CN101235356B - Culture medium for biologically synthesizing heparinase - Google Patents
Culture medium for biologically synthesizing heparinase Download PDFInfo
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- CN101235356B CN101235356B CN2007100369285A CN200710036928A CN101235356B CN 101235356 B CN101235356 B CN 101235356B CN 2007100369285 A CN2007100369285 A CN 2007100369285A CN 200710036928 A CN200710036928 A CN 200710036928A CN 101235356 B CN101235356 B CN 101235356B
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- heparinase
- substratum
- heparin
- concentration
- flavobacterium
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Abstract
The invention discloses a culture medium which utilizes Flavobacterium heparinum ATCC 13125 as bacterial to biologically synthesize heparinase, which comprises carbon source, nitrogen source, amino acid and heparin. The culture medium is characterized in that nitrogen source is composed by NH4Cl, soybean tryptone and tryptone. In the culture medium of the invention, the thallus yield of flavobacteriun heparinum can reach to 15g/L (wet weight). Enzyme production potency can reach 2,000U/L through adopting the culture medium of the invention to synthesize heparinase, and the production cost is low, the control is simple, and scale production is easy to achieve.
Description
Technical field
The present invention relates to a kind of substratum, specifically, relating to a kind of is bacterial classification with heparin Flavobacterium Flavobacterium heparinum ATCC 13125, the substratum of biologically synthesizing heparinase.
Background technology
Heparinase can rupture specifically and have the different sequences of specific modification on heparin and the heparitin chain, produces different oligose fragment.Heparinase has many important use, comprises the elimination of heparin in the body-internal-circulation, the heparin precision architecture determine that the research of anticoagulant heparin mechanism and heparin structure and function prepares low molecular weight heparin and antitumor drug etc.External report adopt that heparin Flavobacterium fermentative production heparinase can reach 1,475U/L tires, and domestic report is also very low.Wherein, " process optimization of heparinase is produced in the fermentation of heparin Flavobacterium " literary composition discloses, can be by extractum carnis, peptone, yeast powder and corn steep liquor etc. as the synthetic heparinase of the organic nitrogen source of substratum but effect and not obvious, wherein adopt following cultivation prescription ((g/L): glucose 8, ammonium chloride 4, dihydrogen phosphate 6, Histidine 0.75, methionine(Met) 0.75, magnesium chloride 0.5; Trace salt 10
-4Mol/L) yield of enzyme can be improved 66% than original.But in the prior art, because tiring of the synthetic heparinase of heparin Flavobacterium fermenting organism is generally not high, becoming the major obstacle that fermentation method large-scale production heparinase is adopted in restriction, is a problem that presses for solution.
Summary of the invention
The technical issues that need to address of the present invention are to improve tiring of the heparin Flavobacterium synthetic heparinase of fermenting organism.
For this reason, the invention provides a kind of is bacterial classification with heparin Flavobacterium Flavobacterium heparinumATCC 13125, and the substratum of biologically synthesizing heparinase comprises carbon source, nitrogenous source, amino acid and heparin, it is characterized in that, described nitrogenous source is by NH
4Cl, soy peptone and Tryptones are formed.
Unique distinction of the present invention is the prescription of nitrogenous source, promptly adopts NH simultaneously
4Cl, soy peptone and Tryptones.
Described NH
4The weight percent of Cl, soy peptone and Tryptones is 4: 4～5: 2～3, preferred 4: 4.6: 2.3.
Described NH
4Cl concentration in substratum is generally 2～5g/L, and preferred concentration is 3～4g/L.
Described amino acid is L-His and L-Met, when its concentration is 0.25g/L during to 0.75g/L, can obtain to grow preferably and produce the enzyme effect, and preferred concentration is that 0.4g/L is to 0.6g/L.
The concentration of described heparin be 1g/L to 5g/L, preferred 2g/L is to 3g/L.
The bacterial classification of generation heparinase used herein is the heparin Flavobacterium, and bacterium numbering is ATCC13125, and the bacterial strain that is actually used in production can be autonomous mutagenic obtained heparinase superior strain.
Carbon source of the present invention can include but not limited to glucose, semi-lactosi etc., optimally uses glucose, and this carbon source concentration is 1%～7g/L in nutrient solution, and preferred concentration is 3～5g/L.
It is sodium phosphate salt or potassium phosphate salt that the present invention adopts mixed phosphate, and concentration is 3～7g/L, is preferably 4～6g/L; The present invention adds trace element (NaMoO in nutrient solution
3, CuCl
2, FeCl
2, CoCl
2, MnCl
2, CaCl
2), concentration is 10
-3～10
-5M, optimum are 10
-4M.
Air air flow in the biosynthetic process of the present invention is usually at 0.2～1.5vvm, preferably at 0.5～0.8vvm; Temperature is controlled at 23～28 ℃, preferably at 24 ℃～26 ℃; Can record the high enzymatic activity time at 30～36h, preferably at 32～34h.
The present invention's pH value during the fermentation is controlled at 6.0～7.0, is preferably 6.2～6.8.
Centrifugal (10,000r/min 8min) collects thalline to fermentation ends, with certain volume 0.02mol/L, pH7.0 phosphoric acid buffer washing back suspension cell, carries out ultrasonic disruption in ice bath with cell culture fluid.It is centrifugal then that (14,000r/min 30min) collects supernatant and promptly gets acellular thick enzyme.
Adopt culture medium prescription of the present invention to cultivate the heparin Flavobacterium, thalli growth and product enzyme all are better than the substratum that document is reported, thalline output can reach 15g/L (weight in wet base), and the product enzyme is tired can be up to 2,000U/L.
Embodiment
Embodiment 1
Heparin Flavobacterium (Flavobacterium heparinum, ATCC 13125), 750ml shakes bottle, and nutrient solution consists of: (g/L) glucose 3; NH
4Cl 4; Soy peptone 4.6; Tryptones 2.3; L-Met 0.5; L-His 0.5; Mixed phosphate 5; Heparin sodium 2; Trace element 10
-4M (NaMoO
3, CuCl
2, FeCl
2, CoCl
2, MnCl
2, CaCl
2), pH7.0,48 hours ages of seed liquor kind, 10% inoculation, liquid amount 90ml/750ml cultivated 32 hours for 25 ℃.Centrifugal (10,000r/min 8min) collects thalline, with certain volume 0.02mol/L, pH7.0 phosphoric acid buffer washing back suspension cell, carries out ultrasonic disruption in ice bath with cell culture fluid.Broken liquid is centrifugal, and (14,000r/min 30min) collects supernatant and promptly gets acellular crude enzyme liquid.Can obtain the thalline and 1 of 15g/L (weight in wet base), the heparinase that 768U/L tires.
Embodiment 2
Heparin Flavobacterium (Flavobacterium heparinum, ATCC 13125), 750ml shakes bottle, and nutrient solution consists of: (g/L) glucose 3; NH
4Cl 4; Soy peptone 4; Tryptones 2; L-Met 0.25; L-His 0.25; Mixed phosphate 5; Heparin sodium 2; Trace element 10
-5M (NaMoO
3, CuCl
2, FeCl
2, CoCl
2, MnCl
2, CaCl
2), pH7.0,48 hours ages of seed liquor kind, 10% inoculation, liquid amount 90ml/750ml cultivated 32 hours for 25 ℃.Centrifugal (10,000r/min 8min) collects thalline, with certain volume 0.02mol/L, pH7.0 phosphoric acid buffer washing back suspension cell, carries out ultrasonic disruption in ice bath with cell culture fluid.Broken liquid is centrifugal, and (14,000r/min 30min) collects supernatant and promptly gets acellular crude enzyme liquid.Can obtain the thalline of 14g/L (weight in wet base) and about 1, the heparinase that 550U/L tires.
Embodiment 3
Heparin Flavobacterium (Flavobacterium heparinum, ATCC 13125), 750ml shakes bottle, and nutrient solution consists of: (g/L) glucose 3; NH
4Cl 4; Soy peptone 4; Tryptones 3; L-Met 0.25; L-His 0.25; Mixed phosphate 5; Heparin sodium 2; Trace element 10
-5M (NaMoO
3, CuCl
2, FeCl
2, CoCl
2, MnCl
2, CaCl
2), pH7.0,48 hours ages of seed liquor kind, 10% inoculation, liquid amount 90ml/750ml cultivated 32 hours for 25 ℃.Centrifugal (10,000r/min 8min) collects thalline, with certain volume 0.02mol/L, pH7.0 phosphoric acid buffer washing back suspension cell, carries out ultrasonic disruption in ice bath with cell culture fluid.Broken liquid is centrifugal, and (14,000r/min 30min) collects supernatant and promptly gets acellular crude enzyme liquid.Can obtain the thalline of 15g/L (weight in wet base) and about 1, the heparinase that 550U/L tires.
Embodiment 4
Heparin Flavobacterium (Flavobacterium heparinum, ATCC 13125), 750ml shakes bottle, and nutrient solution consists of: (g/L) glucose 5; NH
4Cl 4; Soy peptone 5; Tryptones 2; L-Met 0.75; L-His 0.75; Mixed phosphate 5; Heparin sodium 2; Trace element 10
-4M (NaMoO
3, CuCl
2, FeCl
2, CoCl
2, MnCl
2, CaCl
2), pH7.0,48 hours ages of seed liquor kind, 10% inoculation, liquid amount 90ml/750ml cultivated 32 hours for 25 ℃.Centrifugal (10,000r/min 8min) collects thalline, with certain volume 0.02mol/L, pH7.0 phosphoric acid buffer washing back suspension cell, carries out ultrasonic disruption in ice bath with cell culture fluid.Broken liquid is centrifugal, and (14,000r/min 30min) collects supernatant and promptly gets acellular crude enzyme liquid.Can obtain the thalline and 1 of 13g/L (weight in wet base), the heparinase that 650U/L tires.
Embodiment 5
Heparin Flavobacterium (Flavobacterium heparinum, ATCC 13125), 750ml shakes bottle, and nutrient solution consists of: (g/L) glucose 5; NH
4Cl 4; Soy peptone 5; Tryptones 3; L-Met 0.75L-His 0.75; Mixed phosphate 5; Heparin sodium 2; Trace element 10
-4M (NaMoO
3, CuCl
2, FeCl
2, CoCl
2, MnCl
2, CaCl
2), pH7.0,48 hours ages of seed liquor kind, 10% inoculation, liquid amount 90ml/750ml, 25C cultivated 32 hours.Centrifugal (10,000r/min 8min) collects thalline, with certain volume 0.02mol/L, pH7.0 phosphoric acid buffer washing back suspension cell, carries out ultrasonic disruption in ice bath with cell culture fluid.Broken liquid is centrifugal, and (14,000r/min 30min) collects supernatant and promptly gets acellular crude enzyme liquid.Can obtain the thalline and 1 of 14g/L (weight in wet base), the heparinase that 620U/L tires.
Embodiment 6
Heparin Flavobacterium (Flavobacterium heparinum, ATCC 13125), 750ml shakes bottle, and nutrient solution consists of: (g/L) glucose 5; NH
4Cl 4; Soy peptone 4.5; Tryptones 2; L-Met 0.75; L-His 0.75; Mixed phosphate 5; Heparin sodium 2; Trace element 10
-4M (NaMoO
3, CuCl
2, FeCl
2, CoCl
2, MnCl
2, CaCl
2), pH7.0,48 hours ages of seed liquor kind, 10% inoculation, liquid amount 90ml/750ml cultivated 32 hours for 25 ℃.Centrifugal (10,000r/min 8min) collects thalline, with certain volume 0.02mol/L, pH7.0 phosphoric acid buffer washing back suspension cell, carries out ultrasonic disruption in ice bath with cell culture fluid.Broken liquid is centrifugal, and (14,000r/min 30min) collects supernatant and promptly gets acellular crude enzyme liquid.Can obtain the thalline and 1 of 13g/L (weight in wet base), the heparinase that 600U/L tires.
Embodiment 7
Heparin Flavobacterium (Flavobacterium heparinum, ATCC 13125), 750ml shakes bottle, and nutrient solution consists of: (g/L) glucose 5; NH
4C14 soy peptone 4.5; Tryptones 3; L-Met 0.75; L-His 0.75; Mixed phosphate 5; Heparin sodium 2; Trace element 10
-4M (NaMoO
3, CuCl
2, FeCl
2, CoCl
2, MnCl
2, CaCl
2), pH7.0,48 hours ages of seed liquor kind, 10% inoculation, liquid amount 90ml/750ml cultivated 32 hours for 25 ℃.Centrifugal (10,000r/min 8min) collects thalline, with certain volume 0.02mol/L, pH7.0 phosphoric acid buffer washing back suspension cell, carries out ultrasonic disruption in ice bath with cell culture fluid.Broken liquid is centrifugal, and (14,000r/min 30min) collects supernatant and promptly gets acellular crude enzyme liquid.Can obtain the thalline and 1 of 14.5g/L (weight in wet base), the heparinase that 610U/L tires.
Embodiment 8
Heparin Flavobacterium (Flavobacterium heparinum, ATCC 13125), 750ml shakes bottle, and nutrient solution consists of: (g/L) glucose 5; NH
4Cl 4; Soy peptone 4.5; Tryptones 2.5; L-Met 0.75; L-His 0.75; Mixed phosphate 5; Heparin sodium 2; Trace element 10
-4M (NaMoO
3, CuCl
2, FeCl
2, CoCl
2, MnCl
2, CaCl
2), pH7.0,48 hours ages of seed liquor kind, 10% inoculation, liquid amount 90ml/750ml cultivated 32 hours for 25 ℃.Centrifugal (10,000r/min 8min) collects thalline, with certain volume 0.02mol/L, pH7.0 phosphoric acid buffer washing back suspension cell, carries out ultrasonic disruption in ice bath with cell culture fluid.Broken liquid is centrifugal, and (14,000r/min 30min) collects supernatant and promptly gets acellular crude enzyme liquid.Can obtain the thalline and 1 of 15g/L (weight in wet base), the heparinase that 720U/L tires.
Embodiment 9
Glucose in the use semi-lactosi alternative embodiment 1 is tested, and is surplus with embodiment 1, can obtain the thalline and 2 of 13g/L (weight in wet base), the heparinase that 000U/L tires.
Embodiment 10
Glucose in the use semi-lactosi alternative embodiment 2 is tested, and is surplus with embodiment 1, can obtain the thalline and 1 of 10g/L (weight in wet base), the heparinase that 500U/L tires.
Embodiment 11
Glucose in the use semi-lactosi alternative embodiment 3 is tested, and is surplus with embodiment 1, can obtain the thalline and 1 of 14g/L (weight in wet base), the heparinase that 840U/L tires.
Embodiment 12
Heparin Flavobacterium (Flavobacterium heparinum, ATCC 13125), the 5L fermentor tank, nutrient solution consists of: (g/L) glucose 3; NH
4Cl 4; Soy peptone 4.6; Tryptones 2.3; L-Met 0.5; L-His 0.5; Mixed phosphate 5; Heparin sodium 2; Trace element 10
-4M (NaMoO
3, CuCl
2, FeCl
2, CoCl
2, MnCl
2, CaCl
2), 48 hours ages of seed liquor kind, 10% inoculation, air air flow 0.5vvm; Temperature is controlled at 25 ℃; Fermenting process pH value is controlled at 6.5 to 7.0, and bubble enemy stream adds fermentation time 32h.Centrifugal (10,000r/min 8min) collects thalline to fermentation ends, with certain volume 0.02mol/L, pH7.0 phosphoric acid buffer washing back suspension cell, carries out ultrasonic disruption in ice bath with cell culture fluid.Broken liquid is centrifugal, and (14,000r/min 30min) collects supernatant and promptly gets acellular crude enzyme liquid.Can obtain the thalline of 13～15g/L (weight in wet base) and the heparinase that about 1700U/L tires.
Embodiment 13
Heparin Flavobacterium (Flavobacterium heparinum, ATCC 13125), the 5L fermentor tank, nutrient solution consists of: (g/L) semi-lactosi 3; NH
4Cl 4; Soy peptone 4.6; Tryptones 2.3; L-Met 0.5; L-His 0.5; Mixed phosphate 5; Heparin sodium 2; Trace element 10
-4M (NaMoO
3, CuCl
2, FeCl
2, CoCl
2, MnCl
2, CaCl
2), 48 hours ages of seed liquor kind, 10% inoculation, air air flow 0.5vvm; Temperature is controlled at 25 ℃; Fermenting process pH value is controlled at 6.5 to 7.0, and bubble enemy stream adds fermentation time 32h.Centrifugal (10,000r/min 8min) collects thalline to fermentation ends, with certain volume 0.02mol/L, pH7.0 phosphoric acid buffer washing back suspension cell, carries out ultrasonic disruption in ice bath with cell culture fluid.Broken liquid is centrifugal, and (14,000r/min 30min) collects supernatant and promptly gets acellular crude enzyme liquid.Can obtain the thalline of 11～13g/L (weight in wet base) and about 1, the heparinase that 600U/L tires.
Embodiment 14
Heparin Flavobacterium (Flavobacterium heparinum, ATCC 13125), the 15L fermentor tank, nutrient solution consists of: (g/L) glucose 3; NH
4Cl 4; Soy peptone 4.6; Tryptones 2.3; L-Met 0.5; L-His 0.5; Mixed phosphate 5; Heparin sodium 2; Trace element 10
-4M (NaMoO
3, CuCl
2, FeCl
2, CoCl
2, MnCl
2, CaCl
2), 48 hours ages of seed liquor kind, 10% inoculation, air air flow 0.5vvm; Temperature is controlled at 25 ℃; Fermenting process pH value is controlled at 6.5 to 7.0, and bubble enemy stream adds fermentation time 32h.Centrifugal (10,000r/min 8min) collects thalline to fermentation ends, with certain volume 0.02mol/L, pH7.0 phosphoric acid buffer washing back suspension cell, carries out ultrasonic disruption in ice bath with cell culture fluid.Broken liquid is centrifugal, and (14,000r/min 30min) collects supernatant and promptly gets acellular crude enzyme liquid.Can obtain the thalline and 1,600～1 of 12～15g/L (weight in wet base), the heparinase that 700U/L tires.
Embodiment 15
Heparin Flavobacterium (Flavobacterium heparinum, ATCC 13125), the 15L fermentor tank, nutrient solution consists of: (g/L) semi-lactosi 3; NH
4Cl 4; Soy peptone 4.6; Tryptones 2.3; L-Met 0.5; L-His 0.5; Mixed phosphate 5; Heparin sodium 2; Trace element 10
-4M (NaMoO
3, CuCl
2, FeCl
2, CoCl
2, MnCl
2, CaCl
2), 48 hours ages of seed liquor kind, 10% inoculation, air air flow 0.5vvm; Temperature is controlled at 25 ℃; Fermenting process pH value is controlled at 6.5 to 7.0, and bubble enemy stream adds fermentation time 32h.Centrifugal (10,000r/min 8min) collects thalline to fermentation ends, with certain volume 0.02mol/L, pH7.0 phosphoric acid buffer washing back suspension cell, carries out ultrasonic disruption in ice bath with cell culture fluid.Broken liquid is centrifugal, and (14,000r/min 30min) collects supernatant and promptly gets acellular crude enzyme liquid.Can obtain the thalline and 1,700～1 of 10～14g/L (weight in wet base), the heparinase that 900U/L tires.
Claims (11)
1. one kind is bacterial classification with heparin Flavobacterium Flavobacterium heparinum ATCC 13125, and the substratum of biologically synthesizing heparinase comprises carbon source, nitrogenous source, amino acid and heparin, it is characterized in that, described nitrogenous source is by NH
4Cl, soy peptone and Tryptones are formed, and described NH
4The weight percent of Cl, soy peptone and Tryptones is 4: 4～5: 2～3.
2. substratum as claimed in claim 1 is characterized in that, described NH
4The weight percent of Cl, soy peptone and Tryptones is 4: 4.6: 2.3.
3. as the arbitrary described substratum of claim 1 to 2, it is characterized in that described NH
4The concentration of Cl in substratum is 2～5g/L.
4. substratum as claimed in claim 3 is characterized in that, described NH
4The concentration of Cl in substratum is 3～4g/L.
5. substratum as claimed in claim 1 is characterized in that, described amino acid is L-His and L-Met.
6. substratum as claimed in claim 5 is characterized in that the concentration of described L-His and L-Met is 0.25g/L to 0.75g/L.
7. substratum as claimed in claim 6 is characterized in that the concentration of described L-His and L-Met is 0.4g/L to 0.6g/L.
8. substratum as claimed in claim 1 is characterized in that, the concentration of described heparin is that 1g/L is to 5g/L.
9. substratum as claimed in claim 8 is characterized in that, the concentration of described heparin is that 2g/L is to 3g/L.
10. substratum as claimed in claim 1 is characterized in that, described carbon source is glucose or semi-lactosi, and concentration is 1～7g/L.
11. substratum as claimed in claim 10 is characterized in that, described carbon source is a glucose, and concentration is 3～5g/L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007100369285A CN101235356B (en) | 2007-01-29 | 2007-01-29 | Culture medium for biologically synthesizing heparinase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007100369285A CN101235356B (en) | 2007-01-29 | 2007-01-29 | Culture medium for biologically synthesizing heparinase |
Publications (2)
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|---|---|
| CN101235356A CN101235356A (en) | 2008-08-06 |
| CN101235356B true CN101235356B (en) | 2011-05-11 |
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| CN2007100369285A Expired - Fee Related CN101235356B (en) | 2007-01-29 | 2007-01-29 | Culture medium for biologically synthesizing heparinase |
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Families Citing this family (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101886067B (en) * | 2009-05-11 | 2012-03-07 | 深圳市海普瑞药业股份有限公司 | Method for preparing flavobacterium heparinum heparinase I |
| CN106497897A (en) * | 2016-10-26 | 2017-03-15 | 天津科技大学 | A kind of engineered strain construction method for improving Heparinase I activity |
| CN111073921A (en) * | 2019-12-30 | 2020-04-28 | 东营天东制药有限公司 | Preparation method of heparin sodium |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4396762A (en) * | 1981-08-24 | 1983-08-02 | Massachusetts Institute Of Technology | Heparinase derived anticoagulants |
-
2007
- 2007-01-29 CN CN2007100369285A patent/CN101235356B/en not_active Expired - Fee Related
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4396762A (en) * | 1981-08-24 | 1983-08-02 | Massachusetts Institute Of Technology | Heparinase derived anticoagulants |
Non-Patent Citations (4)
| Title |
|---|
| E. C. CERBELAUD ET AL.Sulfur Regulation of Heparinase and Sulfatases in Flavobacterium heparinum.《APPLIED AND ENVIRONMENTAL MICROBIOLOGY》.1986,第51卷(第3期),640-646. * |
| 马小来等.添加核苷对肝素黄杆菌发酵产肝素酶的影响.《微生物学通报》.2005,第32卷(第5期),92-96. * |
| 马小来等.肝素黄杆菌发酵产肝素酶的工艺优化.《华东理工大学学报(自然科学版)》.2005,第31卷(第4期),446－448. * |
| 高宁国等.肝素酶产生菌的筛选及发酵条件.《微生物学报》.1999,第39卷(第1期),64-67. * |
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Effective date of registration: 20160125 Address after: No. 1559 Zhang Heng Road, China Shanghai Free Trade Zone, Shanghai, 1 2B15 Patentee after: Shanghai faith Biotechnology Co. Ltd. Patentee after: Shanghai Institute of pharmaceutical industry Address before: 200040 Beijing West Road, Shanghai, No. 1320 Patentee before: Shanghai Institute of pharmaceutical industry |
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