-mentioned and catalytic capability, and escherichia coli have its most suitable growth culture medium, and generating of the NMN transferring enzyme of separate sources is different with the culture medium of its thalli growth, therefore, screen a kind of be not only suitable for colibacillary thalli growth but also the culture medium improving NMN transferring enzyme can be beneficial to very necessary。
Summary of the invention
The present invention carries out to solve the problems referred to above, it is therefore intended that provides a kind of and is not only suitable for colibacillary thalli growth but also can be beneficial to the fermentation medium improving NMN transferring enzyme and the method adopting this fermentation medium fermentation NMN transferring enzyme。
The technical solution used in the present invention is as follows:
The invention provides a kind of fermentation medium, have the feature that, comprise: concentration is the yeast powder of 20～30g/L, concentration is the tryptone of 8～12g/L, and volume fraction is the glycerol of 3%～4.5%, and concentration is the MgSO of 0.2～0.3g/L4, yeast extract, concentration is the NH of 1.0～1.5g/L4Cl, concentration is the NaCl of 0.4～0.6g/L, and concentration is the KH of 2～3g/L2PO4, concentration is the K of 9～13.5g/L2HPO4And distilled water。
Further, present invention also offers the fermentation process of a kind of nicotinamide mononucleotide. adenosine (NMN) transferring enzyme, above-mentioned fermentation medium is utilized to ferment, it is characterized in that, comprise the following steps: step one, in escherichia coli streak inoculation to solid medium, 12～18h will be cultivated when 37 DEG C, obtain containing colibacillary solid medium；Step 2, takes a certain amount of LB culture medium and is dissolved in distilled water, and regulating its pH value is 7.0, and sterilizing 20min under 121 DEG C of conditions obtains the activating solution that concentration is 25g/L；Step 3, is inoculated in activating solution from picking escherichia coli solid medium in an aseptic environment, and adds the kanamycin sulfate of final concentration of 30 μ g/ml, activates 8～12h, obtain the liquid containing the coli somatic activated under 37 DEG C of conditions；Step 4, accesses the liquid containing the coli somatic of activation in the fermentation medium of sterilizing according to inoculum concentration that volume fraction is 1～5%, is 37 DEG C in temperature, and when shaking speed is 180～200r/min, fermentation culture is to OD600When being 0.5～0.7, add the inducer isopropylthio thiogalactoside (IPTG) of final concentration of 8mmol/L, it it is 25 DEG C in temperature, when shaking speed is 180r/min, inducing culture 8～12h, collects the fermentation liquid obtaining niacinamide-containing mononucleotide adenosine (NMN) transferring enzyme；Step 5, is positioned in centrifuge tube by fermentation liquid, and when 8000～12000r/min, centrifugal 5～10min, takes the precipitation in centrifuge tube and obtain the escherichia coli wet thallus containing nicotinamide mononucleotide. adenosine (NMN) transferring enzyme。
In the fermentation process of nicotinamide mononucleotide. adenosine (NMN) transferring enzyme provided by the invention, can also have a feature in that wherein, in above-mentioned steps one, the preparation method of solid medium is: be dissolved in the conical flask of 500ml by LB culture medium and the agar distilled water that concentration is 20g/L that concentration is 25g/L, adding NaOH adjustment pH is 7.0, under 121 DEG C of conditions, autoclaving 20min, obtains solid medium。
In the fermentation process of nicotinamide mononucleotide. adenosine (NMN) transferring enzyme provided by the invention, can also have a feature in that wherein, the enzyme step being measured alive to nicotinamide mononucleotide. adenosine (NMN) transferring enzyme in escherichia coli wet thallus, this step includes following sub-step: sub-step one, it is 50mmol/L by escherichia coli wet thallus concentration, the phosphate buffered solution of pH7.5 is resuspended, ultrasonication escherichia coli wet thallus, bacterium solution after breaking cellular wall is 4 DEG C in temperature, rotating speed is the centrifugal 20min of 12000r/min, collect the supernatant of bacterium solution, the supernatant of bacterium solution is crude enzyme liquid；Concentration to be the buffer of 1mol/LpH7.5Tris-HCl, concentration the be NMN transferring enzyme of 300mmol/L, concentration are the ATP of 50mmol/L, concentration by sub-step two is the Mn of 50mmol/L2+, volume fraction be 2% ethanol, volume fraction be 53% crude enzyme liquid and the mixing of ethanol dehydrogenase (ADH) that enzyme amount is 7U, obtain mixed solution；Sub-step three, mixed solution is placed in the thermostat water bath of 37 DEG C oscillating reactions 2h, add the EDTA that concentration is 0.155mol/L and react 5min, terminate enzyme reaction, after ice bath cooling when rotating speed 8000r/min centrifugal 1min, take the supernatant of mixed solution and at the light absorption value OD of 340nm place assaying reaction product340, light absorption value OD340Represent NMN transferring enzyme enzyme activity size。
In the fermentation process of nicotinamide mononucleotide. adenosine (NMN) transferring enzyme provided by the invention, it is also possible to have a feature in that wherein, in above-mentioned sub-step three, the condition of ultrasonication is power is 300W, ultrasonic time 4s, every minor tick 6s, ultrasonic number of times 60 times。
The effect of invention and effect
The invention provides a kind of fermentation medium and the method for the NMN transferring enzyme that ferments with this fermentation medium, containing the indispensable element being suitable for Escherichia coli Growth in this fermentation medium, under the effect of derivant IPTG, make escherichia coli good growth and can high yield NMN transferring enzyme in this fermentation medium, this medium component is simple, cost is low, utilization has a extensive future, this fermentation process is simple and practical, it is easy to operation。
Detailed description of the invention
The present invention is expanded on further below in conjunction with embodiment, for concrete grammar used in embodiment or material, those skilled in the art on the basis of the technology of the present invention thinking, can carry out the replacement of routine and select according to existing technology, are not limited solely to the concrete record of the embodiment of the present invention。
The test method used in embodiment if no special instructions, is accordingly to be regarded as conventional method；The material that used, reagent etc., if no special instructions, all commercially obtain。
Embodiment one
1, fermentation medium
Comprising in the fermentation medium of the present embodiment: concentration is the yeast powder of 20g/L, concentration is the tryptone of 8g/L, and volume fraction is the glycerol of 4.5%, and concentration is the MgSO of 0.2g/L4, yeast extract, concentration is the NH of 1.0g/L4Cl, concentration is the NaCl of 0.6g/L, and concentration is the KH of 2g/L2PO4, concentration is the K of 9g/L2HPO4And distilled water。
2, fermentation process
The fermentation medium adopting the present embodiment component content carries out the fermentation of nicotinamide mononucleotide. adenosine (NMN) transferring enzyme, comprises the following steps:
Step one, by escherichia coli streak inoculation to the solid medium (preparation of solid medium: LB culture medium and the agar distilled water that concentration is 20g/L that concentration is 25g/L are dissolved in the conical flask of 500ml, adding NaOH adjustment pH is 7.0, autoclaving 20min under 121 DEG C of conditions) on, cultivate 12～18h when 37 DEG C, obtain containing colibacillary solid medium；
Step 2, takes a certain amount of LB culture medium and is dissolved in distilled water, and regulating its pH value is 7.0, and sterilizing 20min under 121 DEG C of conditions obtains the activating solution that concentration is 25g/L；
Step 3, is inoculated in activating solution from picking escherichia coli solid medium in an aseptic environment, and adds the kanamycin sulfate of final concentration of 30 μ g/ml, activates 8～12h, obtain the liquid containing the coli somatic activated under 37 DEG C of conditions；
Step 4, the liquid containing the coli somatic of activation is accessed in the fermentation medium of sterilizing according to the inoculum concentration that volume fraction is 1.5%, it it is 37 DEG C in temperature, when shaking speed is 180～200r/min, fermentation culture to OD600 be 0.5～0.7 time, add the inducer isopropylthio thiogalactoside (IPTG) of final concentration of 8mmol/L, it it is 25 DEG C in temperature, when shaking speed is 180r/min, inducing culture 8～12h, collects the fermentation liquid obtaining niacinamide-containing mononucleotide adenosine (NMN) transferring enzyme；
Step 5, is positioned in centrifuge tube by fermentation liquid, and when 8000～12000r/min, centrifugal 5～10min, takes the precipitation in centrifuge tube and obtain the escherichia coli wet thallus containing nicotinamide mononucleotide. adenosine (NMN) transferring enzyme。
3, escherichia coli produce the enzyme activity determination method of NMN transferring enzyme
The enzyme of nicotinamide mononucleotide. adenosine (NMN) transferring enzyme in the escherichia coli wet thallus that the present embodiment is obtained is lived and is measured, including following sub-step:
Sub-step one, by resuspended for phosphate buffered solution that escherichia coli wet thallus concentration is 50mmol/L, pH7.5, (power is 300W in ultrasonication, ultrasonic time 4s, interval time 6s, ultrasonic number of times 60 times) escherichia coli wet thallus, the bacterium solution after breaking cellular wall temperature be 4 DEG C, rotating speed be the centrifugal 20min of 12000r/min, collecting the supernatant of bacterium solution, the supernatant of bacterium solution is crude enzyme liquid；
Concentration to be the buffer of 1mol/LpH7.5Tris-HCl, concentration the be NMN transferring enzyme of 300mmol/L, concentration are the ATP of 50mmol/L, concentration by sub-step two is the Mn of 50mmol/L2+, volume fraction be 2% ethanol, volume fraction be 53% crude enzyme liquid and the mixing of ethanol dehydrogenase (ADH) that enzyme amount is 7U, obtain mixed solution；
Sub-step three, mixed solution is placed in the thermostat water bath of 37 DEG C oscillating reactions 2h, add the EDTA that concentration is 0.155mol/L and react 5min, terminate enzyme reaction, after ice bath cooling when rotating speed 8000r/min centrifugal 1min, take the supernatant of mixed solution and at the light absorption value OD of 340nm place assaying reaction product340, light absorption value OD340Represent NMN transferring enzyme enzyme activity size。
In the present embodiment, the vigor of the NMN transferring enzyme fermenting out by the fermentation medium of the present embodiment is 15.23～17.12U/mg。
Embodiment two
1, fermentation medium
Comprising in the fermentation medium of the present embodiment: concentration is the yeast powder of 30g/L, concentration is the tryptone of 12g/L, and volume fraction is the glycerol of 3%, and concentration is the MgSO of 0.3g/L4, yeast extract, concentration is the NH of 1.5g/L4Cl, concentration is the NaCl of 0.4g/L, and concentration is the KH of 2g/L2PO4, concentration is the K of 9g/L2HPO4And distilled water。
2, fermentation process
The fermentation medium adopting the present embodiment component content carries out the fermentation of nicotinamide mononucleotide. adenosine (NMN) transferring enzyme, comprises the following steps:
Step one, by escherichia coli streak inoculation to the solid medium (preparation of solid medium: LB culture medium and the agar distilled water that concentration is 20g/L that concentration is 25g/L are dissolved in the conical flask of 500ml, adding NaOH adjustment pH is 7.0, autoclaving 20min under 121 DEG C of conditions) on, cultivate 12～18h when 37 DEG C, obtain containing colibacillary solid medium；
Step 2, takes a certain amount of LB culture medium and is dissolved in distilled water, and regulating its pH value is 7.0, and sterilizing 20min under 121 DEG C of conditions obtains the activating solution that concentration is 25g/L；
Step 3, is inoculated in activating solution from picking escherichia coli solid medium in an aseptic environment, and adds the kanamycin sulfate of final concentration of 30 μ g/ml, activates 8～12h, obtain the liquid containing the coli somatic activated under 37 DEG C of conditions；
Step 4, the liquid containing the coli somatic of activation is accessed in the fermentation medium of sterilizing according to the inoculum concentration that volume fraction is 1.5%, it it is 37 DEG C in temperature, when shaking speed is 180～200r/min, fermentation culture to OD600 be 0.5～0.7 time, add the inducer isopropylthio thiogalactoside (IPTG) of final concentration of 8mmol/L, it it is 25 DEG C in temperature, when shaking speed is 180r/min, inducing culture 8～12h, collects the fermentation liquid obtaining niacinamide-containing mononucleotide adenosine (NMN) transferring enzyme；
Step 5, is positioned in centrifuge tube by fermentation liquid, and when 8000～12000r/min, centrifugal 5～10min, takes the precipitation in centrifuge tube and obtain the escherichia coli wet thallus containing nicotinamide mononucleotide. adenosine (NMN) transferring enzyme。
3, escherichia coli produce the enzyme activity determination method of NMN transferring enzyme
The enzyme of nicotinamide mononucleotide. adenosine (NMN) transferring enzyme in the escherichia coli wet thallus that the present embodiment is obtained is lived and is measured, including following sub-step:
Sub-step one, by resuspended for phosphate buffered solution that escherichia coli wet thallus concentration is 50mmol/L, pH7.5, (power is 300W in ultrasonication, ultrasonic time 4s, interval time 6s, ultrasonic number of times 60 times) escherichia coli wet thallus, the bacterium solution after breaking cellular wall temperature be 4 DEG C, rotating speed be the centrifugal 20min of 12000r/min, collecting the supernatant of bacterium solution, the supernatant of bacterium solution is crude enzyme liquid；
Concentration to be the buffer of 1mol/LpH7.5Tris-HCl, concentration the be NMN transferring enzyme of 300mmol/L, concentration are the ATP of 50mmol/L, concentration by sub-step two is the Mn of 50mmol/L2+, volume fraction be 2% ethanol, volume fraction be 53% crude enzyme liquid and the mixing of ethanol dehydrogenase (ADH) that enzyme amount is 7U, obtain mixed solution；
Sub-step three, mixed solution is placed in the thermostat water bath of 37 DEG C oscillating reactions 2h, add the EDTA that concentration is 0.155mol/L and react 5min, terminate enzyme reaction, after ice bath cooling when rotating speed 8000r/min centrifugal 1min, take the supernatant of mixed solution and at the light absorption value OD of 340nm place assaying reaction product340, light absorption value OD340Represent NMN transferring enzyme enzyme activity size。
In the present embodiment, the vigor of the NMN transferring enzyme fermenting out by the fermentation medium of the present embodiment is 12.44～16.38U/mg。
Embodiment three
1, fermentation medium
Comprising in the fermentation medium of the present embodiment: concentration is the yeast powder of 20g/L, concentration is the tryptone of 8g/L, and volume fraction is the glycerol of 3%, and concentration is the MgSO of 0.2g/L4, yeast extract, concentration is the NH of 1.5g/L4Cl, concentration is the NaCl of 0.6g/L, and concentration is the KH of 3.0g/L2PO4, concentration is the K of 9.0g/L2HPO4And distilled water。
2, fermentation process
The fermentation medium adopting the present embodiment component content carries out the fermentation of nicotinamide mononucleotide. adenosine (NMN) transferring enzyme, comprises the following steps:
Step one, by escherichia coli streak inoculation to the solid medium (preparation of solid medium: LB culture medium and the agar distilled water that concentration is 20g/L that concentration is 25g/L are dissolved in the conical flask of 500ml, adding NaOH adjustment pH is 7.0, autoclaving 20min under 121 DEG C of conditions) on, cultivate 12～18h when 37 DEG C, obtain containing colibacillary solid medium；
Step 2, takes a certain amount of LB culture medium and is dissolved in distilled water, and regulating its pH value is 7.0, and sterilizing 20min under 121 DEG C of conditions obtains the activating solution that concentration is 25g/L；
Step 3, is inoculated in activating solution from picking escherichia coli solid medium in an aseptic environment, and adds the kanamycin sulfate of final concentration of 30 μ g/ml, activates 8～12h, obtain the liquid containing the coli somatic activated under 37 DEG C of conditions；
Step 4, the liquid containing the coli somatic of activation is accessed in the fermentation medium of sterilizing according to the inoculum concentration that volume fraction is 1.5%, it it is 37 DEG C in temperature, when shaking speed is 180～200r/min, fermentation culture to OD600 be 0.5～0.7 time, add the inducer isopropylthio thiogalactoside (IPTG) of final concentration of 8mmol/L, it it is 25 DEG C in temperature, when shaking speed is 180r/min, inducing culture 8～12h, collects the fermentation liquid obtaining niacinamide-containing mononucleotide adenosine (NMN) transferring enzyme；
Step 5, is positioned in centrifuge tube by fermentation liquid, and when 8000～12000r/min, centrifugal 5～10min, takes the precipitation in centrifuge tube and obtain the escherichia coli wet thallus containing nicotinamide mononucleotide. adenosine (NMN) transferring enzyme。
3, escherichia coli produce the enzyme activity determination method of NMN transferring enzyme
The enzyme of nicotinamide mononucleotide. adenosine (NMN) transferring enzyme in the escherichia coli wet thallus that the present embodiment is obtained is lived and is measured, including following sub-step:
Sub-step one, by resuspended for phosphate buffered solution that escherichia coli wet thallus concentration is 50mmol/L, pH7.5, (power is 300W in ultrasonication, ultrasonic time 4s, interval time 6s, ultrasonic number of times 60 times) escherichia coli wet thallus, the bacterium solution after breaking cellular wall temperature be 4 DEG C, rotating speed be the centrifugal 20min of 12000r/min, collecting the supernatant of bacterium solution, the supernatant of bacterium solution is crude enzyme liquid；
Concentration to be the buffer of 1mol/LpH7.5Tris-HCl, concentration the be NMN transferring enzyme of 300mmol/L, concentration are the ATP of 50mmol/L, concentration by sub-step two is the Mn of 50mmol/L2+, volume fraction be 2% ethanol, volume fraction be 53% crude enzyme liquid and the mixing of ethanol dehydrogenase (ADH) that enzyme amount is 7U, obtain mixed solution；
Sub-step three, mixed solution is placed in the thermostat water bath of 37 DEG C oscillating reactions 2h, add the EDTA that concentration is 0.155mol/L and react 5min, terminate enzyme reaction, after ice bath cooling when rotating speed 8000r/min centrifugal 1min, take the supernatant of mixed solution and at the light absorption value OD of 340nm place assaying reaction product340, light absorption value OD340Represent NMN transferring enzyme enzyme activity size。
In the present embodiment, the vigor of the NMN transferring enzyme fermenting out by the fermentation medium of the present embodiment is 14.31～16.43U/mg。
The effect of embodiment one to embodiment three and effect
Embodiment one to embodiment three provides a kind of fermentation medium and the method for the NMN transferring enzyme that ferments with this fermentation medium, containing the indispensable element being suitable for Escherichia coli Growth in this fermentation medium, under the effect of derivant IPTG, make escherichia coli in this fermentation medium good growth and can high yield NMN transferring enzyme, before non-screening and optimizing。Enzyme is lived and is significantly improved, and this medium component is simple, cost is low, utilization has a extensive future, and this fermentation process is simple and practical, it is easy to operation。
Above example is only the basic explanation under present inventive concept, does not limit the invention。And according to any equivalent transformation that technical scheme is made, belong to protection scope of the present invention。
Claims (5)
1. a fermentation medium, it is characterised in that comprise:
Concentration is the yeast powder of 20～30g/L, and concentration is the tryptone of 8～12g/L, and volume fraction is the glycerol of 3%～4.5%, and concentration is the MgSO of 0.2～0.3g/L4, yeast extract, concentration is the NH of 1.0～1.5g/L4Cl, concentration is the NaCl of 0.4～0.6g/L, and concentration is the KH of 2～3g/L2PO4, concentration is the K of 9～13.5g/L2HPO4And distilled water。
2. a fermentation process for nicotinamide mononucleotide. adenosine (NMN) transferring enzyme, utilizes the fermentation medium described in claim 1 to ferment, it is characterised in that to comprise the following steps:
Step one, in escherichia coli streak inoculation to solid medium, will cultivate 12～18h, obtains containing colibacillary solid medium when 35～45 DEG C；
Step 2, takes a certain amount of LB culture medium and is dissolved in distilled water, and regulating its pH value is 6.0～8.0, and sterilizing 15～30min under 121 DEG C of conditions obtains the activating solution that concentration is 20～30g/L；
Step 3, it is inoculated in described activating solution from picking escherichia coli described solid medium in an aseptic environment, and add the kanamycin sulfate of final concentration of 20～40 μ g/ml, under 35～45 DEG C of conditions, activate 8～12h, obtain containing the liquid of the coli somatic activated；
Step 4, the liquid of the described coli somatic containing activation is accessed in the described fermentation medium of sterilizing according to the inoculum concentration that volume fraction is 1～5%, being 35～45 DEG C in temperature, when shaking speed is 180～200r/min, fermentation culture is to OD600When being 0.5～0.7, add the inducer isopropylthio thiogalactoside (IPTG) of final concentration of 5～10mmol/L, it it is 20～30 DEG C in temperature, when shaking speed is 150～200r/min, inducing culture 8～12h, collects the fermentation liquid obtaining niacinamide-containing mononucleotide adenosine (NMN) transferring enzyme；
Step 5, is positioned in centrifuge tube by described fermentation liquid, and when 8000～12000r/min, centrifugal 5～10min, takes the precipitation in centrifuge tube and obtain the escherichia coli wet thallus containing nicotinamide mononucleotide. adenosine (NMN) transferring enzyme。
3. the fermentation process of nicotinamide mononucleotide. adenosine (NMN) transferring enzyme according to claim 2, it is characterised in that:
Wherein, in step, the preparation method of described solid medium is: be dissolved in the conical flask of 500ml by LB culture medium and the agar distilled water that concentration is 20g/L that concentration is 25g/L, adding NaOH adjustment pH is 7.0, autoclaving 20min under 121 DEG C of conditions, obtains described solid medium。
4. the fermentation process of nicotinamide mononucleotide. adenosine (NMN) transferring enzyme according to claim 2, it is characterised in that also include:
Wherein, the enzyme step being measured alive to nicotinamide mononucleotide. adenosine (NMN) transferring enzyme in described escherichia coli wet thallus, this step includes following sub-step:
Sub-step one, by resuspended for phosphate buffered solution that escherichia coli wet thallus concentration is 50mmol/L, pH7.5, escherichia coli wet thallus described in ultrasonication, bacterium solution after breaking cellular wall temperature be 4 DEG C, rotating speed be the centrifugal 20min of 12000r/min, collecting the supernatant of described bacterium solution, the supernatant of described bacterium solution is crude enzyme liquid；
Concentration to be the buffer of 1mol/LpH7.5Tris-HCl, concentration the be NMN transferring enzyme of 300mmol/L, concentration are the ATP of 50mmol/L, concentration by sub-step two is the Mn of 50mmol/L2+, volume fraction be 2% ethanol, volume fraction be 53% described crude enzyme liquid and the mixing of ethanol dehydrogenase (ADH) that enzyme amount is 7U, obtain mixed solution；
Sub-step three, it is placed in the thermostat water bath of 37 DEG C by described mixed solution oscillating reactions 2h, add the EDTA that concentration is 0.155mol/L and react 5min, terminate enzyme reaction, after ice bath cooling when rotating speed 8000r/min centrifugal 1min, take the supernatant of described mixed solution and at the light absorption value OD of 340nm place assaying reaction product340, described light absorption value OD340Represent NMN transferring enzyme enzyme activity size。
5. the fermentation process of nicotinamide mononucleotide. adenosine (NMN) transferring enzyme according to claim 4, it is characterised in that:
Wherein, in sub-step three, the condition of described ultrasonication is power is 300W, ultrasonic time 4s, every minor tick 6s, ultrasonic number of times 60 times。
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| CN111635917A (en) * | 2020-06-11 | 2020-09-08 | 辽宁天华生物药业有限公司 | Preparation method of beta-nicotinamide ribodinucleotide |
| CN112625988A (en) * | 2020-12-22 | 2021-04-09 | 江苏诚信药业有限公司 | Escherichia coli fermentation medium, fermentation culture method and application |
| CN112852678A (en) * | 2021-03-08 | 2021-05-28 | 泓博元生命科技（深圳）有限公司 | Enterobacter gondii for producing nicotinamide mononucleotide and application thereof |
| CN117925502A (en) * | 2024-03-21 | 2024-04-26 | 山东沃奇农业开发有限公司 | Shinyleaf yellowhorn tissue culture solution |
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| CN112625988A (en) * | 2020-12-22 | 2021-04-09 | 江苏诚信药业有限公司 | Escherichia coli fermentation medium, fermentation culture method and application |
| CN112852678A (en) * | 2021-03-08 | 2021-05-28 | 泓博元生命科技（深圳）有限公司 | Enterobacter gondii for producing nicotinamide mononucleotide and application thereof |
| CN117925502A (en) * | 2024-03-21 | 2024-04-26 | 山东沃奇农业开发有限公司 | Shinyleaf yellowhorn tissue culture solution |
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