CN101230374A - Preparation process of coenzyme A - Google Patents
Preparation process of coenzyme A Download PDFInfo
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- CN101230374A CN101230374A CNA2008100492496A CN200810049249A CN101230374A CN 101230374 A CN101230374 A CN 101230374A CN A2008100492496 A CNA2008100492496 A CN A2008100492496A CN 200810049249 A CN200810049249 A CN 200810049249A CN 101230374 A CN101230374 A CN 101230374A
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Abstract
The invention discloses a preparation technique of coenzyme A, which is divided into two parts including fermenting, and extracting and purifying. The invention is characterized in that enzyme at the part of fermenting can be synthesized into a reaction stage, and precursors, such as hydrochloric acid-cysteine, calcium pantothenate and trinosin, the quantity of which are known, are added in batches into enzyme reaction liquid. At the reduction stage of the part of extracting and purifying, saturated alkali solution can be used for adjusting pH value, and mercaptoethanol can be used as reducing agent to conduct the reduction. Compared with the prior art, the invention improves the purity of the coenzyme A, and the potency of the coenzyme A contained in 1 mg is no less than 300 units, 50 units higher than the national standard. The storage conditions fixed according to the inspection results of the stability of the invention are shielding light, sealed and kept frozen at minor 20 DEG C; and the determined period of validity is 18 months, 6 months longer than the national standard.
Description
(1) technical field
The present invention relates to a kind of enzymatic synthesis process of coenzyme A.
(2) background technology
1954, Hoagland proposed that the biosynthetic approach of coenzyme A is in the animal tissues:
Levintow has also proposed identical biosynthetic pathway.
Nineteen fifty-nine, Brown determines that the biosynthetic main path of coenzyme A is:
The first step is a most important step in the coenzyme A biosynthesizing.
Initial coenzyme A mainly in the yeast cell extraction separation obtain, later on also through the complete synthesis preparation of chemistry, but two methods are all undesirable, productive rate is low or manufacturing process is complicated.And the development and use of Production by Microorganism Fermentation coenzyme A are expected to provide a large amount of inexpensive coenzyme As to satisfy the demand.
The coenzyme A fermentation starts from nineteen fifty.In 32 ℃ of cultivation 88h, fermentation unit is very low, only 5u/ml with streptomyces fradiae (Streptomyces fradiae) for Deveries etc.Thereafter research, particularly in the nearly all microorganism cells of discovery such as Ogata and Shimizu the coenzyme A synthetase series is arranged all, and the strongest with the activity of two strain Brevibacterium ammoniagenes (Brevibacterium ammoniagenes IFO 12071 and IFO 12072), fermentation unit can reach 413u/ml.Shimizu etc. have studied the zymotechnique of coenzyme A in great detail with Brevibacterium ammoniagenes.
The coenzyme A preparation were established of reported in literature:
1, with the yeast is the formulation of raw material
China is adopting the synthetic coenzyme A of new yeast cake fermentation also to obtain certain achievement, but on extraction and purification process, does not have radical change, and data is introduced vigor loss 20～30%, calculates by fresh yeast cake, and yield only is 3000 units/kg.In addition, adopt the zinc amalgam reduction in this purifying process, this method is complicated operation not only, and labour intensity is big, and yield is low, and influences the healthy of direct labor and cause environmental pollution.
2, with the pork liver be the GMA extraction method of raw material
Low, the purity difference of this process recovery ratio, data are introduced and are just won coenzyme A acetone powder 2 * 104 units in every kg liver, and tiring is 120 units/mg, purity 50%, and the finished product color and luster is yellow.
GMA-LD-601 combination process total recovery is 30%.
3, produce ammonia rod bacillus biological synthesis process
Originally, extract coenzyme A,, make output be subjected to certain restriction because of content is few through broken wall with somatic cells such as yeast, geotrichum candidums.Studied in 1974 and successfully add pantothenic acid and make precursor, use the synthetic coenzyme A of Brevibacterium ammoniagenes fermentation, and formally put into production in 1976, cost descends 4 times than extraction process, and quality is brought up to 100 units/more than the milligram by 50 unit/milligrams.
(3) summary of the invention
The objective of the invention is to overcome some defectives that prior art exists, a kind of coenzyme A preparation method is provided.
The preparation technology that Sodium ATP regeneration system by microorganism carries out the synthetic coenzyme A of enzymatic can be divided into the two parts and promptly ferment and extract purifying.Fermentation part comprises microbial strains seed selection, fermentation condition and enzymatic reaction condition.The key of this part relates to the selection of use bacterial classification and the condition of enzymatic reaction.The bacterial classification that can carry out the synthetic coenzyme A of enzymatic has a lot, as yeast, geotrichum candidum bacterium, quarter butt genus bacillus, pseudomonas, product ammonia rod bacillus etc.From bibliographical information both domestic and external, it is the highest wherein producing the output that ammonia rod bacillus carries out the synthetic coenzyme A of enzymatic by fermentation, and what adopt in our technology also is to produce ammonia rod bacillus.The condition of enzymatic reaction is meant and adds the synthetic needed raw material of coenzyme A that when microbial fermentation arrives to a certain degree promptly precursor substance comprises cysteine hydrochloride, calcium pantothenate, Sodium ATP etc.
Extract purification part and comprise absorption, concentrated, sieve chromatography, reduction, precipitation.Key in this part technology is selecting for use of separation and purification post and reduction reaction, and what adopt on the document is that activated carbon adsorption post absorption and positive post, cloudy post separate.
In order to realize purpose of the present invention, the present invention is according to following technical scheme.A kind of coenzyme A enzymatic synthesis process comprises fermentation part and extracts purification part;
(1) fermentation part
Selected coenzyme A is produced bacterial classification through fermentation culture, detects pH value, OD value, residual sugar and microscopy and should not have assorted bacterium;
In enzyme reaction solution, drop into the precursor substance cysteine hydrochloride in batches, calcium pantothenate, Sodium ATP.
Enzyme reaction temperature is controlled between 29 ℃～31 ℃ and carries out, general reaction time control 18～22 hours, and air flow control is 1: 0.5, stirs, and obtains reaction solution;
Reaction solution acidifying, press filtration obtain the press filtration clear liquid.
(2) extract purification part
The press filtration clear liquid of step (1) gained selects for use DA210-B type non-polar macroporous resin to carry out fractionation by adsorption, and wash-out obtains parting liquid,
Add in the parting liquid with the L-halfcystine and be complexing agent, drop into the cuprous ion reaction and form the mercapto alkoxide, respectively with getting concentrated solution behind diluted acid and purified water washing, the decopper(ing);
Concentrated solution after adopting G-25 type dextrane gel resin to decopper(ing) carries out separation and purification, utilizes nucleic acid-protein detector 280nm wavelength to detect, and collects coenzyme A and effective constituent;
Concentrated solution is used saturated highly basic again, regulates pH value to 4.5～5.0 as potassium hydroxide, sodium hydroxide or lithium hydroxide solution, and preferred lithium hydroxide solution is regulated pH value.With the mercaptoethanol is reductive agent, and temperature is controlled at 34～36 ℃, and the time was controlled at 10～14 hours;
Concentrated solution precipitates coenzyme A control with nitric acid-acetone mixed solution again, and pH is 2.5, and mixeding liquid temperature should be controlled 5～7 ℃, filters, and gets the wet product of coenzyme A, dry then.
The present invention is compared with prior art: improved the purity of coenzyme A, every 1mg contains tiring of coenzyme A and all is not less than 300 units, exceeds 50 units than national standard; This product study on the stability result is decided to be shading with storage requirement, and sealing is-20 ℃ of freezing preservations; And the validity period time limit is defined as 18 months, exceed 6 months than national standard.
(4) embodiment
Among the preparation technology's of coenzyme A fermentation and the extraction purifying two parts, the present invention is only to enzymatic building-up reactions stage of fermentation part, separation, enriching stage, the reduction phase of getting purification part improve, other parts are still prolonged the known technology with former preparation technology, therefore, known preparation technology not to be described in detail among the embodiment.
Embodiment 1
1, spawn culture.Cultured AS1.925 is produced ammonia rod bacillus species move into fermentor cultivation, culture temperature is controlled at 28～30 ℃, and air flow quantity 1: 1 was cultivated 20 hours, and cultivate terminal point control: pH should be 6.2～6.3, and OD is 2.0～2.2; Get the fermentor tank bacterial classification.
2, enzymatic building-up reactions.Before dropping into the known precursors material in zero hour, add broken wall agent Morpan BB earlier, added precursor substance then respectively at 2 hours, 3 hours, 4 hours, 5 hours.The total charging capacity weight percent of precursor substance is 0.1% hydrochloric acid L-halfcystine, 0.07% calcium pantothenate, 0.2% Sodium ATP, and precursor substance is divided equally into five parts, and gradation adds; Temperature of reaction is controlled at 29～31 ℃, and the control of general reaction time is about 20 hours, and air flow control is 1: 0.5, stirs, and the control terminal point: it is 300～350u/ml that survey is tired, and gets reaction solution.
3, pre-treatment.Reaction solution vitriol oil acidifying is regulated pH 1.5～1.8, and filter press obtains the press filtration clear liquid, and feed is made in the filter cake oven dry.
4, separate, concentrate.With DA210-B type non-polar macroporous resin absorption cleaner liquid, flow rate control 1/3 dress column volume/hour, saturated resin; Rare nitric acid washing with 0.03%, flow rate control 1/3 dress column volume/hour, must wash the back resin; With cholamine is the desorbing agent desorption, flow rate control 1/3 dress column volume/hour, collect effluent liquid pH in 2.0～7.0 parts, parting liquid.Concentrated solution is tired and is controlled at 2500～3000u/ml, and 1/10 of the about parting liquid I of volume volume gets concentrated solution.
5, salify, decopper(ing), concentrated, separation.With the L-halfcystine is complexing agent, drops into Red copper oxide in the concentrated solution of step 4 gained, the sulfuric acid complexing, and salify after the hydrolysis washs the coenzyme A mantoquita with diluted acid and purified water respectively behind the salify.The mantoquita precipitation is made pulpous state with the purified water of 1.7 times of complex liquid volumes, regulates pH to 3.2-3.3, H
2S decopper(ing) 6-8 hour.Concentrated solution is tired and is controlled at 25000-35000u/ml, concentrates the back volume and is about 1/10 of original volume, gets concentrated solution.Concentrated solution selects for use G25 type dextrane gel resin purification to separate, and flow velocity is 1/20 dress column volume, and the collection of separated flow fluid is collected effective constituent according to control indication nucleic acid-protein detector and self-registering instrument demonstration.Concentrate once more, control concentrated solution potency unit is at 58000～62000u/ml, and volume is controlled at original volume about 1/8.
6, reduction, concentrated.The concentrated solution that step 5 obtains is at last regulated pH to 4.5～5.0 with saturated lithium hydroxide solution, is reductive agent with the mercaptoethanol, and the ratio that adds the 15ml mercaptoethanol in per 100 units adds, and temperature is controlled at 34～36 ℃, and the time was controlled at 12 hours; The concentrated solution potency unit is controlled at 58000～62000u/ml.
7, precipitation, drying.Precipitation coenzyme A nitric acid-acetone mixed solution, its pH is controlled to be 2.5, and mixeding liquid temperature should be controlled 5～7 ℃, filters, and product must wet; With the discolour silica gel is siccative, and the loft drier temperature is controlled at 25 ℃ ± 1 ℃, vacuum-drying, and the time is controlled to be 48 hours, gets dry product.
Embodiment 2: the enzymatic building-up reactions stage adds the influence of precursor substance to the coenzyme A potency unit step by step.
According to embodiment 1 coenzyme A preparation method, the enzymatic building-up reactions stage all add by 1 time respectively required before the scale of construction, divide and add the required preceding scale of construction for 2 times, 3 times, 4 times, 5 times, 6 times, be prepared into coenzyme A sample 1,2,3,4,5,6, detect the potency unit that every 1ml contains coenzyme A by the coenzyme A statutory standards, concrete detected result is as follows:
| Sample | Sample 1 | Sample 2 | Sample 3 | Sample 4 | Sample 5 | Sample 6 |
| Every 1ml contains tiring of coenzyme A | 282 | 295 | 310 | 312 | 317 | 280 |
According to testing data as can be known: the coenzyme A enzymatic building-up reactions stage adds precursor step by step, can improve the potency unit of coenzyme A, obviously is better than property adding precursor 1 time.
Embodiment 3: separation phase is selected the influence of different model macroporous resin to coenzyme A yield and purity for use
Sample 1:, use DA201-A type macroporous resin adsorption to separate at the extraction and purification process separation phase according to the coenzyme A preparation method of embodiment 1.
Sample 2:, use DA201-B type macroporous resin adsorption to separate at the extraction and purification process separation phase according to the coenzyme A preparation method of embodiment 1.
Sample 3:, use CAD type macroporous resin adsorption to separate at the extraction and purification process separation phase according to the coenzyme A preparation method of embodiment 1.
By detecting the potency unit that contains coenzyme A among fermenation raw liquid and the every 1ml of elutriant by the coenzyme A statutory standards, calculate its adsorption rate, eluting rate and separation front and back purity thereof simultaneously, test-results is as follows:
| Sample | The macroporous resin model | Fermenation raw liquid purity | Adsorption rate | Eluting rate | Elutriant purity |
| 1 | DA201-A | 0.7％ | 80％ | 60％ | 2.8％ |
| 2 | DA201-B | 0.7％ | 98％ | 80％ | 3.0％ |
| 3 | CAD | 0.7％ | 70％ | 70％ | 2.9％ |
Test-results shows: it is all higher to select for use DA201-B type macroporous resin to carry out its adsorption rate of fractionation by adsorption, eluting rate, elutriant purity.
Embodiment 4: reducing process uses the influence of different highly basic to the coenzyme A potency unit.
Sample 1:, use potassium hydroxide to regulate pH value at reducing process according to the coenzyme A preparation method of embodiment 1.
Sample 2:, use lithium hydroxide to regulate pH value at reducing process according to the coenzyme A preparation method of embodiment 1.
Sample 3:, use sodium hydroxide to regulate pH value at reducing process according to the coenzyme A preparation method of embodiment 1.
Detect the potency unit that every 1ml contains coenzyme A by the coenzyme A statutory standards, sample 1, sample 2, sample 3 concrete detected results are as follows:
| Sample | Sample 1 | Sample 2 | Sample 3 |
| Every 1ml contains tiring of coenzyme A | 308 | 317 | 301 |
According to testing data as can be known: coenzyme A reductase technology uses strong base solutions such as potassium hydroxide, lithium hydroxide, sodium hydroxide to regulate pH value, and wherein lithium hydroxide can obviously improve the potency unit of coenzyme A.
Embodiment 5: reducing process of the present invention and bibliographical information 3 described reducing process are relatively.
Sample 1: the coenzyme A preparation method according to embodiment 1 makes sample 1, reducing process is regulated pH to 4.5～5.0 with saturated lithium hydroxide solution, with the mercaptoethanol is reductive agent, the ratio that adds the 15ml mercaptoethanol in per 100 units adds, temperature is controlled at 34～36 ℃, and the time was controlled at 12 hours; The concentrated solution potency unit is controlled at 58000～62000u/ml.
Sample 2: make sample 2 according to bibliographical information 3 described product ammonia rod bacillus biological synthesis process.
Detect the potency unit that every 1ml contains coenzyme A by the coenzyme A statutory standards, sample 1, sample 2 concrete detected results are as follows:
| Sample | Sample 1 | Sample 2 |
| Every 1ml contains tiring of coenzyme A | 318 | 256 |
Claims (7)
1. the preparation technology of a coenzyme A comprises fermentation and extracts the purifying two parts, and fermentation part is divided into microbial strains seed selection, strain fermentation and enzymatic building-up reactions; Extract that purification part is divided into fractionation by adsorption, concentrates, sieve chromatography, reduction, precipitation, it is characterized in that the enzymatic building-up reactions stage in fermentation part, the precursor substance that adds known quantities in enzyme reaction solution in batches is hydrochloric acid-halfcystine, calcium pantothenate, Sodium ATP.
2. the preparation technology of a kind of coenzyme A as claimed in claim 1 is characterized in that the enzymatic building-up reactions stage in fermentation part, divides the precursor substance that adds known quantities for 3 times, 4 times or 5 times in enzyme reaction solution.
3. the preparation technology of a kind of coenzyme A as claimed in claim 1 is characterized in that the enzymatic building-up reactions stage in fermentation part, divides the precursor substance that adds known quantities for 5 times in enzyme reaction solution.
4. the preparation technology of a kind of coenzyme A as claimed in claim 1 is characterized in that at the separation, the enriching stage that extract purification part, adopts DA210-B type non-polar macroporous resin to carry out fractionation by adsorption.
5. the preparation technology of a kind of coenzyme A as claimed in claim 1 is characterized in that: at the reduction phase that extracts purification part, with saturated strong base solution adjusting pH value, be that reductive agent reduces with the mercaptoethanol.
6. the preparation technology of a kind of coenzyme A as claimed in claim 2 is characterized in that regulating PH at the reduction phase that extracts purification part with saturated potassium hydroxide, sodium hydroxide or lithium hydroxide solution, and value is that reductive agent reduces with the mercaptoethanol.
7. the preparation technology of a kind of coenzyme A as claimed in claim 2 is characterized in that regulating pH value at the reduction phase that extracts purification part with saturated lithium hydroxide solution, is that reductive agent reduces with the mercaptoethanol.
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| CNA2008100492496A CN101230374A (en) | 2008-02-20 | 2008-02-20 | Preparation process of coenzyme A |
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| CNA2008100492496A CN101230374A (en) | 2008-02-20 | 2008-02-20 | Preparation process of coenzyme A |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102321708A (en) * | 2011-08-08 | 2012-01-18 | 济南维尔康生化制药有限公司 | Preparation technique of coenzyme A |
| CN112075634A (en) * | 2020-09-27 | 2020-12-15 | 何淑珍 | Anti-aging compound nutrient containing coenzyme and bioactive hydrogen and preparation method and application thereof |
-
2008
- 2008-02-20 CN CNA2008100492496A patent/CN101230374A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102321708A (en) * | 2011-08-08 | 2012-01-18 | 济南维尔康生化制药有限公司 | Preparation technique of coenzyme A |
| CN112075634A (en) * | 2020-09-27 | 2020-12-15 | 何淑珍 | Anti-aging compound nutrient containing coenzyme and bioactive hydrogen and preparation method and application thereof |
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Open date: 20080730 |