CN108504705A - The biochemistry of palmitoyl coenzyme A synthesizes and purification process - Google Patents
The biochemistry of palmitoyl coenzyme A synthesizes and purification process Download PDFInfo
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- CN108504705A CN108504705A CN201810190829.0A CN201810190829A CN108504705A CN 108504705 A CN108504705 A CN 108504705A CN 201810190829 A CN201810190829 A CN 201810190829A CN 108504705 A CN108504705 A CN 108504705A
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- 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
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- C12P19/30—Nucleotides
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- C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
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- C07H19/207—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine adenine dinucleotide or nicotinamide-adenine dinucleotide
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- C12P7/00—Preparation of oxygen-containing organic compounds
- C12P7/64—Fats; Fatty oils; Ester-type waxes; Higher fatty acids, i.e. having at least seven carbon atoms in an unbroken chain bound to a carboxyl group; Oxidised oils or fats
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Abstract
The invention discloses a kind of synthesis of the biochemistry of palmitoyl coenzyme A and purification process.The biochemical synthesis methods of palmitoyl coenzyme A of the present invention, including：It sequentially adds ATP sodium salts, coacetylase, palmitic acid and acetyl-CoA-synthetase in kaliumphosphate buffer, adjusts pH, constant volume is reacted, and collecting reaction product to obtain the final product.The present invention further purifies reaction product, including：Reaction product is crossed into nickel column and removes deproteinized, ether extraction is added in the product after removing protein, the water phase spent ion exchange resin isolated removes inorganic impurity, and the freeze-drying of gained collection liquid obtains palmitoyl coenzyme A crude product；The crude product is added in absolute ethyl alcohol and is stirred, solid is filtered out, is dried in vacuo to obtain the final product.The present invention synthesizes palmitoyl coenzyme A by external enzymatic reaction, only needs a step biochemical reaction, and the reaction time is short, efficient, simply controllable to product purification steps, and product purity is higher, lays the foundation for the large-scale production of palmitoyl coenzyme A.
Description
Technical field
The present invention relates to a kind of synthesis of the biochemistry of palmitoyl coenzyme A and its purification process, belong to palmitoyl coenzyme A
Preparation field.
Background technology
Palmitoyl coenzyme A (Palmitoyl Coenzyme A, Palmityl-CoA) has extensively in research and production
Application.As a kind of important research reagents, palmitoyl coenzyme A is commonly used in the relevant research of some lipids, such as cell
Phospholipid surface synthesis, the synthesis of cell surface glycolipids, the palmitoylation modification of albumen, cell signalling etc..Industry is raw
In production, palmitoyl coenzyme A is often taken as substrate, is used for acyl-CoA oxidase vitality test.In addition, palmitoyl coenzyme A is also normal
It is used for the measurement of some acyltransferase activities.Therefore, searching process is simple, cost of material is reasonable, product quality is high, suitable
The palmitoyl coenzyme A synthesis of large-scale production has great importance with purifying process.
At present about the synthesis of palmitoyl coenzyme A, originally there is scholar to pass through coacetylase and palmitic anhydride reaction to prepare palm
Acyl coenzyme A, since coacetylase autoactivation point is more, reaction selectivity is poor, and by-product is more, and reaction yield is low, and product purification is tired
It is difficult.Later, United States Patent (USP) US5519128 prepared palm fibre by the condensation reaction of palmityl N- succinimides and silanization coacetylase
Palmitic acid acyl coenzyme A, this pure chemistry synthetic method process is complicated, and cost is higher.Chinese patent CN103074400B mentions one kind
The palmitoyl coenzyme A that high added value is produced using the method for Escherichia coli fermentation, is had great advantage in terms of cost control, but
It is that ingredient in tunning is excessively complicated, the purity of subsequent purification complex process, product only has about 40%-50%.It is Chinese special
Sharp CN106543254A introduces thioesters displacement reaction after palmitic anhydride reaction, and this method substantially increases the purity of product,
But reaction process is time-consuming longer, primary first-order equation at least needs 21h, and each reaction step needs the side reaction controlled more,
The uncertain factor of reaction increases.
Therefore, develop a kind of reaction time it is short, it is efficient by external enzymatic reaction synthesize palmitoyl coenzyme A new side
Then method purifies product using chemical method, product purity is higher, will be established for the large-scale production of palmitoyl coenzyme A
Fixed basis.
Invention content
The main object of the present invention is to provide a kind of biochemical synthesis methods of palmitoyl coenzyme A, and this method passes through body
Outer enzymatic reaction synthesizes palmitoyl coenzyme A, only needs a step biochemical reaction, reaction time short；The further utilizationization of the present invention
Method purifies reaction product, and purification step is simply controllable, and product purity is higher.
To achieve the above object, the technical solution used in the present invention includes：
The present invention discloses a kind of biochemical synthesis methods of palmitoyl coenzyme A first, includes the following steps：In phosphoric acid
ATP sodium salts, coacetylase, palmitic acid and acetyl-CoA-synthetase are sequentially added in potassium buffer solution, adjusts pH, and constant volume is reacted
Mixed liquor is reacted, collecting reaction product to get.
Wherein, the ATP sodium salts, coacetylase, palmitic acid, acetyl-CoA-synthetase addition be：In 800ml potassium phosphates
In buffer solution, ATP sodium salts 0.507g, coacetylase 0.767g, palmitic acid 0.512g, acetyl-CoA-synthetase 0.38KU is added.Institute
It is ATP2Na to state ATP sodium salts.
A concentration of 0.05M of the kaliumphosphate buffer, pH value 7.5.
The adjustment pH is adjustment pH to 7.5.The constant volume is to be settled to the total of reaction mixture with kaliumphosphate buffer
Volume is 1L；Wherein, a concentration of 0.05M of the kaliumphosphate buffer, pH value 7.5.
The condition of the reaction includes：37 DEG C of reaction 2h；Preferably, 2h is reacted in 37 DEG C of water-baths.
The biochemistry synthetic method of palmitoyl coenzyme A of the present invention only needs a step biochemical reaction, you can prepare
Palmitoyl coenzyme A, reaction time are short, it is only necessary to which 2h can carry out primary first-order equation, efficient.
The biological synthesis method of palmitoyl coenzyme A of the present invention further includes：Reaction product is purified；Wherein, institute
The step of stating purifying include：(1) reaction product is crossed into nickel column, collected column liquid, obtain the product after removing protein；(2) egg will be removed
Ether extraction is added in product after white, removes organic impurities, isolates water phase；(3) water phase spent ion exchange resin is removed into nothing
Machine impurity, gained collection liquid freeze-drying, obtains palmitoyl coenzyme A crude product；(4) palmitoyl coenzyme A crude product is added to absolute ethyl alcohol
In, it stirs, filtering obtains solid, is dried in vacuo to get palmitoyl coenzyme A.
The present invention removes the acetyl-CoA-synthetase in reaction product using Ni columns.Wherein, step (1) further includes：By nickel
Column is first rinsed with the distilled water of 5 times of column volumes, then uses the kaliumphosphate buffer balance nickel column of 5 times of column volumes, then will reaction production
Object crosses nickel column.Wherein, a concentration of 0.05M of the kaliumphosphate buffer of balance nickel column, pH value 7.5.It is described to collect column
Liquid is to start to collect column liquid after crossing half of column volume of reaction product, stops collecting after excessively complete reaction product；Then it uses successively
The kaliumphosphate buffer of the imidazoles containing 0.5M, water, 20% ethyl alcohol cross nickel column.
Product after removing protein is added isometric ether and extracted by step (2).The amount of step (4) described absolute ethyl alcohol is
5 times of palmitoyl coenzyme A crude product volume, to be stirred at room temperature 2 hours, the vacuum drying is to be dried in vacuo at room temperature for the stirring
3-5 hours.
Room temperature of the present invention is 10-30 DEG C.
The yield of the method for the present invention synthesis palmitoyl coenzyme A reaches 49.75%, and product purity reaches 95% (HPLC).
The palmitoyl coenzyme A that the present invention is synthesized is used for acyl-CoA oxidase enzyme activity determination, the results show that of the invention
The palmitoyl coenzyme A of synthesis can successfully detect acyl-CoA oxidase enzyme activity, and test map is linearly good, degree of reaction spirit
It is quick.
Technical solution of the present invention compared with prior art, has the advantages that：
The present invention synthesizes palmitoyl coenzyme A by external enzymatic reaction, only needs a step biochemical reaction, reaction time
It is short, it is only necessary to which that 2h can carry out primary first-order equation, and efficiency is higher, save manpower.The present invention purifies product using chemical method,
Each definite ingredients in reaction product, purification step is simply controllable, and product purity is higher, is the large-scale production of palmitoyl coenzyme A
It lays the foundation.
Description of the drawings
Fig. 1 is the reaction principle of present invention synthesis palmitoyl coenzyme A；
Fig. 2 is that the palmitoyl coenzyme A that the present invention synthesizes is used for acyl-CoA oxidase enzyme activity determination verification test result；
Wherein, A is testing result of the enzyme dilution as blank control；B is to replace list with 20 enzyme solutions of the μ l containing acyl-CoA oxidase
The testing result of pure enzyme dilution.
Specific implementation mode
The biochemistry of 1 palmitoyl coenzyme A of embodiment synthesizes and purification process
1, test material
1) palmitic acid (AR, Aladdin company)
2) ATP2Na (AR, Sigma company)
3) coacetylase (AR, Roche company)
4) dipotassium hydrogen phosphate (AR, traditional Chinese medicines reagent)
5) potassium dihydrogen phosphate (AR, traditional Chinese medicines reagent)
6) acetyl-CoA-synthetase (Roche companies)
7) absolute ethyl alcohol (AR, traditional Chinese medicines reagent)
8) ether (AR, traditional Chinese medicines reagent)
2, test method and result
(1) palmitoyl coenzyme A synthetic reaction
Configure 0.05M, the kaliumphosphate buffer of pH7.5.Sequentially add 0.507g's in the kaliumphosphate buffer of 800ml
The acetyl-CoA-synthetase of ATP sodium salts, the coacetylase of 0.767g, the palmitic acid of 0.512g and 0.38KU adjusts pH to 7.5, uses
In kaliumphosphate buffer constant volume to the beaker of 1L, beaker is then put into 2h in 37 DEG C of water-baths, synthetic reaction completes (reaction original
Reason is shown in Fig. 1).
(2) product purification
Nickel column is first rinsed with the distilled water of 5 times of column volumes, later with the kaliumphosphate buffer of 5 times of column volumes (0.05M,
PH7.5) balance nickel column crosses synthetic reaction product, starts to collect after crossing half of column volume of reaction product, stop after excessively complete reaction product
Only collect.Then successively with the kaliumphosphate buffer of the imidazoles containing 0.5M, water, 20% ethyl alcohol cross nickel column.PAGE detects removing protein
Effect, the liquid collected is the product after removing protein when crossing reaction product.
Isometric ether extraction is added in product after removing protein, removes organic impurities, isolates water phase, water phase carries out
Ion exchange resin removes inorganic impurity, and gained collection liquid moves into freeze dryer freeze-drying, and it is palm acyl coenzyme to obtain off-white powder
The crude product is added in the absolute ethyl alcohol of 5 times of amounts and is stirred at room temperature 2 hours, filters out solid, at room temperature very by A crude product 0.850g
Dry 3~5 hours of sky is to get to off-white powder palmitoyl coenzyme A 0.5g, (yield (yield) 49.75%, HPLC
95%).
1 application verification of test example is tested
Palmitoyl coenzyme A (preparation of embodiment 1) is used for acyl-CoA oxidase enzyme activity determination verification test.
(1) solution allocation
Dilution：The dipotassium hydrogen phosphate of the potassium dihydrogen phosphate and 22.8g of 2.3g is added in 800 ml deionized waters, adjusts
PH is to 7.6 constant volumes to 1L；
Reagent A：The Tris of 24.23g is added in 800ml water, adjusts pH to 8.0, constant volume to 1L；
Reagent B：15 mMs of 4-1AA solution；
Reagent C：0.2% phenol solution；
Reagent D：The POD solution of 50U/ml；
Reagent E：The palmitoyl coenzyme A solution of 5mg/ml.
(2) operating procedure
Operating procedure is shown in Table 1.
1 operating procedure of table
(1) each 100 μ l of A, B, C, D, E, 300 μ l of water are sequentially added, is added in quartz colorimetric utensil and (pays attention to liquid level),
It mixes well, 37 DEG C of incubation reactions to 300s.
(2) in 300s, 20 μ l enzyme dilutions are added, are sufficiently mixed uniformly, 37 DEG C the reaction was continued to 500s；Record the
OD in 360s-420s500Light absorption value changes delta Ab/min per minute.
(3) mixed liquor initial absorbance OD5000.08 should be just less than.Δ Ab/min should be less than 0.03, and (display enzyme activity is less than
20U/L), otherwise enzyme dilution may be contaminated, and be needed to change；Either cuvette is contaminated, need to be with cleaning again.
(4) if meeting the requirements, Enzyme activity assay can be carried out, enzyme dilution is shown in Fig. 2 as blank control, testing result
(A)。
(5) (1)-(4) step is repeated, replaces simple enzyme dilute with 20 enzyme solutions of the μ l containing acyl-CoA oxidase in experimental group
Liquid is released, testing result is shown in Fig. 2 (B).
The results show that the palmitoyl coenzyme A that the present invention synthesizes can successfully detect acyl-CoA oxidase enzyme activity, inspection
Mapping spectral line is good, and degree of reaction is sensitive.
Obviously, the above embodiments are merely examples for clarifying the description, and does not limit the embodiments.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of variation or
It changes.There is no necessity and possibility to exhaust all the enbodiments.And it is extended from this it is obvious variation or
It changes still within the protection scope of the invention.
Claims (10)
1. a kind of biochemical synthesis methods of palmitoyl coenzyme A, which is characterized in that include the following steps：It is buffered in potassium phosphate
ATP sodium salts, coacetylase, palmitic acid and acetyl-CoA-synthetase are sequentially added in liquid, adjust pH, constant volume obtains reaction mixture,
Reacted, collecting reaction product to get.
2. biochemical synthesis methods described in accordance with the claim 1, which is characterized in that the ATP sodium salts, coacetylase, palm
Acid, acetyl-CoA-synthetase addition be：In 800ml kaliumphosphate buffers, ATP sodium salts 0.507g, coacetylase is added
0.767g, palmitic acid 0.512g, acetyl-CoA-synthetase 0.38KU.
3. according to biochemical synthesis methods as claimed in claim 1 or 2, it is characterised in that：The ATP sodium salts are ATP
2Na。
4. according to biochemical synthesis methods as claimed in claim 1 or 2, it is characterised in that：The kaliumphosphate buffer it is dense
Degree is 0.05M, pH value 7.5.
5. biochemical synthesis methods described in accordance with the claim 1, it is characterised in that：The adjustment pH is adjustment pH to 7.5；
The constant volume is：It is settled to 1L with kaliumphosphate buffer；Wherein, a concentration of 0.05M of the kaliumphosphate buffer, pH value
It is 7.5.
6. biochemical synthesis methods described in accordance with the claim 1, which is characterized in that the condition of the reaction includes：37℃
React 2h；Preferably, 2h is reacted in 37 DEG C of water-baths.
7. biochemical synthesis methods described in accordance with the claim 1, which is characterized in that further include：Reaction product is carried out pure
Change；Wherein, the step of purifying includes：
(1) reaction product is crossed into nickel column, collected column liquid, obtain the product after removing protein；(2) product after removing protein is added
Ether extracts, and removes organic impurities, isolates water phase；(3) water phase spent ion exchange resin is removed into inorganic impurity, gained is collected
Liquid is lyophilized, and obtains palmitoyl coenzyme A crude product；(4) palmitoyl coenzyme A crude product is added in absolute ethyl alcohol, is stirred, filtering obtains
To solid, it is dried in vacuo to get palmitoyl coenzyme A.
8. biochemical synthesis methods according to claim 7, which is characterized in that step (1) further includes：Nickel column is first used
The distilled water of 5 times of column volumes rinses, and then with the kaliumphosphate buffer balance nickel column of 5 times of column volumes, then reaction product is crossed nickel
Column；
The column liquid of collecting is to start to collect column liquid after crossing half of column volume of reaction product, is stopped after excessively complete reaction product
It collects；
Wherein, a concentration of 0.05M of the kaliumphosphate buffer, pH value 7.5.
9. biochemical synthesis methods according to claim 7, it is characterised in that：Step (2) is by the product after removing protein
Isometric ether extraction is added.
10. biochemical synthesis methods according to claim 7, it is characterised in that：The amount of step (4) described absolute ethyl alcohol
It it is 5 times of palmitoyl coenzyme A crude product volume, to be stirred at room temperature 2 hours, the vacuum drying is dry for vacuum at room temperature for the stirring
It is 3-5 hours dry.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103074400A (en) * | 2012-12-19 | 2013-05-01 | 北京利德曼生化股份有限公司 | Preparation method of palmitoyl coenzyme A |
| CN106543254A (en) * | 2016-11-07 | 2017-03-29 | 北京利德曼生化股份有限公司 | The chemical synthesis process of palmitoyl coenzyme A potassium salt |
| CN107083412A (en) * | 2017-06-13 | 2017-08-22 | 刘超 | A kind of application of middle short chain acyl CoA synthase |
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2018
- 2018-03-08 CN CN201810190829.0A patent/CN108504705A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103074400A (en) * | 2012-12-19 | 2013-05-01 | 北京利德曼生化股份有限公司 | Preparation method of palmitoyl coenzyme A |
| CN106543254A (en) * | 2016-11-07 | 2017-03-29 | 北京利德曼生化股份有限公司 | The chemical synthesis process of palmitoyl coenzyme A potassium salt |
| CN107083412A (en) * | 2017-06-13 | 2017-08-22 | 刘超 | A kind of application of middle short chain acyl CoA synthase |
Non-Patent Citations (1)
| Title |
|---|
| J. BAR-TANA等: "Palmitoyl-Coenzyme A Synthetase", 《BIOCHEM. J.》 * |
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