CN1070534C - Method for separating and refining polyhydroxy fatty acid ester in bacteria cell from bacteria - Google Patents
Method for separating and refining polyhydroxy fatty acid ester in bacteria cell from bacteria Download PDFInfo
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- CN1070534C CN1070534C CN98100266A CN98100266A CN1070534C CN 1070534 C CN1070534 C CN 1070534C CN 98100266 A CN98100266 A CN 98100266A CN 98100266 A CN98100266 A CN 98100266A CN 1070534 C CN1070534 C CN 1070534C
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- Prior art keywords
- polyhydroxyalkanoate
- fatty acid
- bacterium
- particle
- separation
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Links
- 241000894006 Bacteria Species 0.000 title claims abstract description 28
- 238000000034 method Methods 0.000 title claims description 18
- 235000014113 dietary fatty acids Nutrition 0.000 title claims description 14
- 239000000194 fatty acid Substances 0.000 title claims description 14
- 229930195729 fatty acid Natural products 0.000 title claims description 14
- -1 fatty acid ester Chemical class 0.000 title claims description 14
- 238000007670 refining Methods 0.000 title 1
- 239000002245 particle Substances 0.000 claims abstract description 18
- 239000005014 poly(hydroxyalkanoate) Substances 0.000 claims description 46
- 229920000903 polyhydroxyalkanoate Polymers 0.000 claims description 39
- 239000003945 anionic surfactant Substances 0.000 claims description 25
- FRXSZNDVFUDTIR-UHFFFAOYSA-N 6-methoxy-1,2,3,4-tetrahydroquinoline Chemical compound N1CCCC2=CC(OC)=CC=C21 FRXSZNDVFUDTIR-UHFFFAOYSA-N 0.000 claims description 22
- 238000005406 washing Methods 0.000 claims description 13
- 238000000926 separation method Methods 0.000 claims description 12
- 238000000605 extraction Methods 0.000 claims description 10
- 238000000746 purification Methods 0.000 claims description 9
- 239000000243 solution Substances 0.000 claims description 9
- 238000006068 polycondensation reaction Methods 0.000 claims description 7
- 239000007787 solid Substances 0.000 claims description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 5
- 239000003637 basic solution Substances 0.000 claims description 3
- 239000012535 impurity Substances 0.000 claims description 3
- 238000001035 drying Methods 0.000 claims description 2
- 238000001914 filtration Methods 0.000 claims description 2
- 239000007790 solid phase Substances 0.000 claims description 2
- 108091005658 Basic proteases Proteins 0.000 claims 1
- 108091005804 Peptidases Proteins 0.000 abstract description 22
- 102000035195 Peptidases Human genes 0.000 abstract description 11
- 238000006243 chemical reaction Methods 0.000 abstract description 11
- 238000004519 manufacturing process Methods 0.000 abstract description 9
- 238000012545 processing Methods 0.000 abstract description 6
- 238000000855 fermentation Methods 0.000 abstract description 5
- 230000004151 fermentation Effects 0.000 abstract description 5
- 239000002994 raw material Substances 0.000 abstract description 4
- 238000012805 post-processing Methods 0.000 abstract description 2
- 125000000129 anionic group Chemical group 0.000 abstract 1
- 235000019833 protease Nutrition 0.000 abstract 1
- 239000004094 surface-active agent Substances 0.000 abstract 1
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 15
- 238000001556 precipitation Methods 0.000 description 15
- 210000004027 cell Anatomy 0.000 description 12
- 239000004365 Protease Substances 0.000 description 11
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 description 11
- 239000008399 tap water Substances 0.000 description 11
- 235000020679 tap water Nutrition 0.000 description 11
- 230000001143 conditioned effect Effects 0.000 description 6
- 239000007788 liquid Substances 0.000 description 6
- 102000004190 Enzymes Human genes 0.000 description 5
- 108090000790 Enzymes Proteins 0.000 description 5
- 241000588724 Escherichia coli Species 0.000 description 5
- 230000001580 bacterial effect Effects 0.000 description 5
- DAJSVUQLFFJUSX-UHFFFAOYSA-M sodium;dodecane-1-sulfonate Chemical compound [Na+].CCCCCCCCCCCCS([O-])(=O)=O DAJSVUQLFFJUSX-UHFFFAOYSA-M 0.000 description 4
- 241000588986 Alcaligenes Species 0.000 description 3
- 230000000977 initiatory effect Effects 0.000 description 3
- 239000000203 mixture Substances 0.000 description 3
- 239000011734 sodium Substances 0.000 description 3
- 241000193033 Azohydromonas lata Species 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 2
- 239000013543 active substance Substances 0.000 description 2
- GVGUFUZHNYFZLC-UHFFFAOYSA-N dodecyl benzenesulfonate;sodium Chemical compound [Na].CCCCCCCCCCCCOS(=O)(=O)C1=CC=CC=C1 GVGUFUZHNYFZLC-UHFFFAOYSA-N 0.000 description 2
- 238000009434 installation Methods 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 229940080264 sodium dodecylbenzenesulfonate Drugs 0.000 description 2
- 101710093543 Probable non-specific lipid-transfer protein Proteins 0.000 description 1
- 239000002585 base Substances 0.000 description 1
- 229920000704 biodegradable plastic Polymers 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 210000002421 cell wall Anatomy 0.000 description 1
- 230000000694 effects Effects 0.000 description 1
- 238000003912 environmental pollution Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000012262 fermentative production Methods 0.000 description 1
- 238000010353 genetic engineering Methods 0.000 description 1
- 159000000011 group IA salts Chemical class 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 230000003834 intracellular effect Effects 0.000 description 1
- 150000002500 ions Chemical class 0.000 description 1
- 238000005215 recombination Methods 0.000 description 1
- 230000006798 recombination Effects 0.000 description 1
- 238000003756 stirring Methods 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 229920001169 thermoplastic Polymers 0.000 description 1
- 239000004416 thermosoftening plastic Substances 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The present invention belongs to the technical field of biological engineering downstream post processing. The present invention comprises: an anionic surface active agent is used for processing and fermentation under an alkaline condition to obtain bacterium mycelium, and ploy-hydroxy alkanoate particles contained in the bacterium mycelium are centrifugally extracted; ploy-hydroxy alkanoate products obtained in the previous step are processed by proteinase; the obtained ploy-hydroxy alkanoate products are collected and dried. The present invention has the advantages of cheap raw material utilization, milden reaction condition and little production equipment investment, and the present invention is suitable for the requirements of large-scaleindustrialization production and greatly reduces the production cost of ploy-hydroxy alkanoate.
Description
The invention belongs to post-processing technology field, biotechnology downstream.
Polyhydroxyalkanoate (Poly-β-Hydroxyalkanoates is called for short PHA) is an inclusion in a kind of bacterium born of the same parents, has the characteristic of thermoplastics.Simultaneously because biodegradability and bio-compatible that it had make it that application potential be arranged in every respect.But owing to its form with inclusion is present in the bacterial body, complicated component makes its purification extremely difficult.Existing downstream aftertreatment purifying technique, perhaps utilize organic solvent (as chloroform, methylene dichloride etc.) to carry out extracting, the enzyme of perhaps taking heat to be used in combination the multiple pricing costliness decomposes non-polyhydroxyalkanoate composition in the cell to reach the purpose of purification.Complex manufacturing, the facility investment height, the raw materials cost costliness causes the polyhydroxyalkanoate product price too high, makes this coming biodegradable plastic be difficult to promote the use of.The present patent application people found through experiments, earlier with negatively charged ion alkalescence solution-treated bacterium thalline, separation and Extraction polyhydroxyalkanoate particle is further handled with proteolytic enzyme and is removed residual albumen impurity on the degranulation, can obtain highly purified polyhydroxyalkanoate product.Because this technological reaction mild condition does not have particular requirement to equipment, the raw material cheapness is fit to the requirement that large-scale industrialization is produced, and greatly reduces the production cost of polyhydroxyalkanoate.
The objective of the invention is to propose to use cheap anion surfactant and a spot of proteolytic enzyme, utilize the existing installation of general fermentation plant, from the tunning of bacterium, extract purifying polyhydroxyalkanoate product, product cost is greatly reduced.
The present invention proposes a kind of method of separating interior polyhydroxyalkanoate in the purification bacterium born of the same parents from the bacterium thalline, it is characterized in that may further comprise the steps: 1) earlier with anion surfactant basic solution agitator treating bacterium thalline; The result makes the bacterium broken wall and its cell wall is degraded to small shreds, discharges entocyte simultaneously.2) the poly-hydroxy fatty acid fat particle of separation and Extraction solid phase; Remove most of non-polyhydroxyalkanoate (PHA) composition that produces in the fermentation; 3) use the said particle of the further agitator treating of enzyme solution of basic protein again, remove residual albumen impurity, further improve its purity and reduce its albumen foreign matter content greatly, make it satisfy the further requirement of processing; 4) separation and Extraction purifying solid polycondensation hydroxy fatty acid fat particle; 5) drying obtains high purity powdered form shape poly-hydroxy fatty acid fat prod.The pH value of the said basic solution of the present invention is in the 9-13 scope.Wash temperature can be in 20-100 ℃ of scope.Said separating and extracting method can be the throw out in centrifugal or the filtering separation collection washings.The present invention also further comprises between said second, third step and washes said particle with water, further removes non-polyhydroxyalkanoate composition and adds tensio-active agent with institute, and separation and Extraction solid polycondensation hydroxy fatty acid fat particle.Between said the 4th, the 5th step, also can comprise washing said particle with water, and the solid polycondensation hydroxy fatty acid fat particle that is further purified of separation and Extraction.
The suitable process object of technology of the present invention is extensive, can handle the multiple bacterium of polyhydroxyalkanoate and the tunning of variant and genetic engineering recombination strain thereof of containing, and is not high to the polyhydroxyalkanoate content requirement of thalline.
The present invention can be according to the process object difference, in the anionic surfactant treatment process, select corresponding anion surfactant kind, suitable treatment condition (for example the concentration of the processing of solution, the amount ratio of tensio-active agent, the pH value condition of processing and the temperature of processing).The alkaline condition of anionic surfactant solution can obtain by adding various bases and alkaline salt.The present invention also can select suitable reaction conditions (as activity, pH value, temperature and time) according to the characteristic of use proteolytic enzyme, produces purity to improve, and reduces production costs.
The present invention compares with existing purifying technique, has following characteristics: (1) not with an organic solvent, facility investment is few, environmental pollution is little; (2) the anion surfactant low price of Shi Yonging; (3) proteolytic enzyme of Shi Yonging is more cheap relatively, and consumption seldom, and raw materials cost is low; (4) product purity height; (5) can adopt the existing installation of common fermentation factory to produce.
Polyhydroxyalkanoate product by explained hereafter of the present invention has the purity height, and protein content is little, the characteristics that molecular weight product is high.Being fit to further processing uses.
Embodiment one: extract polyhydroxyalkanoate from the thalline of vickers nitrogen-fixing bacteria (Azotobactor vinelandii).
Bacterial classification: vickers nitrogen-fixing bacteria UWD (Azotobactor vinelandii UWD)
Polyhydroxyalkanoate in the dry cell weight (PHA) content: 50%;
Anion surfactant: sodium laurylsulfonate;
Anion surfactant washings concentration: 0.4-0.7% (w/v);
Anion surfactant washings pH value: 11;
Anion surfactant wash temperature: 40 ℃;
Anion surfactant washing time: 30 minutes;
The anion surfactant consumption becomes the ratio of branch: 1/10 (w/w) with non-polyhydroxyalkanoate in the dry cell weight;
Proteolytic enzyme: (it is about 50 that enzyme is lived, 000Unit/ml) for 2709 Sumizyme MP liquid;
Protease treatment pH value: 11;
Protease treatment temperature: 40 ℃;
The protease treatment time: 30 minutes;
The proteolytic enzyme consumption becomes the ratio of branch with non-polyhydroxyalkanoate in the initiating cell dry weight: 5, and 000Unit/g;
Reactive system alkaline conditioner: NaOH.
The technological process of production of present embodiment is as follows:
By fermentation and the centrifugal vickers nitrogen-fixing bacteria UWD thalline that obtains containing polyhydroxyalkanoate (PHA) about 50%.Get about 1000g (equivalent dry weight) thalline, add sodium laurylsulfonate 50g, tap water 10L makes it to remain on about 11 with NaOH solution conditioned reaction system pH.Keep temperature agitator treating 30 minutes more than 40 ℃.Add 5L tap water and 50ml Sumizyme MP liquid again, the conditioned reaction system pH makes it to remain on about 11.Keep temperature to stir 30 minutes for about 40 ℃, centrifugal collecting precipitation adds the tap water agitator treating, the recentrifuge collecting precipitation.The oven dry precipitation promptly gets Powdered high purity polyhydroxyalkanoate (PHA) product.
The polyhydroxyalkanoate that makes (PHA) product purity is greater than 96%, and protein content is less than 0.5%.Has good workability.Embodiment two: extract poly-hydroxyl from the thalline of genetically engineered recombinant escherichia coli (E.coli) with PHA synthesis capability
Fatty acid ester.
Bacterial classification: genetically engineered recombinant escherichia coli (E.coli)
Polyhydroxyalkanoate in the dry cell weight (PHA) content: 70%;
Anion surfactant: sodium laurylsulfonate;
Anion surfactant wash concentration: 0.65-0.90% (w/v);
Anion surfactant washing pH value: 11;
Anion surfactant wash temperature: 60 ℃;
Anion surfactant washing time: 30 minutes;
The anion surfactant consumption becomes the ratio of branch: 1/8 (w/w) with non-polyhydroxyalkanoate in the dry cell weight;
Proteolytic enzyme: 2709 Sumizyme MP liquid (it is about 50 that enzyme is lived, and 000Unit/ml, optimum reaction conditions are 40 ℃ of temperature, pH value 11);
Protease treatment pH value: 11;
Protease treatment temperature: 40 ℃;
The protease treatment time: 1 hour;
The proteolytic enzyme consumption becomes the ratio of branch with non-polyhydroxyalkanoate in the initiating cell dry weight: 3, and 000Unit/g;
Reactive system alkaline conditioner: Na
2CO
3
The technological process of production of present embodiment is as follows:
Obtain the thalline that polyhydroxyalkanoate (PHA) content accounts for dry cell weight 70%, centrifugal collection thalline by fermentative production bacterial classification (genetically engineered recombinant escherichia coli).Get about 500g (equivalent dry weight) thalline, add sodium laurylsulfonate 18.75g, tap water 2.5L adds Na in solution
2CO
3The conditioned reaction system pH makes it to remain on about 11.Keep temperature agitator treating 30 minutes more than 60 ℃.Centrifugal collecting precipitation added the tap water agitator treating 5 minutes, the recentrifuge collecting precipitation.Add Sumizyme MP liquid 9ml in precipitation, tap water 1L adds Na
2CO
3The conditioned reaction system pH makes it to remain on about 11.Kept 40 ℃ of left and right sides agitator treatings of temperature 1 hour, centrifugal collecting precipitation added the tap water agitator treating 5 minutes, the recentrifuge collecting precipitation.The oven dry precipitation promptly gets Powdered high purity polyhydroxyalkanoate (PHA) product.
The polyhydroxyalkanoate that makes (PHA) product purity is greater than 97%, and protein content is less than 0.2%.Has good workability.Embodiment three: extract polyhydroxyalkanoate from the thalline of huge Alcaligenes (Alcaligenes latus).
Bacterial classification: huge Alcaligenes DSM (Alcaligenes latus DSM)
Polyhydroxyalkanoate in the dry cell weight (PHA) content: 60%;
Anion surfactant: Sodium dodecylbenzene sulfonate;
Anion surfactant wash concentration: 0.5% (w/v);
Anion surfactant washing pH value: 12;
Anion surfactant wash temperature: 80 ℃;
Anion surfactant washing time: 1 hour;
The anion surfactant consumption becomes the ratio of branch: 1/5 (w/w) with non-polyhydroxyalkanoate in the dry cell weight;
Proteolytic enzyme: high-temperature alkaline liquid of protease (it is about 100 that enzyme is lived, and 000Unit/ml, optimum reaction conditions are 60 ℃ of temperature, pH value 10);
Protease treatment pH value: 10;
Protease treatment temperature: 60 ℃; The protease treatment time: 1 hour;
The proteolytic enzyme consumption becomes the ratio of branch with non-polyhydroxyalkanoate in the initiating cell dry weight: 4, and 000Unit/g;
Reactive system alkaline conditioner: NaOH.
The technological process of production of present embodiment is as follows:
Obtain the thalline that polyhydroxyalkanoate (PHA) content accounts for dry cell weight 60%, centrifugal collection thalline by the huge Alcaligenes DSM that ferments.Get about 500g (equivalent dry weight) thalline, add Sodium dodecylbenzene sulfonate 40g, tap water 8L makes it to remain on about 12 with NaOH solution conditioned reaction system pH.Keep temperature agitator treating 1 hour more than 80 ℃.Centrifugal collecting precipitation added the tap water agitator treating 10 minutes, the recentrifuge collecting precipitation.Add high-temperature alkaline liquid of protease 8ml in precipitation, tap water 4L makes it to remain on about 10 with NaOH solution conditioned reaction system pH.Kept 60 ℃ of left and right sides agitator treatings of temperature 1 hour, centrifugal collecting precipitation added the tap water agitator treating 10 minutes, the recentrifuge collecting precipitation.The oven dry precipitation promptly gets Powdered high purity polyhydroxyalkanoate (PHA) product.
The polyhydroxyalkanoate that makes (PHA) product purity is greater than 94%, and protein content is less than 0.6%.Has good workability.
Claims (5)
- One kind from the bacterium thalline, separate in the purification bacterium born of the same parents in the method for polyhydroxyalkanoate, it is characterized in that may further comprise the steps: 1) earlier with anion surfactant basic solution agitator treating bacterium thalline, wash temperature is 20-100 ℃; 2) the poly-hydroxy fatty acid fat particle of separation and Extraction solid phase; 3) be the said particle of the further agitator treating of alkaline protease solution of 9-13 with the pH value again, remove residual albumen impurity; 4) the solid polycondensation hydroxy fatty acid fat particle of separation and Extraction purifying; 5) drying obtains Powdered poly-hydroxy fatty acid fat prod.
- 2. as claimed in claim 1 a kind of from the bacterium thalline, separate in the purification bacterium born of the same parents in the method for polyhydroxyalkanoate, it is characterized in that said separating and extracting method is centrifugal or filtering separation is collected throw out in the washings.
- 3. a kind of method of from the bacterium thalline, separating interior polyhydroxyalkanoate in the purification bacterium born of the same parents as claimed in claim 1 or 2, it is characterized in that also further between said second, third step, comprising washing said particle with water, and separation and Extraction solid polycondensation hydroxy fatty acid fat particle.
- 4. a kind of method of from the bacterium thalline, separating interior polyhydroxyalkanoate in the purification bacterium born of the same parents as claimed in claim 1 or 2, it is characterized in that between said the 4th, the 5th step, comprise washing said particle with water, and the solid polycondensation hydroxy fatty acid fat particle that is further purified of separation and Extraction.
- 5, a kind of method of from the bacterium thalline, separating interior polyhydroxyalkanoate in the purification bacterium born of the same parents as claimed in claim 3, it is characterized in that between said the 4th, the 5th step, comprise washing said particle with water, and the solid polycondensation hydroxy fatty acid fat particle that is further purified of separation and Extraction.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN98100266A CN1070534C (en) | 1998-01-23 | 1998-01-23 | Method for separating and refining polyhydroxy fatty acid ester in bacteria cell from bacteria |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN98100266A CN1070534C (en) | 1998-01-23 | 1998-01-23 | Method for separating and refining polyhydroxy fatty acid ester in bacteria cell from bacteria |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1190674A CN1190674A (en) | 1998-08-19 |
| CN1070534C true CN1070534C (en) | 2001-09-05 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN98100266A Expired - Fee Related CN1070534C (en) | 1998-01-23 | 1998-01-23 | Method for separating and refining polyhydroxy fatty acid ester in bacteria cell from bacteria |
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Families Citing this family (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| AU2003292860B2 (en) | 2003-12-19 | 2010-03-04 | Tianan Biologic Material Co., Ltd. Ningbo | A method for Separating, Extracting and Purifying Poly- beta -hydroxyalkanoates (PHA's) directly from Bacterial Fermentation Broth |
| CA2579721C (en) * | 2004-09-13 | 2012-11-27 | Metabolix, Inc. | Single solvent polymer extraction methods |
| CN109517156A (en) * | 2019-01-02 | 2019-03-26 | 清华大学 | A kind of purification process of polyhydroxyalkanoate |
| CN112813112B (en) * | 2021-01-07 | 2022-11-15 | 上海碧州环保能源科技有限公司 | Non-methanation process with PHA production as guide |
| CN115058461B (en) * | 2022-06-20 | 2024-05-28 | 宁波天安生物材料有限公司 | Method for directly separating and purifying polyhydroxyalkanoate from fermentation broth |
| CN115807044B (en) * | 2022-11-09 | 2023-10-13 | 华南理工大学 | Method for efficiently extracting and purifying high-purity polyhydroxyalkanoate |
| CN115786411B (en) * | 2023-01-09 | 2023-06-23 | 北京微构工场生物技术有限公司 | Extraction method of polyhydroxyalkanoate |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1104683A (en) * | 1993-12-29 | 1995-07-05 | 中国科学院成都生物研究所 | Preparation of betal-polyhydroxybutyrate |
| CN1152943A (en) * | 1994-06-01 | 1997-06-25 | 普罗克特和甘保尔公司 | Process for recovering polyhydroxyalkanoates using centrifugal fractionation |
-
1998
- 1998-01-23 CN CN98100266A patent/CN1070534C/en not_active Expired - Fee Related
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1104683A (en) * | 1993-12-29 | 1995-07-05 | 中国科学院成都生物研究所 | Preparation of betal-polyhydroxybutyrate |
| CN1152943A (en) * | 1994-06-01 | 1997-06-25 | 普罗克特和甘保尔公司 | Process for recovering polyhydroxyalkanoates using centrifugal fractionation |
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| Publication number | Publication date |
|---|---|
| CN1190674A (en) | 1998-08-19 |
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