CN101659973A - Method for preparing low molecular chondroitin sulfate - Google Patents
Method for preparing low molecular chondroitin sulfate Download PDFInfo
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- CN101659973A CN101659973A CN200810146857A CN200810146857A CN101659973A CN 101659973 A CN101659973 A CN 101659973A CN 200810146857 A CN200810146857 A CN 200810146857A CN 200810146857 A CN200810146857 A CN 200810146857A CN 101659973 A CN101659973 A CN 101659973A
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Abstract
The invention relates to a method for producing low molecular weight chondroitin sulfate, which comprises the following steps of: (1) cracking high molecular weight chondrotn sulfate by using acid mucopolysaccharide hydrolase; (2) immediately filtering formed low molecular chondroitin sulfate by using an ultrafiltration membrane with a cut-off molecular weight of 5000 Da to obtain obtain the produced low molecular chondroitin sulfate. The invention has the advantages of high product recovery rate and high product quality.
Description
Technical field
What the present invention relates to is a kind of method of biological technical field, specifically, is a kind of method for preparing low molecular chondroitin sulfate.
Background technology
Chondroitin sulfate is the acidic mucopolysaccharide material that extracts from animal cartilage; can improve lipase activity and quicken that high triglyceride resolves into lipid acid and clarifies blood in the chylomicron; also having antithrombotic forms; the protection cartilage degradation; prevent senile joint degenerative disease; keep articulation health, effects such as rheumatism.Low-molecular weight chondroitin sulfate has than the better oral absorption rate of natural molecule amount chondroitin sulfate, not only be used to cure the disease in the U.S. as medicine, carry out disease prevention as high-grade nutritious supplementary more, the consumer group spreads to common people from patient, oral dosage is increased to 1.2 grams from 0.8 gram, and demand is very big.Scripture this product
The acid hydrolysis process that low-molecular weight chondroitin sulfate production is commonly used, the poor specificity of this method, desulfurization acid groups rate height, the rate that manufactures a finished product is low, is not suitable for scale operation and uses.
Find that by prior art documents Chinese patent " preparation method of low-molecular weight chondroitin sulfate " (03114375.X).This paper is raw materials for production by utilizing animal cartilage and/or commercially available common macromole chondroitin sulfate, after conventional production process, acid hydrolysis processing, use ultra-filtration membrane special process such as carry out that molecular weight is held back, concentrated, can obtain molecular weight and be lower than 10,000 daltonian chondroitin sulfates.Hold back though this process using ultra-filtration membrane carries out molecular weight, the low-molecular weight chondroitin sulfate by film still is in the acid-reaction environment and is continued degraded, unresolved production efficiency problem.Therefore, be necessary to develop the method for high-level efficiency, low cost production low-molecular weight chondroitin sulfate.
Summary of the invention
The objective of the invention is to overcome the deficiencies in the prior art, a kind of low-molecular weight chondroitin sulfate preparation method is provided, make its cost low, efficient height, efficient production low-molecular weight chondroitin sulfate in enormous quantities.
The present invention is achieved by the following technical solutions, the present invention is with acidic mucopolysaccharide enzymatic lysis high molecular chondroitin sulfate, simultaneously the low-molecular weight chondroitin sulfate that generates is in time removed and prevent its further cracking, thereby obtain the low-molecular weight chondroitin sulfate of high yield.
The present invention includes following steps:
(1) the 50mM Tris/HCl that the acidic mucopolysaccharide enzyme is added contains the 50mM sodium-acetate, and pH 8.0, sulfur acid chrondroitin concentration of substrate 1mg/ml.The molecular weight of raw material sulphuric acid chrondroitin is 10000-60000Da;
The acidic mucopolysaccharide enzyme refers to Unidasa, chondrosulphatase, heparinase cracking stock chondroitin sulfate in the described step (1);
Described raw material sulphuric acid chrondroitin refers to that material purity can be from 36-95% from the chondroitin sulfate of various animal cartilage preparations.
(2) the low-molecular weight chondroitin sulfate molecular weight cut-off that generates of enzymolysis is that the ultra-filtration membrane of 5000Da in time removes, thereby obtains the low-molecular weight chondroitin sulfate product.
In the step of stating (2), reaction conditions is pH7.5-9.0, and sodium-acetate concentration is the damping fluid of 0.05-0.2M, temperature 25-45 ℃.
The present invention had both overcome that the molecular weight distribution of chondroitin sulfate acid hydrolysis process preparing product is wide, and the shortcoming that the product yield is low can prepare low-molecular weight chondroitin sulfate efficiently, in enormous quantities, and yield is more than 80%.
Embodiment
Embodiment 1
Getting the 10g molecular-weight average is the chondroitin sulfate crude product of 30000Da, be dissolved in 1000ml 50mM Tris/HCl and 50mM sodium-acetate buffer, pH 8.0, add streptomyces Unidasa 0.1g, under 30 ℃ of conditions, be incubated 20min, the 2h that circulates in molecular weight cut-off is the ultrafiltration system of 3000Da then collects filtered solution and analyzes chondroitin sulfate molecular-weight average 3600Da, the product yield 8g between the molecular weight distribution 3000-5000Da.
Embodiment 2
Getting the 10g molecular-weight average is the chondroitin sulfate crude product of 30000Da, be dissolved in 1000ml 50mM Tris/HCl and 50mM sodium-acetate buffer, pH 7.5, add proteus vulgaris chondrosulphatase ABC 0.2g, under 30 ℃ of conditions, be incubated 20min, the 2h that circulates in molecular weight cut-off is the ultrafiltration system of 3000Da then collects filtered solution and analyzes chondroitin sulfate molecular-weight average 4200Da, the product yield 7g between the molecular weight distribution 3000-5000Da.
Embodiment 3
Getting the 10g molecular-weight average is the chondroitin sulfate crude product of 30000Da, be dissolved in 1000ml 50mM Tris/HCl and 50 mM sodium-acetate buffers, pH 8.5, add heparin flavobacterium heparinum enzyme 0.1g, under 30 ℃ of conditions, be incubated 20min, the 2h that circulates in molecular weight cut-off is the ultrafiltration system of 3000Da then collects filtered solution and analyzes chondroitin sulfate molecular-weight average 3800Da, the product yield 6g between the molecular weight distribution 3000-5000Da.
Embodiment 4
Getting the 10g molecular-weight average is the chondroitin sulfate crude product of 25000Da, be dissolved in 1000ml 50mM Tris/HCl and 50mM sodium-acetate buffer, pH 8.5, add Testis Caprae seu Ovis Unidasa 0.3g, under 30 ℃ of conditions, be incubated 20min, the 2h that circulates in molecular weight cut-off is the ultrafiltration system of 3000Da then collects filtered solution and analyzes chondroitin sulfate molecular-weight average 4500Da, the product yield 6.5g between the molecular weight distribution 3000-5000Da.
Embodiment 5
Getting the 10g molecular-weight average is the chondroitin sulfate crude product of 30000Da, be dissolved in 1000ml 50mM Tris/HCl and 50mM sodium-acetate buffer, pH 8.0, add bull testis Unidasa 0.1g, under 25 ℃ of conditions, be incubated 20min, the 2h that circulates in molecular weight cut-off is the ultrafiltration system of 3000Da then collects filtered solution and analyzes chondroitin sulfate molecular-weight average 4500Da, the product yield 8.5g between the molecular weight distribution 3000-5000Da.
Embodiment 6
Getting the 10g molecular-weight average is the chondroitin sulfate crude product of 30000Da, be dissolved in 1000ml 50mM Tris/HCl and 50mM sodium-acetate buffer, pH 8.0, add Sphingobacterium sp. heparinase 0.2g, under 35 ℃ of conditions, be incubated 20min, the 2h that circulates in molecular weight cut-off is the ultrafiltration system of 3000Da then collects filtered solution and analyzes chondroitin sulfate molecular-weight average 3500Da, the product yield 6.3g between the molecular weight distribution 3000-5000Da.
Embodiment 7
Getting the 10g molecular-weight average is the chondroitin sulfate crude product of 30000Da, be dissolved in 1000ml 50mM Tris/HCl and 50mM sodium-acetate buffer, pH 8.0, add heparin Flavobacterium chondrosulphatase C 0.25g, under 45 ℃ of conditions, be incubated 20min, the 2h that circulates in molecular weight cut-off is the ultrafiltration system of 3000Da then collects filtered solution and analyzes chondroitin sulfate molecular-weight average 4200Da, the product yield 5.0g between the molecular weight distribution 3000-5000Da.
Embodiment 8
Getting the 10g molecular-weight average is the chondroitin sulfate crude product of 30000Da, be dissolved in 1000ml 50mM Tris/HCl and 50mM sodium-acetate buffer, pH 8.0, add heparin Flavobacterium chondrosulphatase AC 0.5g, under 40 ℃ of conditions, be incubated 20min, the 2h that circulates in molecular weight cut-off is the ultrafiltration system of 3000Da then collects filtered solution and analyzes chondroitin sulfate molecular-weight average 4600Da, the product yield 5.5g between the molecular weight distribution 3000-5000Da.
Embodiment 9
Getting the 10g molecular-weight average is the chondroitin sulfate crude product of 30000Da, be dissolved in 1000ml 50mM Tris/HCl damping fluid, pH 8.0, add heparin Flavobacterium chondrosulphatase B 0.3g, under 30 ℃ of conditions, be incubated 20min, the 2h that circulates in molecular weight cut-off is the ultrafiltration system of 3000Da then collects filtered solution and analyzes chondroitin sulfate molecular-weight average 3600Da, the product yield 6.6g between the molecular weight distribution 3000-5000Da.
Embodiment 10
Getting the 10g molecular-weight average is the chondroitin sulfate crude product of 30000Da, be dissolved in 1000ml 50mM Tris/HCl 100mM sodium-acetate buffer, pH 8.0, add golden yellow Arthrobacter chondrosulphatase AC 0.1g, under 30 ℃ of conditions, be incubated 20min, the 2h that circulates in molecular weight cut-off is the ultrafiltration system of 3000Da then collects filtered solution and analyzes chondroitin sulfate molecular-weight average 4100Da, the product yield 7.6g between the molecular weight distribution 3000-5000Da.
Claims (12)
1. low-molecular weight chondroitin sulfate production method, comprise (1) usefulness acidic mucopolysaccharide lytic enzyme cracking high molecular chondroitin sulfate, (2) are that the ultra-filtration membrane of 3000Da obtains low-molecular weight chondroitin sulfate with the timely filtering of low-molecular weight chondroitin sulfate that generates with molecular weight cut-off.
2. low-molecular weight chondroitin sulfate production method according to claim 1 is characterized in that, used acidic mucopolysaccharide lytic enzyme is a heparin flavobacterium heparinum enzyme.
3. low-molecular weight chondroitin sulfate production method according to claim 1 is characterized in that, used acidic mucopolysaccharide lytic enzyme is proteus vulgaris chondrosulphatase ABC.
4. according to the described low-molecular weight chondroitin sulfate production method of claim 1, it is characterized in that used acidic mucopolysaccharide lytic enzyme is golden yellow Arthrobacter chondrosulphatase AC.
5. according to the described low-molecular weight chondroitin sulfate production method of claim 1, it is characterized in that used acidic mucopolysaccharide lytic enzyme is heparin Flavobacterium chondrosulphatase AC.
6. according to the described low-molecular weight chondroitin sulfate production method of claim 1, it is characterized in that used acidic mucopolysaccharide lytic enzyme is heparin Flavobacterium chondrosulphatase B.
7. according to the described low-molecular weight chondroitin sulfate production method of claim 1, it is characterized in that used acidic mucopolysaccharide lytic enzyme is heparin Flavobacterium chondrosulphatase C.
8. low-molecular weight chondroitin sulfate production method according to claim 1 is characterized in that, used acidic mucopolysaccharide lytic enzyme is the bull testis Unidasa.
9. low-molecular weight chondroitin sulfate production method according to claim 1 is characterized in that, used acidic mucopolysaccharide lytic enzyme is the Testis Caprae seu Ovis Unidasa.
10. low-molecular weight chondroitin sulfate production method according to claim 1 is characterized in that, used acidic mucopolysaccharide lytic enzyme is the Sphingobacterium sp. heparinase.
11. low-molecular weight chondroitin sulfate production method according to claim 1, reaction conditions are pH7.5-9.0, sodium-acetate concentration is the damping fluid of 0.05-0.2M.
12. low-molecular weight chondroitin sulfate production method according to claim 1, temperature of reaction 25-45 ℃.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103103174A (en) * | 2011-11-11 | 2013-05-15 | 清华大学 | Chondrosulphatase AC fusion protein, and coding gene and construction method thereof |
| CN103602711A (en) * | 2013-11-08 | 2014-02-26 | 青岛贝尔特生物科技有限公司 | Preparation method of low-molecular chondroitin sulfate for treating myocarditis |
| WO2015066893A1 (en) * | 2013-11-08 | 2015-05-14 | 青岛贝尔特生物科技有限公司 | Method of preparing low-molecular chondroitin sulfate for treating myocarditis |
| CN104725532A (en) * | 2015-03-23 | 2015-06-24 | 清华大学 | Method for precisely and quantitatively controlling chondroitin sulfate and dermatan sulfate contents of heparin/heparinoid |
| CN105441471A (en) * | 2015-10-28 | 2016-03-30 | 北京电子科技职业学院 | Preparation method and application of chondroitin sulfate ABC enzyme fusion protein |
| CN107460179A (en) * | 2017-09-22 | 2017-12-12 | 青岛农业大学 | A kind of polysaccharide degrading enzyme and its encoding gene and application |
| CN109517777A (en) * | 2018-11-27 | 2019-03-26 | 常熟理工学院 | One plant of withered grass gemma genetic engineering bacterium and its preparing the application in micromolecule hyaluronic acid |
| CN109651529A (en) * | 2018-11-14 | 2019-04-19 | 宁波绿之健药业有限公司 | A kind of chondroitin sulfate preparation method of high bioavilability |
| CN109988739A (en) * | 2018-11-27 | 2019-07-09 | 常熟理工学院 | The method that one-step method efficiently prepares small molecule chondroitin sulfate and micromolecule hyaluronic acid |
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2008
- 2008-08-26 CN CN200810146857A patent/CN101659973A/en active Pending
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103103174B (en) * | 2011-11-11 | 2014-07-23 | 清华大学 | Chondrosulphatase AC fusion protein, and coding gene and construction method thereof |
| CN103103174A (en) * | 2011-11-11 | 2013-05-15 | 清华大学 | Chondrosulphatase AC fusion protein, and coding gene and construction method thereof |
| CN103602711B (en) * | 2013-11-08 | 2016-05-18 | 青岛贝尔特生物科技有限公司 | A kind of preparation method who treats myocarditic low molecular chondroitin sulfate |
| WO2015066893A1 (en) * | 2013-11-08 | 2015-05-14 | 青岛贝尔特生物科技有限公司 | Method of preparing low-molecular chondroitin sulfate for treating myocarditis |
| CN103602711A (en) * | 2013-11-08 | 2014-02-26 | 青岛贝尔特生物科技有限公司 | Preparation method of low-molecular chondroitin sulfate for treating myocarditis |
| CN104725532A (en) * | 2015-03-23 | 2015-06-24 | 清华大学 | Method for precisely and quantitatively controlling chondroitin sulfate and dermatan sulfate contents of heparin/heparinoid |
| CN105441471A (en) * | 2015-10-28 | 2016-03-30 | 北京电子科技职业学院 | Preparation method and application of chondroitin sulfate ABC enzyme fusion protein |
| CN105441471B (en) * | 2015-10-28 | 2018-10-19 | 北京电子科技职业学院 | Chondroitin sulfate A (CSA) BC enzyme fusion proteins preparation method and applications |
| CN107460179A (en) * | 2017-09-22 | 2017-12-12 | 青岛农业大学 | A kind of polysaccharide degrading enzyme and its encoding gene and application |
| CN109651529A (en) * | 2018-11-14 | 2019-04-19 | 宁波绿之健药业有限公司 | A kind of chondroitin sulfate preparation method of high bioavilability |
| CN109651529B (en) * | 2018-11-14 | 2020-10-30 | 宁波绿之健药业有限公司 | Preparation method of chondroitin sulfate with high bioavailability |
| CN109517777A (en) * | 2018-11-27 | 2019-03-26 | 常熟理工学院 | One plant of withered grass gemma genetic engineering bacterium and its preparing the application in micromolecule hyaluronic acid |
| CN109988739A (en) * | 2018-11-27 | 2019-07-09 | 常熟理工学院 | The method that one-step method efficiently prepares small molecule chondroitin sulfate and micromolecule hyaluronic acid |
| CN109517777B (en) * | 2018-11-27 | 2021-07-09 | 常熟理工学院 | Bacillus subtilis genetically engineered bacterium and application thereof in preparation of micromolecular hyaluronic acid |
| CN109988739B (en) * | 2018-11-27 | 2021-10-19 | 常熟理工学院 | Method for efficiently preparing micromolecular chondroitin sulfate and micromolecular hyaluronic acid by one-step method |
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Open date: 20100303 |