CN107955791A - It is a kind of can a step be catalyzed nicotinamide adenine dinucleotide strain and its screening technique and application - Google Patents
It is a kind of can a step be catalyzed nicotinamide adenine dinucleotide strain and its screening technique and application Download PDFInfo
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Abstract
Present invention is disclosed it is a kind of can a step be catalyzed nicotinamide adenine dinucleotide strain and its screening technique and application.The strain of the present invention can be applied to prepare nicotinamide adenine dinucleotide, using niacinamide ribose as substrate solution, it is added to using the cell crude extract of the strain as catalyst in substrate, add atriphos propylhomoserin, convert 18 it is small when after, nicotinamide adenine dinucleotide is formed in standard solution.Strain provided by the invention can a step be catalyzed to obtain nicotinamide adenine dinucleotide (NAD), yield is high.
Description
Technical field
The invention belongs to technical field of bioengineering, more particularly to one kind to utilize microbial cell or cell crude extract enzymatic
The method for synthesizing nicotinamide adenine dinucleotide.
Background technology
Nicotinamide adenine dinucleotide（Nicotinamide adenine dinucleotide, abridge NAD, is also referred to as
For Coenzyme I）Be a kind of basic redox coenzyme, play the pivotal role of core in cell, participate in cellular material metabolism,
The key foundation physiological activities such as energy synthesis, respiration.In vitro in the biocatalytic reaction of reduction-oxidation type, many oxygen
The apoenzyme for changing reduction reaction also needs the participation ability normal reaction of various coenzyme.NAD is a kind of relatively conventional coenzyme, mesh
There are about 700 kinds of enzymes to be catalyzed using coenzyme NAD known to preceding.Therefore, reacted in the industrial biocatalytic of redox class
In, nicotinamide adenine dinucleotide it is industrially prepared it is particularly important that.
At present, the method for preparation nicotinamide adenine dinucleotide (NAD) has a variety of, including chemical synthesis, biology enzyme are urged
Change and saccharomycetes to make fermentation extraction etc..Chemical synthesis by chemical industry it is intrinsic seriously polluted, condition is harsh, and yield is not in addition
Height, synthesis cost are higher.For the method for saccharomycetes to make fermentation extraction, since nicotinamide adenine dinucleotide (NAD) is in yeast
Content in bacterium is too low, therefore this method has significant limitation on industrial production scale.Biological enzyme is because it is solid
Some high efficiency, it is environmentally protective the advantages that be increasingly becoming the main stream approach of industrially prepared nicotinamide adenine dinucleotide (NAD).
Biological enzyme, which prepares nicotinamide adenine dinucleotide (NAD), mainly includes following key step：
The first step：With niacinamide ribose (nicotinamide riboside, NR) for starting material, in niacinamide ribokinase
Under the catalysis of (nicotinamide riboside kinase, NRK), nicotinamide mononucleotide is obtained
(nicotinamidemononucleotide,NMN).The reaction process reaction equation is as follows：
Second step：Nicotinamide mononucleotide (nicotinamidemononucleotide, NMN) is in nicotinamide mononucleotide adenosine
It is and another under the action of transferase (nicotinamidemononucleotide adenylyltransferase, NMNAT)
Precursor atriphos (ATP) reacts, generation nicotinamide adenine dinucleotide (NAD).
But above-mentioned nicotinamide adenine dinucleotide (NAD) biological enzyme preparation method is related to multistep reaction, and yield
It is not high.
The content of the invention
In view of the prior art is there are drawbacks described above, it is an object of the invention to provide it is a kind of in high yield prepare nicotinoyl amine gland
The method of purine dinucleotides (NAD), it is specific to provide the strain screened in a kind of soil, can be with niacinamide ribose
(nicotinamideriboside, NR) is starting material, and a step is catalyzed to obtain nicotinamide adenine dinucleotide (NAD).
The purpose of the present invention, will be achieved by the following technical programs：
It is a kind of can a step be catalyzed nicotinamide adenine dinucleotide strain, the strain is DHP02, and Classification And Nomenclature is deformation
Bacillus Proteus sp., are preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address：China,
Beijing, Beijing institute of microbiology of the academy of sciences, deposit number CGMCC NO：13721, March 3 2017 preservation time.
Preferably, it is described it is a kind of can a step be catalyzed the strain of nicotinamide adenine dinucleotide is preparing nicotinoyl amine gland
Application on purine dinucleotides.
Preferably, it is described it is a kind of can a step be catalyzed the strain of nicotinamide adenine dinucleotide is preparing nicotinoyl amine gland
Application on purine dinucleotides, includes the following steps,
Using niacinamide ribose as substrate solution, it is added in substrate, is added as catalyst using the cell crude extract of the strain
Atriphos propylhomoserin, conversion 18 it is small when after, nicotinamide adenine dinucleotide is formed in standard solution.
Preferably, cell crude extract described above is made for strain progress LB culture medium enrichment cultures, the LB trainings
Base horn of plenty culture medium is supported, is formulated and is：Peptone 1%, yeast extract 0.5%, sodium chloride 0.5%.
Preferably, it is a kind of can a step be catalyzed nicotinamide adenine dinucleotide bacteria selection method, it is including as follows
Step,
S1, systematic sampling domestication step, in the earth that carries out fetching earth at niacinamide ribose synthesis for a long time as sample point, add inorganic salts
Fluid nutrient medium and niacinamide ribose solution, and regulation system pH is 6.6-7.2, domestication culture forms soil supension in 7 days；
S2, solid screening and culturing medium making step, agar powder is added in the culture medium in S1 and carries out high-temperature sterilization postcooling extremely
It is spare that room temperature forms film solid media；
S3, bacterium solution application step, by the soil supension after being tamed in S1 be coated in the film solid media in S2 and carry out culture 48
Hour；
S4, inoculation step, the good single bacterium colony of random selection separation, carries out inoculation picking, is seeded to inorganic in S1 in S3
In salt fluid nutrient medium；
S5, strain separating step, choose the fluid nutrient medium progress strain separating purifying that can grow microorganism in S3；
S6, the fluid nutrient medium taking-up that microorganism will can be grown in S4, repeat the application step of S3, are placed in insulating box culture；
The continuous passage culture of the formation of S7, bacterium colony, repetition S4-S6 steps, progress solid-liquid-solid ", until solid
It is consistent that the cellular morphology that the colonial morphology being observed visually unanimously or under microscope is observed is formed in culture medium.
Preferably, the formula of inorganic salt liquid culture medium is in the S1：
Beneficial effects of the present invention：Strain provided by the invention can a step be catalyzed to obtain nicotinamide adenine dinucleotide (NAD),
Yield is high.
Just attached drawing in conjunction with the embodiments below, is described in further detail the embodiment of the present invention, so that of the invention
Technical solution is more readily understood, grasps.
Brief description of the drawings
Fig. 1：The high performance liquid chromatography reaction collection of illustrative plates of control group.
Fig. 2：Obtained by screening in the present invention strain be catalyzed to obtain nicotinamide adenine dinucleotide high performance liquid chromatography it is anti-
Answer collection of illustrative plates.
Embodiment
Embodiment 1
The factory of niacinamide ribose (nicotinamideriboside, NR) synthesis is being carried out for a long time, selects what sewer passed through
5 sample points of soil, average each point take pedotheque 5g, add the minimal medium and niacinamide ribose of 200mL
(nicotinamideriboside, NR) solution, the pH for adjusting whole system are 6.8, domestication culture 7 days.
The minimal medium is to be used as sole carbon with niacinamide ribose (nicotinamideriboside, NR)
The liquid screening medium in source.Its composition proportion and the dosage of niacinamide ribose, concentration etc. are shown in Table 1.
It is described domestication condition of culture be：The soil liquid for having added minimal medium and niacinamide ribose is loaded
In 500mL sterilizing triangular flasks, sealed, be placed in the constant-temperature table incubator of lucifuge with eight layers of gauze of sterilizing, culture temperature is set
30 DEG C of degree, the rotating speed of 150rpm carries out domestication culture.
Table 1：Tame culture medium prescription：
.Embodiment 2：
Make solid screening and culturing medium：On the basis of the Liquid Culture based formulas of table 1,2% agar powder, 121 DEG C of high temperature are added
Sterilizing 25 minutes after, placement be cooled to 45 DEG C or so it is spare.The glass dish of diameter 90mm is taken, above-mentioned culture is poured into plate
Base 20mL.Room temperature is continued cool to until culture medium is in curdled appearance.It is placed in spare in 4 DEG C of refrigerators.
The solid medium is as primary carbon source with niacinamide ribose (nicotinamideriboside, NR)
Solid screening and culturing medium.
Embodiment 3
Bacterium solution is coated with：The 200 μ L of soil supension tamed in Example 1, the solid screening training being spread evenly across in embodiment 2
Support in base, be placed in constant incubator and cultivate, 30 DEG C of set temperature, when the tablet inversion culture 48 after coating is small.
Embodiment 4
Observation embodiment 3 in cultivated 48 it is small when solid culture primary surface.Random selection separates good single bacterium colony, with inoculation
Pin picking, is seeded in fluid nutrient medium described in embodiment 1 and cultivates respectively, sets rotating speed 150rpm, 30 DEG C of temperature.
Embodiment 5
The situation that fluid nutrient medium is grown in observation embodiment 4, sampling carries out micro- sem observation, for that can grow a large amount of microorganisms
Fluid nutrient medium carry out next step strain separating purification work.
Embodiment 6
The fluid nutrient medium that a large amount of microorganisms are grown in embodiment 5 is taken out into 200 μ L, according to the method in embodiment 3, is applied to
Solid culture primary surface in embodiment 2.It is placed in constant incubator and cultivates, 30 DEG C of set temperature, the tablet after coating is inverted
Cultivate 48 it is small when.
Embodiment 7
The step of repeating embodiment 4 to embodiment 6, carries out the continuous passage culture of " solid-liquid-solid ", until solid
The cellular morphology that the colonial morphology being observed visually under body culture medium unanimously or is under the microscope observed is consistent.
Screening technique is used in this patent：Using niacinamide ribose (nicotinamideriboside, NR) as culture
Sole carbon source in base, the microorganism that can be grown out in this culture medium, is that can utilize niacinamide ribose in theory
(nicotinamideriboside, NR) is further metabolized.And the agar component in solid medium, it is also possible to
Used by microorganism as carbon source.Therefore the once liquid without other carbon source materials is added between solid culture twice to train
Support, above-mentioned possibility can be excluded, ensure the purebred microorganism energy metabolism niacinamide ribose that screening obtains to greatest extent
(nicotinamideriboside, NR).
Embodiment 8
By above step, 1 plant of pure culture has been finally obtained, LB culture medium enrichment cultures are carried out to this microorganism.The LB
Culture medium horn of plenty culture medium, is formulated and is：Peptone 1%, yeast extract 0.5%, sodium chloride 0.5%.Condition of culture：30 DEG C,
40 it is small when, rotating speed 150rpm.Cultivate 40 it is small when after, under the conditions of 4 DEG C, with 12000rpm centrifuge 10 minutes carry out microorganism collection.
After centrifugation, using the 0.1mol/L of 3 times of weight, the phosphate buffer of pH7.0 carries out thalline resuspension.
The preparation method of the phosphate buffer is：Weigh disodium hydrogen phosphate 21.84g, sodium dihydrogen phosphate dihydrate
6.08g, is dissolved in 1000ml deionized waters.
Thus obtained bacteria suspension deposits in 4 DEG C, 1 hour to after sufficiently cool, by using the pressure of 12000 psig
By suspension by the homogenizer equipped with two level homogenate valve member, its content is discharged from cell, immediately by cell after rupture
Homogenate is cooled to 4 DEG C.Gained homogenate further in centrifuge under conditions of 4 DEG C with 12000rpm, centrifuge 30 minutes
The supernatant clarified.
Embodiment 9
Prepare the substrate solution of niacinamide ribose (nicotinamideriboside, NR), concentration 20g/L.Take 0.5mL's
Above-mentioned clarification thalline crude extract is added in 150mL niacinamide ribose (nicotinamideriboside, NR) standard solution,
Add atriphos propylhomoserin（ATP）, lucifuge, 30 DEG C, 100 rpm in constant-temperature table incubator, conversion 18 it is small when after, survey
Determine the growing amount of nicotinamide adenine dinucleotide in standard solution (NAD).Meanwhile separately take same niacinamide ribose
(nicotinamideriboside, NR) standard solution 150mL, adds the sterile purified water of 0.5mL, is put into together same
In incubator, conversion reaction is carried out by same condition, operation repetitive reaction is control group.It is small that 18 have been carried out in conversion reaction
Shi Hou, sampling detection obtains following high performance liquid chromatography reaction collection of illustrative plates, respectively as shown in Fig. 2, Fig. 1.
HPLC testing conditions are as follows：
Mobile phase A：10mM ammonium acetates, Mobile phase B：Methanol,
Wavelength：260nm, column temperature：23 DEG C, flow velocity：0.5mL/min.
Chromatographic column：5 μm of 4.6 × 250mm of Shimadzu C18 columns
Gradient elution：
As can be seen from Figure, for experimental group in 21 minutes characteristic peaks for nicotinamide adenine dinucleotide (NAD) occur, its was dense
Spend for 8.8g/L.The peak area of substrate niacinamide ribose (nicotinamideriboside, NR) reduces 66.3%.Control
Group, in 20.903 minutes characteristic peaks without nicotinamide adenine dinucleotide (NAD), and substrate niacinamide ribose
The peak area of (nicotinamideriboside, NR) is unchanged.
Experimental data demonstrate screening obtained one plant of energy using niacinamide ribose (nicotinamideriboside,
NR microorganism).The cell crude extract of the microorganism have a step by niacinamide ribose (nicotinamideriboside,
NR) it is converted into the ability of nicotinamide adenine dinucleotide (NAD).
The present invention still has numerous embodiments, all technical sides formed using equivalents or equivalent transformation
Case, is within the scope of the present invention.
Claims (6)
1. it is a kind of can a step be catalyzed nicotinamide adenine dinucleotide strain, it is characterised in that：The strain is DHP02,
It is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, address：China, Beijing, Beijing academy of sciences are micro-
Biological study institute, deposit number CGMCC NO：13721, March 3 2017 preservation time.
2. it is as claimed in claim 1 it is a kind of can a step be catalyzed the strain of nicotinamide adenine dinucleotide is preparing niacinamide
Application on adenine-dinucleotide.
3. it is as claimed in claim 2 it is a kind of can a step be catalyzed the strain of nicotinamide adenine dinucleotide is preparing niacinamide
Application on adenine-dinucleotide, it is characterised in that：Include the following steps,
Using niacinamide ribose as substrate solution, it is added in substrate, is added as catalyst using the cell crude extract of the strain
Atriphos propylhomoserin, conversion 18 it is small when after, nicotinamide adenine dinucleotide is formed in standard solution.
4. it is as claimed in claim 3 it is a kind of can a step be catalyzed the strain of nicotinamide adenine dinucleotide is preparing niacinamide
Application on adenine-dinucleotide, it is characterised in that：The cell crude extract carries out the enrichment training of LB culture mediums for the strain
Support and be made, the LB culture mediums horn of plenty culture medium is formulated and is：Peptone 1%, yeast extract 0.5%, sodium chloride 0.5%.
5. it is as claimed in claim 1 it is a kind of can a step be catalyzed nicotinamide adenine dinucleotide bacteria selection method,
It is characterized in that：Include the following steps,
S1, systematic sampling domestication step, in the earth that carries out fetching earth at niacinamide ribose synthesis for a long time as sample point, add inorganic salts
Fluid nutrient medium and niacinamide ribose solution, and regulation system pH is 6.6-7.2, domestication culture forms soil supension in 7 days；
S2, solid screening and culturing medium making step, agar powder is added in the culture medium in S1 and carries out high-temperature sterilization postcooling extremely
It is spare that room temperature forms film solid media；
S3, bacterium solution application step, by the soil supension after being tamed in S1 be coated in the film solid media in S2 and carry out culture 48
Hour；
S4, inoculation step, the good single bacterium colony of random selection separation, carries out inoculation picking, is seeded to inorganic in S1 in S3
In salt fluid nutrient medium；
S5, strain separating step, choose the fluid nutrient medium progress strain separating purifying that can grow microorganism in S3；
S6, the fluid nutrient medium taking-up that microorganism will can be grown in S4, repeat the application step of S3, are placed in insulating box culture；
The continuous passage culture of the formation of S7, bacterium colony, repetition S4-S6 steps, progress solid-liquid-solid ", until solid
It is consistent that the cellular morphology that the colonial morphology being observed visually unanimously or under microscope is observed is formed in culture medium.
6. it is as claimed in claim 5 it is a kind of can a step be catalyzed nicotinamide adenine dinucleotide bacteria selection method,
It is characterized in that：The formula of inorganic salt liquid culture medium is in the S1：
。
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110643587A (en) * | 2019-10-29 | 2020-01-03 | 杭州唯泰生物药业有限公司 | Method for preparing nicotinamide adenine dinucleotide phosphate by enzyme method |
| 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 |
| WO2020258111A1 (en) * | 2019-06-27 | 2020-12-30 | 邦泰合盛生物科技（深圳）有限公司 | Enzymatic method for industrial production of nad |
| CN113637715A (en) * | 2021-08-12 | 2021-11-12 | 安徽瑞邦生物科技有限公司 | Method for efficiently converting nicotinamide into nicotinic acid strains |
| CN114945677A (en) * | 2019-10-11 | 2022-08-26 | 国立大学法人静冈大学 | Nicotinamide ribose-producing lactic acid bacterium, and nicotinamide mononucleotide and nicotinamide ribose-producing lactic acid bacterium |
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2020258111A1 (en) * | 2019-06-27 | 2020-12-30 | 邦泰合盛生物科技（深圳）有限公司 | Enzymatic method for industrial production of nad |
| CN112437813A (en) * | 2019-06-27 | 2021-03-02 | 邦泰生物工程(深圳)有限公司 | Method for industrially producing NAD (nicotinamide adenine dinucleotide) by enzyme method |
| CN112437813B (en) * | 2019-06-27 | 2023-03-03 | 邦泰生物工程(深圳)有限公司 | Method for industrially producing NAD (nicotinamide adenine dinucleotide) by enzyme method |
| CN114945677A (en) * | 2019-10-11 | 2022-08-26 | 国立大学法人静冈大学 | Nicotinamide ribose-producing lactic acid bacterium, and nicotinamide mononucleotide and nicotinamide ribose-producing lactic acid bacterium |
| CN110643587A (en) * | 2019-10-29 | 2020-01-03 | 杭州唯泰生物药业有限公司 | Method for preparing nicotinamide adenine dinucleotide phosphate by enzyme method |
| 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 |
| CN113637715A (en) * | 2021-08-12 | 2021-11-12 | 安徽瑞邦生物科技有限公司 | Method for efficiently converting nicotinamide into nicotinic acid strains |
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