CN109628528A - A kind of synthetic method of cordycepin - Google Patents
A kind of synthetic method of cordycepin Download PDFInfo
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- CN109628528A CN109628528A CN201811483813.5A CN201811483813A CN109628528A CN 109628528 A CN109628528 A CN 109628528A CN 201811483813 A CN201811483813 A CN 201811483813A CN 109628528 A CN109628528 A CN 109628528A
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- cordycepin
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- C—CHEMISTRY; METALLURGY
- 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
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/26—Preparation of nitrogen-containing carbohydrates
- C12P19/28—N-glycosides
- C12P19/38—Nucleosides
- C12P19/40—Nucleosides having a condensed ring system containing a six-membered ring having two nitrogen atoms in the same ring, e.g. purine nucleosides
Abstract
Disclosed by the invention is a kind of synthetic method of cordycepin, and this method includes harvesting mature fruiting bodies of cordyceps militaris, is quickly pulverized with after liquid nitrogen flash freezer；Extraction buffer is added, stirring is extracted, is centrifuged, and the retention concentration of supernatant ultrafiltration membrane is taken；Displacement trapped fluid is diluted with Extraction buffer, concentration obtains enzyme mixation；Substrate cocktail is added into enzyme mixation, obtains enzyme reaction system；Enzyme reaction system is put in reaction under preference temperature and obtains cordycepin, synthetic method of the present invention avoids the long time of culture fruiting bodies of cordyceps militaris, and strain is not easy to pollute, and fermentation time is short；Synthetic ingredient is simple, and purification process is simple；Realize the external bio-mimetic syntheses of cordycepin；Green non-pollution, it is safe and healthy；Enzyme reaction mixture can reuse, and avoid wasting；It is low in cost, it is easy to spread.
Description
Technical field
The present invention relates to a kind of synthetic methods, more specifically say, are related to a kind of synthetic method of cordycepin, belong to biology
Chemical field field.
Background technique
Cordycepin is the major physiological active constituent of Cordyceps militaris, is a kind of nucleosides material, by adenine and deoxypentose
Composition has very big market prospects in terms of health care.The preparation of cordycepin mainly has chemical synthesis and biosynthesis
Two ways, due to current chemical synthesis cordycepin high production cost, synthesis technology is complicated, and yield is low, and product purification is more difficult,
So cordycepin is mainly prepared by biological synthesis process.Biological synthesis process prepares approach there are two types of cordycepins at present: first is that solid is trained
It supports base culture and obtains cordyceps militaris sporocarp, then therefrom extract；Second is that by solution culture fermentation cordyceps mycelium, from fermentation liquid
In directly extract.But either all to there is the reaction time long for liquid fermentation or solid fermentation, equipment requirement is high, isolates and purifies
The disadvantage of complex steps difficulty.
Summary of the invention
In order to solve above-mentioned prior art problem, the present invention, which provides, has simple process, and efficiently, product purification is simple for preparation
A kind of synthetic method of cordycepin of equal technical characterstics.
To achieve the goals above, the present invention is achieved by the following technical solutions:
A kind of synthetic method of cordycepin, it is characterised in that the synthetic method includes the following steps:
A Extraction buffer) is prepared: by 100-150mM potassium acetate, 10-30mM magnesium acetate, 1.5-2 μM of iron chloride, 10-
30mM DTT, 10-30mM HEPES-KOH is uniformly mixed, and adjusting pH is 7.2-7.4；
B Substrate cocktail) is prepared: by 100-150mM potassium acetate, 10-30mM magnesium acetate, 1.5-2 μM of iron chloride, 10-
30mM DTT, 5-10mM adenosine, 5-10mM ADP, 10-30mM ATP, 0.5-1mM dATP, 10-30mM HEPES-KOH mixing
Uniformly, adjusting pH is 7.2-7.4；
C it) prepares enzyme mixation: a) harvesting mature fruiting bodies of cordyceps militaris, quickly pulverized with after liquid nitrogen flash freezer；B) it is added
Extraction buffer, 1-2h is extracted in stirring under the conditions of being placed in 4 DEG C, obtains preextraction liquid；C) preextraction liquid is placed under the conditions of 4 DEG C
In centrifuge, 10-20min is centrifuged with 12000rpm revolving speed, take supernatant and retains concentration with ultrafiltration membrane, goes filtrate；D) with mentioning
Buffer dilution displacement trapped fluid is taken, concentration obtains enzyme mixation；
D) the synthesis of cordycepin: by step C) obtain enzyme mixation and step B) obtain Substrate cocktail mix
Reaction solution is formed, 1-10h is stirred to react under the conditions of placing reaction liquid into 25 DEG C, obtains cordycepin.
As an improvement the additional amount of Extraction buffer described in step b) is to harvest fruiting bodies of cordyceps militaris in step a)
3-10 times (v/m) of quality.
As an improvement the specification of ultrafiltration membrane described in step c) is 3-5kD.
As an improvement step D) described in the volume ratio of Substrate cocktail and enzyme mixation be 1:0.5-2.
As an improvement step D) in use magnetic stirrer.
The utility model has the advantages that 1), in existing Cordyceps militaris there is the enzyme of synthesis cordycepin, but specifically reaction metabolic process is difficult to
It controls, the present invention extracts most enzymes in Cordyceps militaris, and cordycepin reaction premise adenosine and synthesis worm is added
The substrates such as the careless necessary purine of element carry out the synthesis of cordycepin, avoid long time and the bacterium of culture fruiting bodies of cordyceps militaris
Kind is easy to pollute, the long problem of fermentation time；
2), due to not having to provide the substrate of synthesis other materials in reaction system, so synthetic ingredient is simple, compared to straight
Meet the method that cordycepin is extracted from fermentation liquid and fruiting bodies of cordyceps militaris, the cordycepin purifying side synthesized using the reaction system
Method is simple and convenient；
3), for the present invention by extracting the enzyme system in Cordyceps militaris, imitating physiological condition realizes the external bionical of cordycepin
Synthesis；
4), the method relative to chemical synthesis cordycepin, cordycepin generated time is short, and process is simple, easily operated, avoids
The use of organic reagent, does not pollute in chemical synthesis, safe and healthy；
5), enzyme reaction mixture provided by the invention can reuse, and avoid wasting；
6) low in cost, with the method for present invention synthesis cordycepin, it is easy to spread.
Detailed description of the invention
Fig. 1 is efficient liquid phase detection cordycepin result figure after reacting in the embodiment of the present invention 3.
Specific embodiment
The present invention will be further described with reference to embodiments.But it should not be construed as limiting the invention.It is not carrying on the back
In the case where from spirit of that invention and essence, to modifications or substitutions made by the method for the present invention, step or condition, this hair is belonged to
Bright range.
Embodiment 1
A kind of synthetic method of cordycepin, it is characterised in that the synthetic method includes the following steps:
A Extraction buffer) is prepared: by 100mM potassium acetate, 20mM magnesium acetate, 2 μM of iron chloride, 30mM DTT, 30mM
HEPES-KOH is uniformly mixed, and adjusting pH is 7.4；
B Substrate cocktail) is prepared: by 100mM potassium acetate, 20mM magnesium acetate, 2 μM of iron chloride, 30mM DTT, 10mM gland
Glycosides, 10mM ADP, 30mM ATP, 2mM dATP, 30mMHEPES-KOH are uniformly mixed, and adjusting pH is 7.4；
C it) prepares enzyme mixation: a) choosing mature fruiting bodies of cordyceps militaris culture 45 days, harvest 10g and with after liquid nitrogen flash freezer
Quickly pulverize；B) 4 DEG C of 30ml of Extraction buffer is added, liquid is transferred to beaker magnetic stirring apparatus under the conditions of 4 DEG C
1h is extracted in stirring, obtains preextraction liquid；C) in the centrifuge under the conditions of preextraction liquid being placed in 4 DEG C, with 12000rpm revolving speed from
Heart 15min takes supernatant and retains concentration with 3kD ultrafiltration membrane, goes filtrate；D) displacement trapped fluid is diluted with Extraction buffer, with
The small molecule compounds such as remaining cordycepin in extracting solution are removed, trapped fluid are concentrated into 10ml, the trapped fluid of acquisition is enzyme
Mixed liquor；
D) the synthesis of cordycepin: taking each 1ml of Substrate cocktail, enzyme mixation, carries out being mixed to get the anti-of synthesis cordycepin
Liquid is answered, is stirred to react 1h under the conditions of placing reaction liquid into 25 DEG C, is extracted reaction solution for liquid chromatographic detection (liquid chromatographic detection item
Part: it is measured by the regulation of NY/T 2116-2012), there is cordycepin synthesis in reaction system, reaction terminates to use reaction solution
3kD ultrafiltration membrane ultrafiltration further can purify to obtain cordyceps sinensis containing small molecules such as cordycepins in filtrate using the method for chromatogram purification
Element, wherein trapped fluid is the enzyme reaction system that can synthesize cordycepin, reusable.
Embodiment 2
A kind of synthetic method of cordycepin, it is characterised in that the synthetic method includes the following steps:
A Extraction buffer) is prepared: by 100mM potassium acetate, 10mM magnesium acetate, 1.5 μM of iron chloride, 10mM DTT, 10mM
HEPES-KOH is uniformly mixed, and adjusting pH is 7.2；
B Substrate cocktail) is prepared: by 100mM potassium acetate, 10mM magnesium acetate, 1.5 μM of iron chloride, 10mM DTT, 5mM gland
Glycosides, 5mM ADP, 10mM ATP, 0.5mM dATP, 10mMHEPES-KOH are uniformly mixed, and adjusting pH is 7.2；
C it) prepares enzyme mixation: a) choosing mature fruiting bodies of cordyceps militaris culture 60 days, harvest fast after 10g liquid nitrogen flash freezer
Speed is pulverized；B) 4 DEG C of 100ml of Extraction buffer is added, liquid is transferred to beaker magnetic stirring apparatus under the conditions of 4 DEG C
2h is extracted in stirring, obtains preextraction liquid；C) in the centrifuge under the conditions of preextraction liquid being placed in 4 DEG C, with 12000rpm revolving speed from
Heart 10min takes supernatant and retains concentration with 5kD ultrafiltration membrane, goes filtrate；D) displacement trapped fluid is diluted with Extraction buffer, with
The small molecule compounds such as remaining cordycepin in extracting solution are removed, trapped fluid are concentrated into 10ml, the trapped fluid of acquisition is enzyme
Mixed liquor；
D) the synthesis of cordycepin: taking 2ml Substrate cocktail and 1ml enzyme mixation, carries out being mixed to get synthesis cordycepin
Reaction solution, be stirred to react 3h under the conditions of placing reaction liquid into 25 DEG C, extract reaction solution for liquid chromatographic detection (liquid chromatogram inspection
Survey condition: it is measured by the regulation of NY/T 2116-2012), there is cordycepin synthesis in reaction system, reaction terminates to react
Liquid 5kD ultrafiltration membrane ultrafiltration further can purify to obtain containing small molecules such as cordycepins in filtrate using the method for chromatogram purification
Cordycepin, wherein trapped fluid is the enzyme reaction system that can synthesize cordycepin, reusable.
Embodiment 3
A kind of synthetic method of cordycepin, it is characterised in that the synthetic method includes the following steps:
A Extraction buffer) is prepared: by 150mM potassium acetate, 30mM magnesium acetate, 2 μM of iron chloride, 10mM DTT, 10mM
HEPES-KOH is uniformly mixed, and adjusting pH is 7.4；
B Substrate cocktail) is prepared: by 150mM potassium acetate, 30mM magnesium acetate, 2 μM of iron chloride, 10mM DTT, 5mM adenosine,
5mM ADP, 10mM ATP, 0.5mM dATP, 10mM HEPES-KOH is uniformly mixed, and adjusting pH is 7.4；
C it) prepares enzyme mixation: a) choosing mature fruiting bodies of cordyceps militaris culture 75 days, harvest 10g and with after liquid nitrogen flash freezer
Quickly pulverize；B) 4 DEG C of 50ml of Extraction buffer is added, liquid is transferred to beaker magnetic stirring apparatus under the conditions of 4 DEG C
2h is extracted in stirring, obtains preextraction liquid；C) in the centrifuge under the conditions of preextraction liquid being placed in 4 DEG C, with 12000rpm revolving speed from
Heart 10min takes supernatant and retains concentration with 5kD ultrafiltration membrane, goes filtrate；D) displacement trapped fluid is diluted with Extraction buffer, with
The small molecule compounds such as remaining cordycepin in extracting solution are removed, trapped fluid are concentrated into 10ml, the trapped fluid of acquisition is enzyme
Mixed liquor；
D) the synthesis of cordycepin: taking 1ml Substrate cocktail and 2ml enzyme mixation, and mixed liquor carries out being mixed to get synthesis
The reaction solution of cordycepin is stirred to react 2h under the conditions of placing reaction liquid into 25 DEG C, as shown in Figure 1, extracting reaction solution for liquid phase color
Spectrum detects (liquid chromatographic detection condition: being measured by the regulation of NY/T 2116-2012), has cordycepin conjunction in reaction system
At reaction terminates reaction solution 5kD ultrafiltration membrane ultrafiltration, containing small molecules such as cordycepins in filtrate, can further use chromatography
The method of purifying purifies to obtain cordycepin, and wherein trapped fluid is the enzyme reaction system that can synthesize cordycepin, reusable.
Embodiment 4
A Extraction buffer) is prepared: by 100mM potassium acetate, 10mM magnesium acetate, 1.5 μM of iron chloride, 10mM DTT, 10mM
HEPES-KOH is uniformly mixed, and adjusting pH is 7.2；
B Substrate cocktail) is prepared: by 100mM potassium acetate, 10mM magnesium acetate, 1.5 μM of iron chloride, 10mM DTT, 5mM gland
Glycosides, 5mM ADP, 10mM ATP, 0.5mM dATP, 10mM HEPES-KOH are uniformly mixed, and adjusting pH is 7.2；
C it) prepares enzyme mixation: a) choosing mature fruiting bodies of cordyceps militaris culture 45 days, harvest 10g and with after liquid nitrogen flash freezer
Quickly pulverize；B) Extraction buffer is added, 1h is extracted in stirring under the conditions of being placed in 4 DEG C, obtains preextraction liquid；C) by preextraction
Liquid be placed in 4 DEG C under the conditions of centrifuge in, with 12000rpm revolving speed be centrifuged 10min, take supernatant and with ultrafiltration membrane retain be concentrated,
Go filtrate；D) displacement trapped fluid is diluted with Extraction buffer, to remove the small molecule compounds such as remaining cordycepin in extracting solution,
Concentration obtains enzyme mixation；
D) the synthesis of cordycepin: by step C) obtain enzyme mixation and step B) obtain Substrate cocktail press 1:0.5
The reaction solution for being mixed to get synthesis cordycepin is carried out, 1h is stirred to react under the conditions of placing reaction liquid into 25 DEG C, extracts reaction solution and be used for
Liquid chromatographic detection (liquid chromatographic detection condition: is measured by the regulation of NY/T2116-2012), has cordyceps sinensis in reaction system
Element synthesis, reaction terminate reaction solution 3kD ultrafiltration membrane ultrafiltration, containing small molecules such as cordycepins in filtrate, can further use
The method of chromatogram purification purifies to obtain cordycepin, and wherein trapped fluid is the enzyme reaction system that can synthesize cordycepin, repeats benefit
With.
Embodiment 5
A Extraction buffer) is prepared: by 125mM potassium acetate, 20mM magnesium acetate, 1.75 μM of iron chloride, 20mM DTT, 20mM
HEPES-KOH is uniformly mixed, and adjusting pH is 7.3；
B Substrate cocktail) is prepared: by 125mM potassium acetate, 20mM magnesium acetate, 1.75 μM of iron chloride, 20mM DTT, 7.5mM
Adenosine, 7.5mM ADP, 20mM ATP, 0.75mM dATP, 20mMHEPES-KOH are uniformly mixed, and adjusting pH is 7.3；
C it) prepares enzyme mixation: a) choosing mature fruiting bodies of cordyceps militaris culture 60 days, harvest 10g and with after liquid nitrogen flash freezer
Quickly pulverize；B) Extraction buffer is added, 1.5h is extracted in stirring under the conditions of being placed in 4 DEG C, obtains preextraction liquid；C) it will mention in advance
In centrifuge under the conditions of taking liquid to be placed in 4 DEG C, 15min is centrifuged with 12000rpm revolving speed, take supernatant and is retained with ultrafiltration membrane dense
Contracting, goes filtrate；D) displacement trapped fluid is diluted with Extraction buffer, to remove the small molecules chemical combination such as remaining cordycepin in extracting solution
Object, concentration obtain enzyme mixation；
D) the synthesis of cordycepin: by step C) obtain enzyme mixation and step B) obtain Substrate cocktail press 1:1.25
The reaction solution for being mixed to get synthesis cordycepin is carried out, 5.5h is stirred to react under the conditions of placing reaction liquid into 25 DEG C, extracts reaction solution use
It (liquid chromatographic detection condition: is measured by the regulation of NY/T2116-2012) in liquid chromatographic detection, has worm in reaction system
Careless element synthesis, reaction terminate reaction solution 4kD ultrafiltration membrane ultrafiltration, containing small molecules such as cordycepins in filtrate, can further adopt
Purified to obtain cordycepin with the method for chromatogram purification, wherein trapped fluid is the enzyme reaction system that can synthesize cordycepin, is repeated
It utilizes.
Embodiment 6
A Extraction buffer) is prepared: by 150mM potassium acetate, 30mM magnesium acetate, 2 μM of iron chloride, 30mM DTT, 30mM
HEPES-KOH is uniformly mixed, and adjusting pH is 7.4；
B Substrate cocktail) is prepared: by 150mM potassium acetate, 30mM magnesium acetate, 2 μM of iron chloride, 30mM DTT, 10mM gland
Glycosides, 10mM ADP, 30mM ATP, 2mM dATP, 30mMHEPES-KOH are uniformly mixed, and adjusting pH is 7.4；
C it) prepares enzyme mixation: a) choosing mature fruiting bodies of cordyceps militaris culture 75 days, harvest 10g and with after liquid nitrogen flash freezer
Quickly pulverize；B) Extraction buffer is added, 2h is extracted in stirring under the conditions of being placed in 4 DEG C, obtains preextraction liquid；C) by preextraction
Liquid be placed in 4 DEG C under the conditions of centrifuge in, with 12000rpm revolving speed be centrifuged 20min, take supernatant and with ultrafiltration membrane retain be concentrated,
Go filtrate；D) displacement trapped fluid is diluted with Extraction buffer, to remove the small molecule compounds such as remaining cordycepin in extracting solution,
Concentration obtains enzyme mixation；
D) the synthesis of cordycepin: by step C) obtain enzyme mixation and step B) obtain Substrate cocktail by 1:2 into
Row is mixed to get the reaction solution of synthesis cordycepin, is stirred to react 10h under the conditions of placing reaction liquid into 25 DEG C, extracts reaction solution for liquid
Phase chromatography detects (liquid chromatographic detection condition: being measured by the regulation of NY/T 2116-2012), has cordyceps sinensis in reaction system
Element synthesis, reaction terminate reaction solution 5kD ultrafiltration membrane ultrafiltration, containing small molecules such as cordycepins in filtrate, can further use
The method of chromatogram purification purifies to obtain cordycepin, and wherein trapped fluid is the enzyme reaction system that can synthesize cordycepin, repeats benefit
With.
Finally it should be noted that present invention is not limited to the above embodiments, there can also be many variations.This field it is general
All deformations that logical technical staff directly can export or associate from present disclosure, are considered as of the invention
Protection scope.
Claims (5)
1. a kind of synthetic method of cordycepin, it is characterised in that the synthetic method includes the following steps:
A Extraction buffer) is prepared: by 100-150mM potassium acetate, 10-30mM magnesium acetate, 1.5-2 μM of iron chloride, 10-30mM
DTT, 10-30mM HEPES-KOH are uniformly mixed, and adjusting pH is 7.2-7.4；
B Substrate cocktail) is prepared: by 100-150mM potassium acetate, 10-30mM magnesium acetate, 1.5-2 μM of iron chloride, 10-30mM
DTT, 5-10mM adenosine, 5-10mM ADP, 10-30mM ATP, 0.5-2mM dATP, 10-30mM HEPES-KOH are uniformly mixed,
Adjusting pH is 7.2-7.4；
C it) prepares enzyme mixation: a) harvesting mature fruiting bodies of cordyceps militaris, quickly pulverized with after liquid nitrogen flash freezer；B) it is added and extracts
Buffer, 1-2h is extracted in stirring under the conditions of being placed in 4 DEG C, obtains preextraction liquid；C) centrifugation under the conditions of preextraction liquid being placed in 4 DEG C
In machine, 10-20min is centrifuged with 12000rpm revolving speed, take supernatant and retains concentration with ultrafiltration membrane, goes filtrate；D) slow with extracting
Fliud flushing dilution displacement trapped fluid, concentration obtain enzyme mixation；
D) the synthesis of cordycepin: by step C) obtain enzyme mixation and step B) obtain Substrate cocktail be mixed to form
Reaction solution is stirred to react 1-10h under the conditions of placing reaction liquid into 25 DEG C, obtain cordycepin.
2. a kind of synthetic method of cordycepin according to claim 1, it is characterised in that: extract buffering described in step b)
The additional amount of liquid is 3-10 times of harvesting fruiting bodies of cordyceps militaris quality in step a).
3. a kind of synthetic method of cordycepin according to claim 1 or 2, it is characterised in that: ultrafiltration described in step c)
The specification of film is 3-5kD.
4. a kind of synthetic method of cordycepin according to claim 1, it is characterised in that: step D) described in substrate mixing
The volume ratio of liquid and enzyme mixation is 1:0.5-2.
5. it is according to claim 1 or 2 synthesis cordycepin method, it is characterised in that: step D) in use magnetic agitation
Device stirring.
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