CN101736048A - Method for producing S-adenosylmethionine - Google Patents

Method for producing S-adenosylmethionine Download PDF

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Publication number
CN101736048A
CN101736048A CN200810225881A CN200810225881A CN101736048A CN 101736048 A CN101736048 A CN 101736048A CN 200810225881 A CN200810225881 A CN 200810225881A CN 200810225881 A CN200810225881 A CN 200810225881A CN 101736048 A CN101736048 A CN 101736048A
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sam
born
fermentation
same parents
enzyme
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徐明波
吴彦卓
陈献华
秦富景
赵晶晶
刘迎
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BEIJING SHUANGLU PHARMACEUTICAL Co Ltd
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BEIJING SHUANGLU PHARMACEUTICAL Co Ltd
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02P—CLIMATE CHANGE MITIGATION TECHNOLOGIES IN THE PRODUCTION OR PROCESSING OF GOODS
    • Y02P20/00—Technologies relating to chemical industry
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    • Y02P20/52—Improvements relating to the production of bulk chemicals using catalysts, e.g. selective catalysts

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Abstract

The invention relates to a method for producing S-adenosylmethionine (SAM), belonging to the production method of SAM. The method is characterized in that high-efficient genetic engineering bacteria which expresses SAM is obtained by the technology for gene engineering; fermentation is carried out by the fermentation technology; SAM and SAM synthetase are expressed in the engineering bacteria, and the SAM and the SAM synthetase in the cell is extracted by breaking cells; and the SAM synthetase extracted in the cell is used to perform extracellular enzymatic synthesis reaction to produce the SAM. Thus, primary fermentation can simultaneously obtain intracellular and extracellular SAM products, thus greatly improving the yield of unit fermentation liquor. The invention provides a new method for producing medical SAM with low cost on a large scale.

Description

A kind of method of producing ademetionine
Technical field
The present invention relates to the production method of ademetionine.
Background technology
Ademetionine (SAM) be present in human body in a organized way with body fluid in a kind of physiologically active molecule.SAM is mainly used in before the treatment liver cirrhosis and intrahepatic cholestasis due to the liver cirrhosis, the Gestation period intrahepatic cholestasis, anti-inflammatory analgesic, anti-spirit depressing, anti-dandruff, antipruritic, subtract and important application also arranged aspect wrinkle, anti-ageing the waiting for a long time.Ademetionine under normal temperature condition water absorbability and the complicacy of unstable and production technique seriously limited the widespread use of ademetionine at medical health field.Therefore, multinomial patent is being declared aspect the stable salify of ademetionine and the production technique by American-European scientific and technological circle.Some representative on medicine material market patents have: as United States Patent (USP) 6635615,5102791,4028183,4369177,4764603 etc.
The national annual requirement of SAM is at least more than 10 tons, and be 50 tons demand every year in the whole world.SAM does not also realize production domesticization, all dependence on import in China.The main production firm of SAM concentrates on Germany and Italy, every kilogram about 8000 yuans of medicinal raw materials.Domestic existing development and research, because of the expression level of fermentation method is low, the enzyme of Enzymatic transformation method and the cost of ATP are very high, so raw material production is failed extensive industrialization so far, mainly by Abbott import China.
At present the ademetionine industrialized producing technology mainly contains two kinds of yeast fermentation method (representation in the born of the same parents) and Enzymatic transformation methods (extracellular enzyme is urged method), and these two kinds of methods all exist different technological difficulties:
Yeast fermentation method (representation in the born of the same parents): the external Wine brewing yeast strains of the expression SAM of special cultivation that adopt pass through to express in the born of the same parents preparation SAM more.The domestic people of having works out the yeast saccharomyces cerevisiae that genetically engineered changes structure, fermentation expression SAM, but express at present the highest 8g/L fermented liquid that also only reaches, and most exploration of only finishing zymotechnique, producing with this method is to express in born of the same parents, because precursor L one methionine(Met) of SAM need see through cell wall, so and be subjected to the interior spatial restriction of born of the same parents to express the very difficult raising of output, yield is low, and the cost height is not suitable for mass preparation.
Enzymatic method (extracellular enzyme is urged method): enzymatic method is many to extract the purifying adenomethionine synthase from cereuisiae fermentum or Other Engineering bacterium, transform preparation SAM by optimizing enzymatic reaction condition then, this method feed stock conversion height, SAM is easy to extract purifying, but the extraction purification procedures complexity of enzyme is in saccharomycetic fragmentation in this method, centrifugal, filter, have the loss of enzyme on concentrated supernatant and the supernatant liquor in the column purification, the uncertain factor that influences enzymic activity simultaneously is more, the cycle of purifying enzyme is longer, cause the activity of enzyme to be subjected to influence, the purifying enzyme amount of this method acquisition is less simultaneously, cause the cost of enzyme very high, also be not suitable for mass preparation SAM.
Summary of the invention
The invention provides to express in a kind of born of the same parents and add the short synthetic method of producing ademetionine of extracellular enzyme.Yield poorly to solve yeast fermentation method (representation in the born of the same parents) expression, yield is low, and cost height and extracellular enzyme are urged the cost height of method enzyme, are not suitable for the problem of mass preparation SAM.The present invention has concentrated the advantage of fermentation method (representation in the born of the same parents) and the short method of extracellular enzyme, utilize one time fermentation can obtain simultaneously to express in the born of the same parents and the short two portions SAM product of extracellular enzyme, improved the output of the fermented liquid SAM of unit greatly, low production cost makes SAM realize the scale operation preparation.
The technical scheme that the present invention takes is:
At first utilize genetic engineering technique to obtain to express the efficient gene engineering bacterium of SAM:
The long 1152bp of nucleotide coding sequence of SAM synthase gene SAM2, coding contains the subunit of 384 amino-acid residues, and molecular weight is 42300.The homology of SAM1 and SAM2 encoding sequence is 83%, and the homology of encoded polypeptide aminoacid sequence illustrates that up to 92% SAM1 and SAM2 are replicator.With the SAM2 gene integration to engineering strain.The gene fragment that will have strong promoter, SAM2 gene and terminator sequence relies on the homologous sequence arm at SAM2 two ends to be incorporated into protogene group SAM2 site, relies on the strong promoter effect to realize the high expression level of SAM2.
Then utilize fermentation technique fermentation, in engineering bacteria, express SAM, extract SAM synthetic enzyme and SAM in the born of the same parents by broken born of the same parents.
Utilize the SAM synthetic enzyme that extracts in the born of the same parents outside born of the same parents, to carry out the short building-up reactions of extracellular enzyme more then and produce SAM, utilize one time fermentation can obtain born of the same parents interior expression SAM and SAM synthetic enzyme simultaneously like this, the SAM synthetic enzyme carries out the short building-up reactions of extracellular enzyme again outside born of the same parents, produce a part of SAM again.Be that one time fermentation can obtain the outer two portions SAM product of born of the same parents in the born of the same parents simultaneously, improved the output of unit fermented liquid greatly, reduced production cost, make SAM realize the scale operation preparation.
Wherein expressing part in the born of the same parents of Huo Deing reaches: be 20.6g, biomass 110g/L in every liter of fermented liquid.Extracellular enzyme promotes production and partly reaches: the pure specific enzyme activity of recombinant adenosine methionine synthase is up to the 10-50lU/ milligram.The enzymatic reaction transformation efficiency reaches more than 95%.SAM concentration reaches and is 12.8g in every liter of enzymatic reaction liquid.
Beer yeast gene recombinant bacterial strain among the present invention, this bacterial classification makes up seed selection by my company by the gene recombination engineering, by my company's technique center preservation, preservation date on March 18th, 2008, this gene recombination saccharomyces cerevisiae carries chemosynthesis adenomethionine synthase gene.The expression of synthetic enzyme is regulated and control by promotor TAC.
Engineering bacteria crushing process among the present invention is to adopt the physical method fragmentation: cytoclasis instrument, efficient homogenizer etc.
Adenomethionine synthase among the present invention is to be that 3000 Hollow Fiber Ultrafiltration post ultrafiltration and concentration extracts by molecular weight cut-off, and yield reaches more than 90%.Adenomethionine synthase can reclaim repeatedly recycling.
Ademetionine synthesis condition among the present invention is: potassium primary phosphate, magnesium chloride, glucose, ATP, inorganic pyrophosphatase make final concentration be: K +Concentration 100-30mmol/L, Mg 2+Concentration 5-30mmol/L, methionine(Met) are 5mmol/L---65mmol/L, and ATP is 5mmol/L---80mmol/L, inorganic pyrophosphatase 0.02mg/L, the pH value is 6.0---7.5, reaction cylinder is warming up to 20 ℃---39 ℃, stir enzymic transformations down, about 10 hours.
The extraction purifying of the ademetionine among the present invention utilizes slightly acidic macroporous acrylic Zeo-karb (D113) ion-exchange to finish.
The present invention has the following advantages and effect:
The present invention at first utilizes genetic engineering technique to obtain to express the efficient gene engineering bacterium of SAM, utilize the fermentation technique fermentation, in engineering bacteria, express SAM and SAM synthetic enzyme, extract SAM and SAM synthetic enzyme in the born of the same parents by broken born of the same parents, utilize the SAM synthetic enzyme that extracts in the born of the same parents outside born of the same parents, to carry out the short building-up reactions of extracellular enzyme more then and produce SAM.Be that one time fermentation can obtain the outer two portions SAM product of born of the same parents in the born of the same parents simultaneously, improved the output of unit fermented liquid greatly, technology is simple, has reduced production cost, makes SAM realize the scale operation preparation, makes SAM can obtain general practical application.
Embodiment:
Experiment material and method:
1, engineering bacteria and reagent:
The beer yeast gene recombinant bacterial strain, this bacterial classification makes up seed selection by my company by the gene recombination engineering, by my company's technique center preservation, preservation date on March 18th, 2008, this gene recombination saccharomyces cerevisiae carries chemosynthesis adenomethionine synthase gene.
Reagent mainly contains: adenosine triphosphate atp AR; The L-methionine(Met); The 99%p-mercaptoethanol; 1,4-butane sodium disulfonate; Tetrahydroxyethyl ethylenediamine tetraacethyl EDTA; Inorganic pyrophosphatase, slightly acidic macroporous acrylic Zeo-karb (D113).Deionizing water and to 1,4-butane sodium disulfonate (content is more than 95%) is for outside our company's self-control, and other all are homemade analytical pure level.
2, fermention medium
Ferment basic salt culture medium (Fermentation Basal Salts Medium, BSM):
85% phosphoric acid 200.25mL,
CaSO4·2H 2O?6.975g，
K 2SO 4136.5g，
MgSO4·7H 2O?111.75g，
KOH?30.9g，
Glycerine 300g,
Supplemented medium:
Feed supplement 1:25%-28% ammoniacal liquor, fermentor tank is controlled it automatically and is added speed, makes the pH value be controlled at certain numerical value.
Feed supplement 2:L-Met 75g, distilled water is settled to 1.25L, 115 ℃ of sterilization 30min.Add PTM115mL again.
Feed supplement 3:PTM148mL
Feed supplement 4: not glycerinated BSM substratum 1.25L adds 300g L-Met, 115 ℃ of sterilization 30min.
3 zymotechniques
3.1 the fermentation fs---adapt to growth phase
3, behind the adding fermention medium, add water to 75L, 121 ℃ of sterilization 30min in the fermentor tank.Be cooled to 37 ℃ of inoculations, stir 3h, rotating speed 500rpm, 600rpm behind the 8h, 650rpm behind the 15h, ventilation is enlarged to 1: 1 gradually.This stage D O and pH continue to descend, and fermentor tank is added ammoniacal liquor control pH5.0 automatically; Improve DO by improving rotating speed and air flow, make it be not less than 20%.
This stage is kept 18-25h, and time length is distinguished to some extent according to the inoculum size difference.This stage biomass reaches about 100g/L, and HPLC detects SAM 0.7g/L.
3.2 fermentation subordinate phase---rapid growth phase
When DO sharply rises suddenly, when rising to 70%-90%, enter subordinate phase by 20%, begin to add feed supplement 2.Feed supplement speed 126mL/h.Rotating speed is brought up to 700rpm.Feed supplement 2 adds DO and promptly can descend rapidly.This stage is added ammoniacal liquor control pH5.0.
This stage thalline is sharply grown, this stage duration 10h, and this moment, biomass reached 210-240g/L, and HPLC detects SAM 3.22g/L.
3.3 the hungry stage of fermentation phase III---carbon source
After feed supplement 2 had been mended, DO can sharply rise again, and entered for the 3rd stage this moment.The hungry 1.5h of carbon source is because the back will change carbon source, in order to allow thalline better adapt to new carbon source.In this stage, DO maintains the high level more than 70%, and pH has certain rising.
The quadravalence section 3.4 ferment---the abduction delivering stage
Behind the hungry 1.5h of carbon source, enter the quadravalence section, add feed supplement 3 and feed supplement 4 simultaneously.Feed supplement 3 flow velocitys are from 10.5mL/h, and every 15min increases 2.1mL/h, and until 25.2mL/h, DO drops to below 20.The back is according to the DO value, adjusts the speed of feed supplement 3, is generally 25.2mL/h-40mL/h, make DO between 20-30 to fermentation ends.Feed supplement 4 flow velocitys are 12.65mL/h, and constant speed is added.The concentration of methyl alcohol is too high, and cell can be poisoned.PH is promoted to 6.0.DO is not less than 15%, when DO is too low, reduces the speed of feed supplement 3, improves dissolved oxygen.
The abduction delivering stage continues 100h left and right sides expression amount and reaches the highest, fermentation ends, following jar.This stage, biomass reached 290-340g/L, about SAM output 21.11g/L when finishing.About whole fermentation process 130h.
The bacterium liquid of afterwards collecting fermenting carries out centrifugal, and rotating speed is 4000Pm, and centrifugal 10 minutes, abandoning supernatant, the thalline of collecting precipitation ,-40 ℃ are frozen.
4, the extraction of adenomethionine synthase preparation
With the thalline more than frozen ten days, suspend with the 20mmol/L phosphoric acid buffer, every gram cell mud is with 2 milliliters of damping fluids, and the bacterium liquid after the suspension utilizes clarifixator to carry out fragmentation, and the pressure that behaviour does is 60MPa, and temperature is controlled at below 15 ℃, circulates twice.Collection suspension carries out centrifugal, and rotating speed is 15000rpm, and the supernatant liquor that contains SAM synthetic enzyme and SAM is collected in centrifugal back.Be 3000 Hollow Fiber Ultrafiltration post ultrafiltration and concentration then with molecular weight cut-off, adenomethionine synthase is trapped in the ultrafiltration and concentration liquid, and ademetionine (SAM) is then in filtered solution.Concentrated solution is as the enzyme liquid of next step enzymatic reaction, and filtered solution is transferred purification procedures and extracted purifying SAM wherein.
5, enzymatic is synthetic
Getting the step concentrated solution is crude enzyme liquid, adds reaction cylinder, adds an amount of purified water, adds potassium primary phosphate, magnesium chloride, glucose, ATP inorganic pyrophosphatase successively and makes final concentration be: K +Concentration 250mmol/L, Mg 2+Concentration 30mmol/L, methionine(Met) is 5mmol/L～65mmol/L, ATP inorganic pyrophosphatase 0.02mg/L is 5mmol/L～80mmol/L, the pH value is 6.0～7.5, reaction cylinder is warming up to 24 ℃～31 ℃, stirs enzymic transformations down, about 10 hours, in reaction process, correspondingly add methionine(Met) and ATP, to keep maximum enzymatic reaction speed according to the process of reaction.At any time do Liquid Detection,, when SAM content reaches the 15g/L conversion fluid in the conversion fluid, can add sulfuric acid and transfer pH3.0 to stop conversion reaction until most of transform (transformation efficiency is more than 90%) of methionine(Met).
After transforming termination, be with reaction volume and diatomaceous ratio: 1: 0.005 amount, add diatomite and go into reaction cylinder, stirred 20 minutes, Plate Filtration is to remove protein.Filter to get filtrate, filtrate added strong-basicity styrene series anion exchange resin (201x7) whip attachment 15 minutes again, and making pH is 4.5, left standstill 20-30 minute, and vacuum filtration filters.
6, the recovery of adenomethionine synthase
Filtrate is 3000 Hollow Fiber Ultrafiltration post with molecular weight cut-off, and ultrafiltration and concentration reclaims adenomethionine synthase.Adenomethionine synthase can reuse.
7, purifying
Getting brokenly on filtered solution behind the bacterium and the filtered solution behind the enzymatic slightly acidic macroporous acrylic Zeo-karb (D113) separator column ion exchange column carries out chromatography and extracts.
To go up operation filtrate and be dissolved in water to become to be diluted to electricity and lead, and transfer pH to add lentamente behind the 5.0-5.5 through good slightly acidic macroporous acrylic Zeo-karb (D113) separator column of acid-alkali treatment aftertreatment less than 5.Detected elutriant in per 30 minutes, and measured its OD value, greater than the aqueous sulfuric acid washing of using pH2.0 at 0.4 o'clock instead 4 hours, per 30 minutes water lotions were done Liquid Detection simultaneously in wavelength 254nm place solution absorption.When only containing ademetionine in the effluent liquid, can stop washing.Then with water for injection preparation 0.3M 1,4-fourth disulfonic acid solution elutes the ademetionine in the separator column, in the process of wash-out, and H +Displacement SAM +, the SAM that replaces +With 1,4-fourth disulfonic acid root knot is closed, and generates 1,4-fourth disulfonic acid ademetionine finished product, and it is unnecessary 1 that elutriant removes with weakly alkaline macroporous acrylic anionite-exchange resin, and 4-fourth disulfonic acid root makes SAM +With 1, the ratio of 4-fourth disulfonic acid root meets the requirements.Elutriant is transferred next step refining step.
8, refining
In elutriant, add the 1g/L medicinal carbon, stir decolouring in 40 minutes, filter, collect filtrate and be placed in the plastic tank, add medicinal alcohol in 1: 6 ratio, place after 48 hours, medicinal alcohol is poured out, add and use the water for injection dissolution precipitation, use filtering with microporous membrane, promptly get the pure product solution of ademetionine.
The lyophilize of pure product solution.Promptly get white powdered ademetionine (SAM) dry powder finished product.
Embodiment one
1, zymotechnique
Utilize 100 liters of ferment tanks, the BSM substratum adds the basic salt culture medium of fermentation
85% phosphoric acid 200.25mL,
CaSO4·2H 2O?6.975g，
K 2SO 4136.5g，
MgSO4·7H 2O?111.75g，
KOH?30.9g，
Glycerine 300g,
After adding fermention medium, add water to 75L, 121 ℃ of sterilization 30min in the fermentor tank.Be cooled to 37 ℃ of inoculations, stir 3h, rotating speed 500rpm, 600rpm behind the 8h, 650rpm behind the 15h, ventilation is enlarged to 1: 1 gradually.This stage D O and pH continue to descend, and fermentor tank is added ammoniacal liquor control pH5.0 automatically; Improve DO by improving rotating speed and air flow, make it be not less than 20%.
When DO sharply rises suddenly, when rising to 70%-90%, enter subordinate phase by 20%, begin to add feed supplement 2.Feed supplement speed 126mL/h.Rotating speed is brought up to 700rpm.Feed supplement 2 adds DO and promptly can descend rapidly.This stage is added ammoniacal liquor control pH5.0.
After feed supplement 2 had been mended, DO can sharply rise again, and entered for the 3rd stage this moment.The hungry 1.5h of carbon source adds feed supplement 3 and feed supplement 4 simultaneously.Feed supplement 3 flow velocitys are from 10.5mL/h, and every 15min increases 2.1mL/h, and until 25.2mL/h, DO drops to below 20.
The abduction delivering stage continues 100h left and right sides expression amount and reaches the highest, fermentation ends, following jar.This stage, biomass reached 290-340g/L, about SAM output 21.11g/L when finishing.
The bacterium liquid of afterwards collecting fermenting carries out centrifugal, and rotating speed is 4000Pm, and centrifugal 10 minutes, abandoning supernatant, the thalline of collecting precipitation ,-40 ℃ are frozen.
2, the extraction of adenomethionine synthase preparation
With the thalline more than frozen ten days, suspend with the 20mmol/L phosphoric acid buffer, every gram cell mud is with 2 milliliters of damping fluids, and the bacterium liquid after the suspension utilizes clarifixator to carry out fragmentation, and the pressure that behaviour does is 60MPa, and temperature is controlled at below 15 ℃, circulates twice.Collection suspension carries out centrifugal, and rotating speed is 15000rpm, and the supernatant liquor that contains SAM synthetic enzyme and SAM is collected in centrifugal back.Be 3000 Hollow Fiber Ultrafiltration post ultrafiltration and concentration then with molecular weight cut-off, adenomethionine synthase is trapped in the ultrafiltration and concentration liquid, and ademetionine (SAM) is then in filtered solution.Concentrated solution is as the enzyme liquid of next step enzymatic reaction, and filtered solution is transferred purification procedures and extracted purifying SAM wherein.
3, enzymatic is synthetic
Getting the step concentrated solution is crude enzyme liquid, adds reaction cylinder, adds an amount of purified water, adds potassium primary phosphate, magnesium chloride, glucose, ATP inorganic pyrophosphatase successively and makes final concentration be: K +Concentration 250mmol/L, Mg 2+Concentration 30mmol/L, methionine(Met) is 5mmol/L～65mmol/L, ATP inorganic pyrophosphatase 0.02mg/L is 5mmol/L～80mmol/L, the pH value is 6.0～7.5, reaction cylinder is warming up to 24 ℃～31 ℃, stirs enzymic transformations down, about 10 hours, in reaction process, correspondingly add methionine(Met) and ATP, to keep maximum enzymatic reaction speed according to the process of reaction.At any time do Liquid Detection,, when SAM content reaches the 15g/L conversion fluid in the conversion fluid, can add sulfuric acid and transfer pH3.0 to stop conversion reaction until most of transform (transformation efficiency is more than 90%) of methionine(Met).
After transforming termination, be with reaction volume and diatomaceous ratio: 1: 0.005 amount, add diatomite and go into reaction cylinder, stirred 20 minutes, Plate Filtration is to remove protein.Filter to get filtrate, filtrate added strong-basicity styrene series anion exchange resin (201x7) whip attachment 15 minutes again, and making pH is 4.5, left standstill 20-30 minute, and vacuum filtration filters.
4, purifying
Getting brokenly on filtered solution behind the bacterium and the filtered solution behind the enzymatic slightly acidic macroporous acrylic Zeo-karb (D113) separator column ion exchange column carries out chromatography and extracts.
To go up operation filtrate and be dissolved in water to become to be diluted to electricity and lead, and transfer pH to add lentamente behind the 5.0-5.5 in good slightly acidic macroporous acrylic Zeo-karb (D113) separator column of acid-alkali treatment aftertreatment to adsorb less than 5.Detected elutriant in per 30 minutes, and measured its OD value, greater than the aqueous sulfuric acid washing of using pH2.0 at 0.4 o'clock instead 4 hours, when only containing SAM in the effluent liquid, can stop washing in wavelength 254nm place solution absorption.With water for injection preparation 0.3M 1,4-fourth disulfonic acid solution elutes the ademetionine in the separator column then, in the process of wash-out, and H +Displacement SAM +, the SAM that replaces +With 1,4-fourth disulfonic acid root knot is closed, generate 1,4-fourth disulfonic acid ademetionine finished product, elutriant removes unnecessary 1 with weakly alkaline macroporous acrylic anionite-exchange resin, 4-fourth disulfonic acid root makes SAM+ and 1, and the ratio of 4-fourth disulfonic acid root meets the requirements, and transfers next step refining step with clean plastic tank splendid attire elutriant.
5, refining
In elutriant, add the 1g/L medicinal carbon, stir decolouring in 40 minutes, filter, collect filtrate and be placed in the plastic tank, add medicinal alcohol in 1: 6 ratio, place after 48 hours, medicinal alcohol is poured out, add and use the water for injection dissolution precipitation, use filtering with microporous membrane, promptly get the pure product solution of ademetionine.
The pure product solution of ademetionine is carried out lyophilize.Promptly get white powdered ademetionine finished product.
Embodiment two enzyme catalysis ademetionine building-up reactions and purification refines.
With 10 liters reaction systems is example,
With the thalline more than frozen ten days, suspend with the 20mmol/L phosphoric acid buffer, every gram cell mud is with 2 milliliters of damping fluids, and the bacterium liquid after the suspension utilizes clarifixator to carry out fragmentation, and the pressure that behaviour does is 60MPa, and temperature is controlled at below 15 ℃, circulates twice.Collection suspension carries out centrifugal, and rotating speed is 15000rpm, and the supernatant liquor that contains SAM synthetic enzyme and SAM is collected in centrifugal back.Be 3000 Hollow Fiber Ultrafiltration post ultrafiltration and concentration then with molecular weight cut-off, adenomethionine synthase is trapped in the ultrafiltration and concentration liquid, and ademetionine (SAM) is then in filtered solution.Concentrated solution is as the enzyme liquid of next step enzymatic reaction, and filtered solution is transferred purification procedures and extracted purifying SAM wherein.
Get and contain SAM synthetic enzyme ultrafiltration and concentration liquid, add reaction cylinder, add an amount of purified water, add potassium primary phosphate, magnesium chloride, glucose, ATP, inorganic pyrophosphatase successively and make final concentration be: K +Concentration 250mmol/L, Mg 2+Concentration 30mmol/L, methionine(Met) is 35mmol/L, ATP is 60mmol/L, inorganic pyrophosphatase 0.02mg/L, and the pH value is 6.5, reaction cylinder is warming up to 31 ℃, stir enzymic transformations down, about 10 hours, until most of transform (transformation efficiency is more than 90%) of methionine(Met), when SAM content reaches the 15g/L conversion fluid in the conversion fluid, can add sulfuric acid and transfer pH3.0 to stop conversion reaction.
After transforming termination, be with reaction volume and diatomaceous ratio: 1: 0.005 amount, add diatomite and go into reaction cylinder, stirred 20 minutes, Plate Filtration is to remove protein.Filter to get filtrate, filtrate added strong-basicity styrene series anion exchange resin (201x7) whip attachment 15 minutes again, and making pH is 4.5, left standstill 20-30 minute, and vacuum filtration filters.Filtrate is 3000 Hollow Fiber Ultrafiltration post with molecular weight cut-off, ultrafiltration, filtered solution.
Get brokenly slightly acidic macroporous acrylic Zeo-karb (D113) separator column on filtered solution behind the bacterium and the filtered solution behind the enzymatic.Detected elutriant in per 30 minutes, and measured its OD value, greater than the aqueous sulfuric acid washing of using pH2.0 at 0.4 o'clock instead 4 hours, stop washing in wavelength 254nm place solution absorption.
After washing stopped, with water for injection preparation 0.3M 1,4-fourth disulfonic acid solution eluted the ademetionine in the separator column, promptly gets 1,4-fourth disulfonic acid ademetionine finished product.In elutriant, add the 1g/L medicinal carbon, stir decolouring in 40 minutes, filter, collect filtrate, add medicinal alcohol, place after 48 hours, medicinal alcohol is poured out in 1: 6 ratio, add and use the water for injection dissolution precipitation, use filtering with microporous membrane, promptly get the pure product solution of ademetionine.
The pure product solution of ademetionine is put into freeze drier carry out lyophilize.Promptly get white powdered ademetionine (SAM) dry powder finished product.

Claims (1)

1. method of producing ademetionine (SAM).It is characterized in that: at first utilize genetic engineering technique to obtain to express the efficient gene engineering bacterium of SAM, utilize the fermentation technique fermentation, in engineering bacteria, express SAM and SAM synthetic enzyme, extract SAM and SAM synthetic enzyme in the born of the same parents by broken born of the same parents, utilize the SAM synthetic enzyme that extracts in the born of the same parents outside born of the same parents, to carry out the short building-up reactions of extracellular enzyme more then and produce SAM.Be that one time fermentation can obtain the outer two portions SAM product of born of the same parents in the born of the same parents simultaneously, improved the output of unit fermented liquid greatly.
CN200810225881A 2008-11-05 2008-11-05 Method for producing S-adenosylmethionine Pending CN101736048A (en)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101979645A (en) * 2010-09-30 2011-02-23 北京凯因科技股份有限公司 Method for preparing adenosylmethionine
CN113499608A (en) * 2021-06-29 2021-10-15 贵州卡本嘉泰生物科技产业发展有限公司 Chromatographic system for enzymatic reaction and control method thereof

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101979645A (en) * 2010-09-30 2011-02-23 北京凯因科技股份有限公司 Method for preparing adenosylmethionine
CN101979645B (en) * 2010-09-30 2012-12-12 北京凯因科技股份有限公司 Method for preparing adenosylmethionine
CN113499608A (en) * 2021-06-29 2021-10-15 贵州卡本嘉泰生物科技产业发展有限公司 Chromatographic system for enzymatic reaction and control method thereof
CN113499608B (en) * 2021-06-29 2022-04-08 贵州卡本嘉泰生物科技产业发展有限公司 Chromatographic system for enzymatic reaction and control method thereof

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Application publication date: 20100616