CN1504454A - Method for purifying mevastatin (forebody of pravastatin) from microorganism fermentation liquor - Google Patents
Method for purifying mevastatin (forebody of pravastatin) from microorganism fermentation liquor Download PDFInfo
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- CN1504454A CN1504454A CNA021510687A CN02151068A CN1504454A CN 1504454 A CN1504454 A CN 1504454A CN A021510687 A CNA021510687 A CN A021510687A CN 02151068 A CN02151068 A CN 02151068A CN 1504454 A CN1504454 A CN 1504454A
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- mevastatin
- organic solvent
- ammonium salt
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Abstract
The invention relates to a process for purifying pravastatin precursor mevastatin from biofermentation liquid, characterized in that, after the fermentation liquor containing mevastatin is filtered, it is processed by hydrolytic enzyme and vacuum concentration, then mevastatin is extracted into hydrophobic organic solvent under acidity condition, the organic solvent is then washed by acidic buffer for vacuum concentration to obtain mevastatin with concentration greater than 300g/L, finally hydrophilic organic solvent is added and mixed evenly, ammonium salt or ammonia are added to convert mevastatin into ammonium salt form for separation by crystallization.
Description
Technical field:
The present invention relates to a kind of from microbial fermentation solution the method for purification of pravastatin precursor mevastatin, can be used for research and production in the blood lipid-lowering medicine, belong to the cardiovascular agent technical field.
Background technology:
Cardiovascular disorder has been one of human main causes of death, and its incidence and mortality all is in all disease prostatitis.Industrialized country cardiovascular disorder occurred and has been very popular in the middle of last century, and the cardiovascular disorder death toll has accounted for half of total death toll.From absolute number, China is the big country of cardiovascular disorder.And along with the improving constantly of living standards of the people, the change of mode of life and food habits makes the incidence of various cardiovascular disordeies just be tending towards rising.The main pathological basis that confirms cardiovascular disorder after deliberation is an atherosclerosis, and hyperlipidemia is to cause atherosclerotic primary factor, so lipid lowerers has caused people's attention in the importance aspect the minimizing cardiovascular disease incidence rate.
In the blood lipid-lowering medicine of numerous types, class hydroxy-methyl-glutaryl coenzyme A (HMG-CoA) reductase inhibitor that last century, early eighties grew up, it is statins, because the high efficiency of their reducing cholesterol, suppress cholesterol synthetic high selectivity and hypotoxicity, become in the cardiovascular agent development the most actively, development is the field the most rapidly.Wherein Pravastatin sodium (pravastatin sodium) is a kind of novel statins antilipemic drugs that developed recently gets up, and is evident in efficacy because its high-efficiency low-toxicity, and advantages such as better tolerance are acknowledged as in such medicine the most rising a kind of.
Pravastatin is to be obtained through the transformed bacteria hydroxylation by its precursor mevastatin (compactin), and mevastatin is produced by microbial fermentation, and its structural formula is:
C
23H
36O
6The quality of mevastatin directly influences the purity of the transformation efficiency and the Pravastatin of transformed bacteria, and it also is the main production cost source of Pravastatin simultaneously.Therefore preparation low cost, high-quality mevastatin are to improving the yield and the purity of Pravastatin, and it is significant to reduce production costs.The preparation method of mevastatin adopts the organic solvent extraction fermented liquid mostly at present, and high temperature refluxes to concentrate down and makes mevastatin closed loop, crystallization purifying under the low temperature then.The mevastatin closed loop of this method preparation is toxic to many Pravastatin transformed bacterias, can't directly add, and production energy consumption is big, and the cost height is unfavorable for the scale operation of Pravastatin.
Summary of the invention:
The purpose of this invention is to provide that a kind of production energy consumption is little, cost is low, yield is high, final production can be directly with the method for purification of pravastatin precursor mevastatin from microbial fermentation solution of Pravastatin.
For realizing above purpose, technical scheme of the present invention provide a kind of from microbial fermentation solution the method for purification of pravastatin precursor mevastatin, the fermented liquid that its characteristics are to contain mevastatin after filtering, handle with lytic enzyme, vacuum concentration then, under acidic conditions, mevastatin is extracted in the hydrophobic organic solvent again, after organic solvent washs with weak acidic buffer, vacuum concentration to mevastatin concentration greater than 300g/L, adding hydrophilic organic solvent mixes, add ammonium salt or ammoniacal liquor at last, make mevastatin be converted to the ammonium salts crystallization and separate out.
It may further comprise the steps:
(1). the fermented liquid that contains mevastatin is 4-10 with acid for adjusting pH value after filtering;
(2). add lytic enzyme in fermented liquid, the lytic enzyme final concentration is 0.1%～10%, and regulating the pH value is 6～10, is incubated 10～20 hours down at 40～60 ℃;
(3). enzyme liberating liquid is cooled to be lower than 30 ℃ at 0.08Mpa, 50～90 ℃ of following concentrating under reduced pressure;
(4). it is 3～5 that concentrated solution is regulated the pH value, adds hydrophobic organic solvent, fully mixes 10～20 minutes, and the volume ratio of organic solvent and concentrated solution is 1: 2;
(5). high speed centrifugation separates organic phase and water, and water can add organic solvent and extract 1～4 time again, abandons water, merges organic phase;
(6). organic phase adds the subacidity damping fluid washing that contains 10～20%NaCl, and the volume ratio of organic phase and damping fluid is 2: 1, and the pH of buffer value is 5～7, and high speed centrifugation water phase separated and organic phase are abandoned water, repeated washing organic phase 1～2 time;
(7). organic phase adds anhydrous Na
2S
O4, stirred 10～20 minutes, filter then, at 0.08MPa, 30～90 ℃ of following vacuum concentration to mevastatin content are 250～350g/L;
(8). concentrated solution is cooled to be lower than 30 ℃, adds hydrophilic organic solvent, and organic solvent final concentration 50～95% adds ammonium salt or ammoniacal liquor while stirring, and mevastatin is separated out with the ammonium salts crystallization;
(9). filter and collect mevastatin ammonium salt crystal, use the hydrophilic organic solvent washing crystal, 40-60 ℃ of following vacuum-drying, HPLC detects mevastatin purity greater than 94%, and yield is greater than 80%;
(10). the mevastatin ammonium salt can further improve purity by recrystallization, and the mevastatin ammonium salt purity that finally obtains is greater than 98%.
Microorganism of the present invention can be Penicillium sp., Penicilliumbrevicompactin, Penicilmyces.sp., Trichoderma longibraiatum, Hyphomyceschrisopomus, the mixture of penicillium citrium one of them or they; Described lytic enzyme can be the mixture of subtilisin, trypsinase, stomach en-one of them or they; Described hydrophobic organic solvent can be the mixture of ethyl acetate, butylacetate, isobutyl acetate, tert.-butyl acetate, propyl acetate, ethyl fumarate, fourth ketone, methylene dichloride, chloroform, tetracol phenixin, ethylene dichloride, toluene one of them or they; Described subacidity damping fluid can be KH
2PO
4, NaH
2PO
4The mixture of one of them or they; Described hydrophilic organic solvent can be the mixture of methyl alcohol, ethanol, propyl alcohol, butanols, primary isoamyl alcohol, octanol, acetone one of them or they; Described ammonium salt or ammoniacal liquor, ammonium salt can be NH
4CL, (NH
4)
2SO
4, NH
4Ac, NH
4NO
3The mixture of one of them or they.
Advantage of the present invention is a mevastatin with ammonium salts crystallization purifying at normal temperatures, and production energy consumption is little, and cost is low, the highly purified mevastatin ammonium salt that yield height, purifying obtain, purity is not less than 98%, to Pravastatin transformed bacteria nontoxicity, can be directly used in Pravastatin production.
Embodiment:
The embodiment of a kind of method that purification of pravastatin precursor U.S.A cuts down from microbial fermentation solution that the present invention is proposed describes in further detail below.
Embodiment 1:
(1) .5L ferment filtrate contains mevastatin 10g, and regulating the pH value with 6N HCl is 8.0;
(2). add 20ml subtilisin solution, 55 ℃ of insulations were stirred 12 hours;
(3). enzyme liberating liquid vacuum concentration under 80 ℃, 0.08MPa is cooled to be lower than 30 ℃ to 50ml;
(4). it is 4.0 that concentrated solution is regulated the pH value with 6N HCl, adds the 25ml ethyl acetate, fully mixes 15 minutes;
(5). high speed centrifugation water phase separated and organic phase, water 25ml ethyl acetate extracting twice again, abandon water, merge organic phase;
(6). organic phase contains the 1%KH of 15%NaCl with 40ml
2PO
4Damping fluid, pH value are 6.5, washing twice, and high speed centrifugation water phase separated and organic phase are abandoned water;
(7). organic phase adds the 0.2g anhydrous Na
2SO
4, stirred 15 minutes, filter, filtrate at 0.08Mpa, 50 ℃ of following vacuum concentration to 3ml;
(8). concentrated solution is cooled to be lower than 30 ℃, adds the 12ml anhydrous propanone, and final concentration is 80%, adds people 0.5ml 5N NH while stirring
4Cl solution is separated out the mevastatin ammonium salt crystallization;
(9). filter and collect mevastatin ammonium salt crystal, use the anhydrous propanone washing crystal, 50 ℃ of following vacuum-dryings, obtain mevastatin ammonium salt crystal 8.8g altogether, it is 93.5% that HPLC detects purity;
(10). the further recrystallization of mevastatin ammonium salt crystal, obtain mevastatin ammonium salt crystal 8.3g, it is 98.5% that HPLC detects purity.
Embodiment 2:
(1) .60L ferment filtrate contains mevastatin 175g, and regulating the pH value with 6N HCl is 8.0;
(2). add the 200ml trypsin solution, 55 ℃ of insulations were stirred 15 hours;
(3). enzyme liberating liquid to 5L, is cooled to be lower than 30 ℃ at 0.08MPa, 80 ℃ of vacuum concentration;
(4). it is 4.0 that concentrated solution is regulated the pH value with 6N HCL, adds the 25L butylacetate, fully mixes 15 minutes;
(5). high speed centrifugation water phase separated and organic phase, water is abandoned water with 25L butylacetate re-extract twice, merges organic phase;
(6). organic phase contains the 1%KH of 15%NaCl with 40L
2PO
4Damping fluid, pH value are 6.5, washing twice, and centrifugation water and organic phase are abandoned water;
(7). organic phase adds the 1g anhydrous Na
2SO
4, stirred 15 minutes, filter, filtrate at 0.08Mpa, 50 ℃ of following vacuum concentration to 550ml;
(8). organic phase is cooled to be lower than 30 ℃, adds the 2200ml dehydrated alcohol, and final concentration is 80%, adds 20ml 2N NH while stirring
4Ac solution is separated out the mevastatin ammonium salt crystallization;
(9). filter and collect mevastatin ammonium salt crystal, use the absolute ethanol washing crystal, 50 ℃ of following vacuum-dryings, obtain mevastatin ammonium salt crystal 150g altogether, it is 94.2% that HPLC detects purity;
(10). the further recrystallization of mevastatin ammonium salt crystal, obtain mevastatin ammonium salt crystal 143g, it is 98.5% that HPLC detects purity.
Claims (7)
1. the method for a purification of pravastatin precursor mevastatin from microbial fermentation solution, it is characterized in that, the fermented liquid that contains mevastatin after filtering, handle with lytic enzyme, vacuum concentration then, under acidic conditions, mevastatin is extracted in the hydrophobic organic solvent again, after organic solvent washs with weak acidic buffer, vacuum concentration to mevastatin concentration greater than 300g/L, adding hydrophilic organic solvent mixes, add ammonium salt or ammoniacal liquor at last, make mevastatin be converted to the ammonium salts crystallization and separate out.
It may further comprise the steps:
(1). the fermented liquid that contains mevastatin is 4-10 with acid for adjusting pH value after filtering;
(2). add lytic enzyme in fermented liquid, the lytic enzyme final concentration is 0.1%～10%, and regulating the pH value is 6～10, is incubated 10～20 hours down at 40～60 ℃;
(3). enzyme liberating liquid is cooled to be lower than 30 ℃ at 0.08Mpa, 50～90 ℃ of following concentrating under reduced pressure;
(4). it is 3～5 that concentrated solution is regulated the pH value, adds hydrophobic organic solvent, fully mixes 10～20 minutes, and the volume ratio of organic solvent and concentrated solution is 1: 2;
(5). high speed centrifugation separates organic phase and water, and water can add organic solvent and extract 1～4 time again, abandons water, merges organic phase;
(6). organic phase adds the subacidity damping fluid washing that contains 10～20%NaCl, and the volume ratio of organic phase and damping fluid is 2: 1, and the pH of buffer value is 5～7, and high speed centrifugation water phase separated and organic phase are abandoned water, repeated washing organic phase 1～2 time;
(7). organic phase adds anhydrous Na
2SO
4, stirred 10～20 minutes, filter then, at 0.08MPa, 30～90 ℃ of following vacuum concentration to mevastatin content are 250～350g/L;
(8). concentrated solution is cooled to be lower than 30 ℃, adds hydrophilic organic solvent, and organic solvent final concentration 50～95% adds ammonium salt or ammoniacal liquor while stirring, and mevastatin is separated out with the ammonium salts crystallization;
(9). filter and collect mevastatin ammonium salt crystal, use the hydrophilic organic solvent washing crystal, 40-60 ℃ of following vacuum-drying, HPLC detects mevastatin purity greater than 94%, and yield is greater than 80%;
(10). the mevastatin ammonium salt can further improve purity by recrystallization, and the mevastatin ammonium salt purity that finally obtains is greater than 98%.
2. according to claim 1 a kind of from microbial fermentation solution the method for purification of pravastatin precursor mevastatin, it is characterized in that, described microorganism can be Penicillium sp., Penicilliumbrevicompactin, Penicilmyces sp., Trichoderma longibraiatum, Hyphomyces chrisopomus, the mixture of penicillium citrium one of them or they;
3. according to claim 1 a kind of from microbial fermentation solution the method for purification of pravastatin precursor mevastatin, it is characterized in that described lytic enzyme can be the mixture of subtilisin, trypsinase, stomach en-one of them or they;
4. according to claim 1 a kind of from microbial fermentation solution the method for purification of pravastatin precursor mevastatin, it is characterized in that described hydrophobic organic solvent can be the mixture of ethyl acetate, butylacetate, isobutyl acetate, tert.-butyl acetate, propyl acetate, ethyl fumarate, fourth ketone, methylene dichloride, chloroform, tetracol phenixin, ethylene dichloride, toluene one of them or they;
5. according to claim 1 a kind of from microbial fermentation solution the method for purification of pravastatin precursor mevastatin, it is characterized in that described subacidity damping fluid can be KH
2PO
4, NaH
2PO
4The mixture of one of them or they;
6. according to claim 1 a kind of from microbial fermentation solution the method for purification of pravastatin precursor mevastatin, it is characterized in that described hydrophilic organic solvent can be the mixture of methyl alcohol, ethanol, propyl alcohol, butanols, primary isoamyl alcohol, octanol, acetone one of them or they;
7. according to claim 1 a kind of from microbial fermentation solution the method for purification of pravastatin precursor mevastatin, it is characterized in that described ammonium salt or ammoniacal liquor, ammonium salt can be NH
4CL, (NH
4)
2SO
4, NH
4Ac, NH
4NO
3The mixture of one of them or they.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA021510687A CN1504454A (en) | 2002-12-05 | 2002-12-05 | Method for purifying mevastatin (forebody of pravastatin) from microorganism fermentation liquor |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA021510687A CN1504454A (en) | 2002-12-05 | 2002-12-05 | Method for purifying mevastatin (forebody of pravastatin) from microorganism fermentation liquor |
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| Publication Number | Publication Date |
|---|---|
| CN1504454A true CN1504454A (en) | 2004-06-16 |
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|---|---|---|---|
| CNA021510687A Pending CN1504454A (en) | 2002-12-05 | 2002-12-05 | Method for purifying mevastatin (forebody of pravastatin) from microorganism fermentation liquor |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102093984A (en) * | 2010-11-23 | 2011-06-15 | 清华大学 | Hybrid enzyme P450sca2-BMR (Brown Mid Rib), and coding gene and application thereof |
| CN102746262A (en) * | 2011-04-22 | 2012-10-24 | 广东蓝宝制药有限公司 | Method for purifying Compactin |
| CN102827123A (en) * | 2012-08-02 | 2012-12-19 | 丽珠集团新北江制药股份有限公司 | Separation and extraction process for Mevastatin |
| CN102875505A (en) * | 2012-08-02 | 2013-01-16 | 丽珠集团新北江制药股份有限公司 | Process for extracting and refining Mevastatin |
-
2002
- 2002-12-05 CN CNA021510687A patent/CN1504454A/en active Pending
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102093984A (en) * | 2010-11-23 | 2011-06-15 | 清华大学 | Hybrid enzyme P450sca2-BMR (Brown Mid Rib), and coding gene and application thereof |
| CN102093984B (en) * | 2010-11-23 | 2012-07-04 | 清华大学 | Hybrid enzyme P450sca2-BMR (Brown Mid Rib), and coding gene and application thereof |
| CN102746262A (en) * | 2011-04-22 | 2012-10-24 | 广东蓝宝制药有限公司 | Method for purifying Compactin |
| CN102746262B (en) * | 2011-04-22 | 2014-06-04 | 广东蓝宝制药有限公司 | Method for purifying Compactin |
| CN102827123A (en) * | 2012-08-02 | 2012-12-19 | 丽珠集团新北江制药股份有限公司 | Separation and extraction process for Mevastatin |
| CN102875505A (en) * | 2012-08-02 | 2013-01-16 | 丽珠集团新北江制药股份有限公司 | Process for extracting and refining Mevastatin |
| CN102827123B (en) * | 2012-08-02 | 2015-04-22 | 丽珠集团新北江制药股份有限公司 | Separation and extraction process for Mevastatin |
| CN102875505B (en) * | 2012-08-02 | 2015-08-05 | 丽珠集团新北江制药股份有限公司 | A kind of extraction and purification process of mevastatin |
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