CN1483829A - Method for producing aclenosylmethionine by enzyme catalysis - Google Patents
Method for producing aclenosylmethionine by enzyme catalysis Download PDFInfo
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- CN1483829A CN1483829A CNA03126834XA CN03126834A CN1483829A CN 1483829 A CN1483829 A CN 1483829A CN A03126834X A CNA03126834X A CN A03126834XA CN 03126834 A CN03126834 A CN 03126834A CN 1483829 A CN1483829 A CN 1483829A
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- adenosylmethionine
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- enzyme catalysis
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- 102000004190 Enzymes Human genes 0.000 title claims abstract description 26
- 238000006555 catalytic reaction Methods 0.000 title claims abstract description 14
- 238000004519 manufacturing process Methods 0.000 title abstract description 11
- MEFKEPWMEQBLKI-AIRLBKTGSA-N S-adenosyl-L-methioninate Chemical compound O[C@@H]1[C@H](O)[C@@H](C[S+](CC[C@H](N)C([O-])=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 MEFKEPWMEQBLKI-AIRLBKTGSA-N 0.000 claims abstract description 61
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 claims abstract description 21
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- 239000002994 raw material Substances 0.000 claims abstract description 3
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- 238000000034 method Methods 0.000 claims description 18
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- 239000010452 phosphate Substances 0.000 claims description 2
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- NWUYHJFMYQTDRP-UHFFFAOYSA-N 1,2-bis(ethenyl)benzene;1-ethenyl-2-ethylbenzene;styrene Chemical compound C=CC1=CC=CC=C1.CCC1=CC=CC=C1C=C.C=CC1=CC=CC=C1C=C NWUYHJFMYQTDRP-UHFFFAOYSA-N 0.000 description 2
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The method for producing adenosylmethionine by enzyme catalysis includes the following steps: (A). utilizing recombinant DNA and enzyme engineering technology and molecualr cloning engineering bacteria constructed by SAM synthetase gene, and making fermentation to prepare adenosylmethionine synthetase enzyme liquor; (B). using methionine of DL-type and adenosine triphosphate as raw material to synthesize adenosylmethionine; and (C). separating and purifying the adenosylmethionine product.
Description
Technical field
The present invention relates to a kind of enzyme catalysis and produce the method for adenosylmethionine, the method for particularly a kind of adenosine methilanin synthase that efficiently expresses with genetic engineering bacterium (EC2.5.1.6) catalysis synthesizing adenosine methilanin.
Background technology
S-adenosylmethionine (SAM) is the intravital main methyl donor of human body and other biological, is body internal protein, fat, Yeast Nucleic Acid and vitamins B
12Methyl is provided, for many ammonia synthesiss in the body provide aminopropyl, for the biosynthesizing of tRNA provides precursor, participating in the biosynthesizing and the metabolism of hormone in vivo, neurotransmitter, nucleic acid, protein and phosphatide, is to safeguard cytolemma normal function and human homergy and healthy indispensable important living matter.SAM also participates in the formation of Triptide, and Triptide is the main antioxidant in the cell, participates in various detoxification processes.S-adenosylmethionine is to safeguard the indispensable important living matter of HUMAN HEALTH.Hepatopathy, dysthymia disorders and sacroiliitis are had clear and definite curative effect, are preventing cancer, cardiovascular disorder and antidotal high-class healthy medicine.
The production method of S-adenosylmethionine introduced in the article that is entitled as " S-adenosine methyl-sulfuric acid synthetic enzyme and the application in S-adenosylmethionine is synthetic thereof " in " medicine biotechnology " calendar year 2001s 8 monthly magazine, comprises chemical synthesis, fermentation method and Enzymatic transformation method.Chemical synthesis adopts S-adenosyl homocysteine and methyl donor CH
3I is synthetic.SAM has (+) and (-) two kinds of configurations, but has only (-) configuration that biological activity is just arranged, and contains a small amount of (+) type SAM in its synthetic product, and is difficult to separate, and causes separation and purification of products cost costliness.
Fermentation method adopts and add the L-methionine(Met) in substratum, with yeast fermentation production (-) type SAM, is the main method of producing SAM the sixties to the eighties.Calculate with the L-methionine(Met), the productive rate of fermentative Production SAM has only 15～30%.And the production cycle is long, is unfavorable for widespread production and the application of SAM.
Advantages such as the Enzymatic transformation method is compared with chemical synthesis, fermentation method, has end product accumulation volume height, separates and purify easily, and reaction time is short and pollution-free are comparatively effective industrialized preparing process.The Enzymatic transformation method mainly utilizes SAM synthetic enzyme catalytic substrate L-methionine(Met) and ATP to generate (-) type SAM.But it is not high that the SAM synthetic enzyme is few at animal and plant and microbe intensive amount, enzyme is lived, and the separation and purification difficulty, therefore is subjected to the restriction of SAM synthetic enzyme vigor with Enzymatic transformation method mass preparation SAM.The development of genetic engineering technique makes the SAM synthetic enzyme that obtains a large amount of high vigor become possibility.Utilize recombinant DNA and enzyme engineering technology, molecular cloning SAM synthase gene makes up efficient engineering bacterium expression system, and the SAM synthetic enzyme of engineering bacterium expression can account for total protein more than 20%, behind the initial gross separation purifying, with the synthetic SAM of the mode of enzymatic reaction.Because it is easy that reorganization SAM synthetic enzyme separates purification, the specific activity height, the finished product purifying is simple, can satisfy medicine fully and produce needs.Though compare with fermentation method and chemical synthesis, the production cost that enzymatic method is produced adenosylmethionine has descended, and the cycle has shortened, but its price is still expensive, limit this medicine production and widespread use, can not satisfy the demand that people increase SAM day by day with the quality of life raising.
Summary of the invention
The object of the present invention is to provide simple, with short production cycle, the method for the adenosine methilanin synthase catalytic production adenosylmethionine that efficiently expresses of using gene engineering bacterium cheaply of a kind of technology.
The present invention includes following steps:
(A) utilize recombinant DNA and enzyme engineering technology, the engineering bacterium fermentation that molecular cloning SAM synthase gene makes up prepares adenosine methilanin synthase enzyme liquid;
(B) methionine(Met) and the Triphosaden with the DL-type is the raw material synthesizing adenosine methilanin;
(C) separation of adenosylmethionine product is purified.
Step of the present invention comprises that also the adenosylmethionine product separates the drying after purifying.Described drying step comprises:
(A) add Vltra tears in the adenosylmethionine salts solution after separation and purification, the quality of the Vltra tears of adding be in the solution adenosylmethionine salts contg (quality) 0.5～50%;
(B) spraying drying is made powder with adenosylmethionine.
Employed damping fluid is that final concentration is the phosphate solution of 0.05～1mol/L in the described enzymic catalytic reaction synthesizing adenosine methilanin, and the pH value of its solution preparation is 8.0.DK110 or HD-2 Zeo-karb are adopted in the separation and purification of adenosylmethionine product.
The present invention adopts the DL-methionine(Met) to replace general L-methionine(Met), can reach identical changing effect, but the price of DL-methionine(Met) only is 10% of a L-methionine(Met).The usage quantity of DL-methionine(Met) is the twice of L-methionine(Met), so its cost is about 20% of L-methionine(Met).The present invention adopts phosphate buffered saline buffer (sodium salt or sylvite) to replace the Tris-HCl damping fluid, transformation efficiency under this damping fluid is more than 95%, with the basic indifference of employing Tris-HCl damping fluid, but the price of phosphate buffered saline buffer only is about 30% of a Tris-HCl damping fluid, and is therefore very favourable to reducing production costs.The chromatography media that the present invention adopts is DK110 and HD-2 weakly acidic cation-exchange resin, this two media is not seen to have when the adenosylmethionine purifying and is used, the two media low price, carrying capacity is big, purification effect good, stability is strong, and its cost performance is better than the import chromatography media greatly.
Because the high-hygroscopicity of adenosylmethionine causes carrying out the processing of subsequent technique.The present invention adopts and adds hydroxymethyl-propyl cellulose (HPMC), and adenosylmethionine is carried out coating or claims microencapsulation to handle, and greatly reduces its water absorbability, for condition has been created in the processing of preparations such as tablet
Embodiment
One, material is prepared
1, engineering bacteria and reagent
Colibacillus engineering carries the gene of adenosine methilanin synthase (2.5.1.6).
Reagent all is homemade analytical pure level.It is standby that each reagent is made into following concentration: 1mol/L phosphoric acid salt (sylvite) damping fluid, and the pH value is 8.0; 3mol/L KCl; 1mol/L MgCl
20.4mol/L ATP; 0.2mol/L DL-methionine(Met); Paratoluenesulfonic acid sodium salt; 99% beta-mercaptoethanol; 0.5mol/L EDTA; Deionized water.HD-2 weakly acidic cation-exchange resin (being called for short HD-2), DK110 macroporous acrylic are weak acid resin (being called for short DK110) (two kinds of resins are the Shanghai China scientific and technological trading company of shake and produce).
2, substratum
A substratum: peptone 10g/L; Yeast powder 5g/L; NaCl 10g/L; Oxidation tsiklomitsin 25 μ g/ml.Peptone, yeast powder and NaCl with deionized water dissolving after, 115 ℃ of sterilizations 20 minutes, add the oxidation tsiklomitsin in when inoculation then and reach 25 μ g/ml.
The B substratum: the agar of adding 1% in the A substratum, promptly make Agar Plating.
3, fermentation
Above-mentioned Agar Plating (B substratum) is gone up the adenosine methionine synthetase gene engineering bacteria of cultivating, provoke single bacterium colony, insert in the 40ml A substratum (containing 25 μ g/ml oxidation tsiklomitsins), 37 ℃ of shaking tables were cultivated 20 hours.
Above bacterium liquid is inoculated in the A substratum of 6L (containing 25 μ g/ml oxidation tsiklomitsins), 37 ℃ of shaking tables were cultivated about 27 hours, surveyed the OD of bacterium liquid
600More than=1.0.
Secondary is cultivated the bacterium liquid of collecting the back carry out centrifugally, rotating speed is 5000rpm, centrifugal 10 minutes, and abandoning supernatant, the thalline of collecting precipitation ,-70 ℃ are frozen.
4, the making of enzyme liquid
The engineering bacteria cell (check back target protein should account for more than 20% of total protein) that fermentation is obtained suspends with 0.1mol/L Tris-HCl damping fluid (pH value is 8.0), every gram cell mud is with 4 milliliters of damping fluids, bacterium liquid after the suspension utilizes clarifixator to carry out fragmentation, working pressure is 60MPa, temperature is controlled at below 35 ℃, circulates twice.Collection suspension carries out centrifugal, and rotating speed is 15000rpm, and temperature is below 2 ℃.It is standby that supernatant liquor is collected in centrifugal back, uses in 24 hours to be stored in 2 ℃, and standing storage should be at-30 ℃.
Two, the enzyme catalysis adenosylmethionine is synthetic, is example with one liter reaction system:
1, adds reagent
Add the above-mentioned reagent for preparing respectively: 100 milliliters of 1mol/L phosphoric acid salt (sylvite) damping fluids (pH8.0); 67 milliliters of 3mol/LKCl; 1mol/LMgCl
252 milliliters; 0.4mol/LATP 25 milliliters; 0.2mol/LDL-120 milliliters of methionine(Met)s; Paratoluenesulfonic acid sodium salt 77 grams; 1 milliliter of 99% beta-mercaptoethanol; 0.5mol/LEDTA 2 milliliters; Deionized water is settled to 1 liter.
2, temperature of reaction and time
Above-mentioned mixed solution is added in the reactor, and it is 35 ℃ that temperature of reaction is set, 15 hours reaction times.
3, reaction solution removes albumen
Add hydrochloric acid in the above-mentioned mixed solution that finishes reaction, final concentration is 0.1mol/L, can make albumen precipitation under this condition.Centrifugal 10 minutes of 15000rpm discards throw out, and it is standby to collect supernatant liquor.
Three, the chromatography purification of adenosylmethionine
1, the processing of chromatography media
New DK110 or HD-2 medium are cleaned 3 column volumes with the hydrochloric acid of 1mol/L respectively, and water washes to pH nearly 7; 0.85mol/L sodium-chlor cleans 3 column volumes, 3 column volumes of water flushing; 1.0mol/L 3 column volumes of sodium hydroxide flushing, the water flushing is to neutral; The hydrochloric acid of 1mol/L is washed 3 column volumes again can be standby.Whenever use up once later on, only needing can be standby with 3 column volumes of hydrochloric acid cleaning of 1mol/L.Flow velocity generally is controlled at 0.04-0.05 column volume.
2, go up sample
With the pH value is the balance that 6.5 phosphoric acid buffer carries out chromatography column, after balance finishes, is added on the chromatography column adenosylmethionine salt that every mL media is adsorbable approximately 5 milligrams with removing the flow velocity of proteic reaction mixture with 0.04 column volume of per minute.
3, flushing
With the pH value is that 6.5 phosphoric acid buffer washes, to 260 wavelength detect do not have absorb till.
4, wash-out
Hydrochloric acid with 0.1-0.20mol/L carries out wash-out, collects the elution peak at 260 wavelength places.The sulfuric acid that adds 2 times of mole numbers of adenosylmethionine in collecting liquid adds the paratoluenesulfonic acid sodium salt with the identical mole number of adenosylmethionine simultaneously.
Collect liquid and be placed on preservation, standby in the 4-8 ℃ of refrigerator.
Four, the drying of adenosylmethionine salt
1, chromatography purification is collected concentrating of liquid
Chromatography purification is collected liquid add in the concentrating under reduced pressure device and carry out reduction vaporization, thickening temperature must not be higher than 40 ℃, and concentrated pressure should be lower than-0.1MPa.Be concentrated into that adenosylmethionine concentration is about 0.05mol/L in the solution.
2, the drying of concentrated solution
In above-mentioned concentrated solution, add quality and be 5% hydroxymethyl-propyl cellulose (HPMC) of adenosylmethionine quality, send to then and carry out spraying drying in the spray-drier.Sticking wall well, does not take place with solution atomization and is as the criterion in sample size, generally follows by the little principle that increases gradually and comes dominant discharge.Inlet temperature generally is controlled at below 200 ℃, and temperature out is about 70-80 ℃.The adenosylmethionine dry powder of collecting should seal cryopreservation.When needing opening, should under the environment of relative humidity 25%, operate.
Claims (6)
1, the method for adenosylmethionine is produced in a kind of enzyme catalysis, comprises the steps:
(A) utilize recombinant DNA and enzyme engineering technology, the engineering bacterium fermentation that molecular cloning SAM synthase gene makes up prepares adenosine methilanin synthase enzyme liquid;
(B) methionine(Met) and the Triphosaden with the DL-type is the raw material synthesizing adenosine methilanin;
(C) separation of adenosylmethionine product is purified.
2, the method for adenosylmethionine is produced in enzyme catalysis according to claim 1, it is characterized in that also comprising that the adenosylmethionine product separates the drying step after purifying.
3, the method for adenosylmethionine is produced in enzyme catalysis according to claim 2, it is characterized in that described drying step comprises:
(A) add Vltra tears in the adenosylmethionine salts solution after separation and purification, the quality of the Vltra tears of adding be in the solution adenosylmethionine salts contg (quality) 0.5～50%;
(B) spraying drying is made powder with adenosylmethionine.
4, the method for adenosylmethionine is produced in enzyme catalysis according to claim 1, it is characterized in that employed damping fluid is that final concentration is the phosphate solution of 0.05～1mol/L in the described enzymic catalytic reaction synthesizing adenosine methilanin, the pH of its solution preparation is 8.0.
5, the method for adenosylmethionine is produced in enzyme catalysis according to claim 1, it is characterized in that adopting DK110 or HD-2 Zeo-karb to realize the separation and purification of described product.
6, the method for adenosylmethionine is produced in enzyme catalysis according to claim 1, and the temperature of reaction that it is characterized in that step B is 30～35 ℃.
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| CN100363499C (en) * | 2006-02-27 | 2008-01-23 | 上海国佳生化工程技术研究中心有限公司 | Novel aden osyl methionine in vitro enzymatic transformation method |
| WO2008034338A1 (en) | 2006-08-31 | 2008-03-27 | Bioright Worldwide Company Limited | S-adenosylmethionine synthetase mutants, the dnas encoding the same and uses of the mutants |
| CN101985616A (en) * | 2010-11-11 | 2011-03-16 | 西北工业大学 | Method for preparing immobilized adenosylmethionine synthetase and adenosylmethionine |
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| CN100363499C (en) * | 2006-02-27 | 2008-01-23 | 上海国佳生化工程技术研究中心有限公司 | Novel aden osyl methionine in vitro enzymatic transformation method |
| WO2008034338A1 (en) | 2006-08-31 | 2008-03-27 | Bioright Worldwide Company Limited | S-adenosylmethionine synthetase mutants, the dnas encoding the same and uses of the mutants |
| US7858351B2 (en) | 2006-08-31 | 2010-12-28 | Geneharbor ( Hong Kong) Technologies, Ltd. | S-adenosylmethionine synthetase mutants, the DNAs encoding the same and uses of the mutants |
| CN102441286B (en) * | 2010-09-30 | 2015-04-15 | 上海张江中药现代制剂技术工程研究中心 | Method for improving melt wall sticking during spray drying of traditional Chinese medicine or food raw material |
| CN102441286A (en) * | 2010-09-30 | 2012-05-09 | 上海张江中药现代制剂技术工程研究中心 | Method for improving melt wall sticking during spray drying of traditional Chinese medicine or food raw material |
| CN101985616A (en) * | 2010-11-11 | 2011-03-16 | 西北工业大学 | Method for preparing immobilized adenosylmethionine synthetase and adenosylmethionine |
| CN101985616B (en) * | 2010-11-11 | 2012-07-04 | 西北工业大学 | Method for preparing immobilized adenosylmethionine synthetase and adenosylmethionine |
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| US9700629B2 (en) | 2011-08-12 | 2017-07-11 | Mitsubishi Gas Chemical Company, Inc. | Composition containing S-adenosyl-L-methionine with excellent storage stability |
| CN103993055A (en) * | 2014-04-22 | 2014-08-20 | 浙江工业大学 | Biosynthesis method of ademetionine |
| CN106191174A (en) * | 2016-08-29 | 2016-12-07 | 北京凯因科技股份有限公司 | A kind of preparation method of enzyme catalysis method S-adenosylmethionine |
| CN113730364A (en) * | 2021-08-03 | 2021-12-03 | 曲阜师范大学 | Preparation method of anti-depression nutritional supplement |
| CN113730364B (en) * | 2021-08-03 | 2022-11-01 | 曲阜师范大学 | Preparation method of anti-depression nutritional supplement |
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