CN102533687A - Method for efficiently expressing recombinant human superoxide dismutase by escherichia coli - Google Patents
Method for efficiently expressing recombinant human superoxide dismutase by escherichia coli Download PDFInfo
- Publication number
- CN102533687A CN102533687A CN2012100425958A CN201210042595A CN102533687A CN 102533687 A CN102533687 A CN 102533687A CN 2012100425958 A CN2012100425958 A CN 2012100425958A CN 201210042595 A CN201210042595 A CN 201210042595A CN 102533687 A CN102533687 A CN 102533687A
- Authority
- CN
- China
- Prior art keywords
- dismutase
- escherichia coli
- superoxide
- liquid
- superoxide dismutase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Landscapes
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a method for efficiently expressing recombinant human superoxide dismutase by escherichia coli. The escherichia coli is used as a strain; the raw materials include yeast powder, peptone, glucose, KH2PO4, (NH4)2HPO4, MgSO4.7H2O, citric acid and ferric citrate; and the human superoxide dismutase is produced through liquid deep fermentation. The method disclosed by the invention has the beneficial effects that: through an improved fermentation way, the superoxide dismutase powder is obtained by two-level seed culture and spray drying; the fermentation technology is simplified; the equipment and time are saved; the high-yield fermentation of the superoxide dismutase is realized; and the method is applicable to large-scale industrial production, greatly lowers the production cost of the superoxide dismutase and has remarkable social and economical benefits.
Description
Technical field
The invention provides a kind of method of escherichia coli high-level expression recombination human source superoxide-dismutase, belong to the fermentation engineering field.
Background technology
Superoxide-dismutase (SOD) is to have the biological intravital superoxide ion of single-minded removing; The oxyradical of balance body, protection human body cell are avoided environmental factorss such as toxic chemical substance, the width of cloth are penetrated, ultraviolet and are destroyed, and the SOD enzyme is anti-ageing; Radioresistance; Anti-inflammatory suppress tumour and cancer, and there is important effect the treatment aspect of autoimmune disease; The SOD enzyme is widely used in industries such as food-drink, makeup as a kind of health care pharmaceutical prod at present, has boundless market.Domestic SOD extracts from animal blood mostly at present, owing to contain various viruses that are difficult to eliminate and external source pollution, European Union promulgated a decree in 1999, forbade that the SOD that from animal blood, extracts is used for the mankind; And the method for from plant, extracting SOD often receives raw-material restriction, and cost is higher; Therefore developing other safer, more economical extractions or compound method, to replace traditional SOD working method will be a trend.Characteristics such as it is simple that the technology of utilizing engineered microbes to prepare SOD has technology, and production cost is low, and is environmentally friendly have become the developing direction of SOD industrialization; At present countries such as Japan, the Canada new biotechnology such as employing recombination that also begins one's study is synthesized SOD; Domestic existing enterprise utilizes biotechnology to produce SOD, but output far can not satisfy the growing market requirement, and Along with people's is familiar with gradually superoxide-dismutase (SOD) and is used, and superoxide-dismutase market possesses opportunity preferably.
Summary of the invention
The present invention is in order to provide a kind of recombination bacillus coli to efficiently express the method for people source FE-SOD.
Technical scheme of the present invention is achieved in that
The present invention efficiently expresses the method for people source FE-SOD for a kind of recombination bacillus coli; Utilize intestinal bacteria as bacterial classification, with yeast powder, peptone, glucose, KHPO
4, (NH
4)
2HPO
4, MgSO
47H
2O, Hydrocerol A, ironic citrate are raw material, carry out deep liquid time fermentative prodn superoxide-dismutase, and fermented liquid carries out solid-liquid separation through tubular-bowl centrifuge, obtains thalline and fermented liquid supernatant liquid; Resulting thalline passes through the clarifixator fragmentation again, thermal treatment, and centrifugal collection supernatant, ultrafiltration, micro-filtration and spraying drying obtain powdery superoxide-dismutase product; Specifically comprise the steps:
1, bacterial classification and seed liquor enlarged culturing
1.1 the used bacterial classification of the present invention is e. coli bl21 (hSOD-pET-28a) recombinant bacterial strain.
1.2 (pH 6.8～7.2 in the 3L triangular flask 600mL first order seed substratum under aseptic condition, to change intestinal bacteria test tube slant bacterial classification over to prepared be contained in; Sterilising conditions: 121 ℃ of sterilization 30min); 250rpm, 37 ± 1 ℃ cultivate 12～14 hours subsequent use.
1.3 bacterial classification is carried out enlarged culturing with the 100L seeding tank
With the yeast powder 480g of technical grade, peptone 900g, 1M phosphoric acid buffer (pH=6.8) 6L, vitamin H 24mg, glucose 600g drop into respectively in the 100L seeding tank, with the tap water constant volume to 60L; Stir; Directly sterilize with steam then, 115 degree insulations 30 minutes are cooled to 37 degree again, insert the 600mL first order seed bacterium liquid of 1.2 gained set by step; Air flow 0.6: 1; 37 ± 1 ℃ of temperature; Cultivate 6h under the condition of pressure tank 0.03～0.05MPa, get the intestinal bacteria secondary seed solution of enlarged culturing.
2, produce superoxide-dismutase with the 1000L ferment tank
With the glucose 5kg of technical grade, the KH of 6.65kg
2PO
4, 2kg (NH
4)
2The MgSO of HPO4,6kg
47H
2O, Hydrocerol A 1.5kg, ironic citrate (III) 0.05kg drop into respectively in the 1000L fermentor tank,, stir to 500L with the tap water constant volume; Directly sterilize with steam then, 115 degree insulations 30 minutes are cooled to 37 degree again, and the sterilization back is by 28%NH
4OH adjusts pH to 7.0, adds liquid microelement (KI of 2.5mg/L, the MnCl of 1.5mg/L of 0.8L again
24H
2The CuCl of O, 1.5mg/L
22H
2The H of O, 3.0mg/L
3BO
3,2.5mg/L Na
2MoO
42H
2Zn (the CH of O, 1.3mg/L
3COO)
22H
2O hydrochloric acid sulphur ammonium 4.5mg/L); The CuSO of 100g
45H
2O, 50g ZnSO
4Insert 60L first order seed nutrient solution; Under the condition of air quantity 1: 0.6～0.8,37 ± 1 ℃ of temperature, pressure tank 0.03～0.05MPa, cultivate, mend glucose after for some time, to OD
600Reach about 40 and begin to induce with IPTG, induce 2 hours after, stream adds micro-CuSO in fermentor tank
45H
2O and ZnSO
4, coinduction 10 hours obtains fermented liquid, utilizes tubular-bowl centrifuge that fermented liquid is carried out solid-liquid separation, and resulting thalline passes through the clarifixator fragmentation again, thermal treatment, centrifugal collection supernatant obtains the superoxide-dismutase clear liquid; So-called air quantity ratio is one cubic metre of fermented liquid and the ratio of the needed air input of PM.
3, the superoxide-dismutase clear liquid that step 2 is obtained adopts the ultra-filtration membrane of 0.22 μ m and the microfiltration membrane of 6k that crude enzyme liquid is filtered and concentrates; Adopt centrifugal spray-drying tower to carry out spraying drying then; 130～140 ℃ of inlet temperatures export 50～60 ℃, obtain powdery superoxide-dismutase embedding product.
4, superoxide dismutase activity is measured
In the ordinary course of things, the SOD enzyme assay can only be used indirect activation measurement; Pyrogallol autoxidation method is adopted in this experiment; Pyrogallol is under alkaline condition, and autoxidation discharges O rapidly
2 -, generating the intermediate product of being with look, reaction beginning afterreaction liquid becomes yellowish brown earlier, changes green behind the several minutes, is transformed into yellow after several hours again, and this is because the result of the continuous oxidation of intermediate of generation; What measure here is the starting stage in the pyrogallol autoxidation process, and after being accumulated in of intermediate was detained 30～45s, linear with the time, general linear session maintained in the scope of 4min, and intermediate goes out to have strong photoabsorption at the 325nm wavelength; When SOD exists, because it can catalysis O
2 -Combine to generate O with H+
2And H
2O
2Thereby, stoped the accumulation of intermediate product, therefore, through calculating the enzymic activity that can obtain SOD.
Enzyme activity unit definition: under 25 ℃ of constant temperatures, in every milliliter of reaction solution, PM suppresses o-phenyl phenol autoxidation rate and reaches 50% enzyme amount and be defined as 1 enzyme activity unit.
Concrete operations are following:
Survey autoxidation speed:
(1) the SOD buffer 4.5ml that gets PH8.2 returns to zero in quartz cuvette 325nm photometry density value.
(2) the SOD buffer4.5ml that gets PH8.2 adds behind the 45mmol/L pyrogallol solution 10ul mixing rapidly in quartz cuvette 325nm wavelength photometry density value in 10mL ep pipe (25 ℃ of water bath heat preservations).
(3) every separated 30s surveys once, if survey 2min, obtains the autoxidation rate ODA of pyrogallol; (ODA/min is best 0.06 ± 0.002) because at this moment error is less, if autoxidation speed height then enriching Hcl; Depend upon circumstances the low SOD buffer that adds of autoxidation speed)
The survey enzyme is lived:
(1) get the SOD buffer 4.5ml that adjusts in 10ml EP pipe (in 25 ℃ of water-baths, being incubated), add enzyme liquid 10ul to be measured and 45mmol/L pyrogallol solution 10ul respectively, behind the mixing rapidly in quartz cuvette, 325nm wavelength photometry density value.
(2) every separated 30s surveys once, surveys 2min altogether, obtains OD value rate of change ODB.
Calculate enzyme activity:
Annotate: ODA is a pyrogallol autoxidation speed
ODB is a sample rate value rate of change
V
1Be the reaction solution TV
V
2Be the working sample TV
Embodiment
Below in more detail technical essential of the present invention is made an explanation through instance, for understanding the present invention better foundation is provided.
Embodiment 1:
1) (pH 6.8～7.2 in the 3L triangular flask 600mL first order seed substratum under aseptic condition, to change intestinal bacteria test tube slant bacterial classification over to prepared be contained in; Sterilising conditions: 121 ℃ of sterilization 30min); 250rpm, 37 ± 1 ℃ cultivate 12～14 hours subsequent use.
2) with the 100L seeding tank bacterial classification is carried out enlarged culturing
With the yeast powder 480g of technical grade, peptone 900g, 1M phosphoric acid buffer (pH=6.8) 6L, vitamin H 24mg, glucose 600g drop into respectively in the 100L seeding tank, with the tap water constant volume to 60L; Stir; Directly sterilize with steam then, 115 degree insulations 30 minutes are cooled to 37 degree again, insert the 600mL first order seed bacterium liquid of 1.2 gained set by step; Air flow 0.6: 1; 37 ± 1 ℃ of temperature; Cultivate 6h under the condition of pressure tank 0.03～0.05MPa, get the intestinal bacteria secondary seed solution of enlarged culturing.
3) produce superoxide-dismutase with the 1000L ferment tank
With the glucose 5kg of technical grade, the KH of 6.65kg
2PO
4, 2kg (NH
4)
2HPO
4,The MgSO of 6kg
47H
2O, Hydrocerol A 1.5kg, ironic citrate (III) 0.05kg drop into respectively in the 1000L fermentor tank,, stir to 500L with the tap water constant volume; Directly sterilize with steam then, 115 degree insulations 30 minutes are cooled to 37 degree again, and the sterilization back is by 28%NH
4OH adjusts pH to 7.0, adds liquid microelement (KI of 2.5mg/L, the MnCl of 1.5mg/L of 0.8L again
24H
2The CuCl of O, 1.5mg/L
22H
2The H of O, 3.0mg/L
3BO
3,2.5mg/L Na
2MoO
42H
2Zn (the CH of O, 1.3mg/L
3COO)
22H
2O hydrochloric acid sulphur ammonium 4.5mg/L); The CuSO45H of 100g
2The ZnSO of O, 50g
4, insert 60L first order seed nutrient solution; Under the condition of air quantity 1: 0.6～0.8,37 ± 1 ℃ of temperature, pressure tank 0.03～0.05MPa, cultivate, mend glucose after for some time, to OD
600Reach about 40 and begin to induce with IPTG, induce 2 hours after, stream adds trace element, CuSO in fermentor tank
45H
2O and ZnSO
4, coinduction 10 hours, the foam that produces in order to eliminate in the fermenting process in the culturing process; In 100L fermentor tank and 1000L fermentor tank, add the skimmer of silicone oil, obtain fermented liquid, utilize tubular-bowl centrifuge that fermented liquid is carried out solid-liquid separation; Resulting thalline passes through the clarifixator fragmentation again; Thermal treatment, centrifugal collection supernatant obtains the superoxide-dismutase clear liquid.The enzyme activity that records superoxide-dismutase is 21723U/mL.
4) the superoxide-dismutase clear liquid that step 2 is obtained adopts the ultra-filtration membrane of 0.22 μ m and the microfiltration membrane of 6k that crude enzyme liquid is filtered and concentrates; Adopt centrifugal spray-drying tower to carry out spraying drying then; 130～140 ℃ of inlet temperatures export 50～60 ℃, obtain powdery superoxide-dismutase embedding product.Record super-oxide
The enzyme activity of dismutase is 2650U/mg.
Claims (5)
1. the method for an escherichia coli high-level expression recombination human source superoxide-dismutase utilizes intestinal bacteria as bacterial classification, with yeast powder, peptone, glucose, KH
2PO
4, (NH
4)
2HPO
4, MgSO
47H
2O, Hydrocerol A, ironic citrate are raw material; Carry out deep liquid time fermentative prodn people source superoxide-dismutase; It is characterized in that through improving fermentation mode and raw material; Realize the high yield fermentation of people source superoxide-dismutase, reduced the production cost of people source superoxide-dismutase greatly, had good economic results in society.
2. according to claim 1; A kind of method of escherichia coli high-level expression recombination human source superoxide-dismutase is characterized in that: intestinal bacteria test tube slant bacterial classification is changed over to (pH6.8～7.2 in the 3L triangular flask 600mL first order seed substratum that are contained in that prepared under aseptic condition; Sterilising conditions: 121 ℃ of sterilization 30min), 250rpm, 37 ± 1 ℃ cultivate 12～14 hours subsequent use.
3. according to claim 1; A kind of method of escherichia coli high-level expression recombination human source superoxide-dismutase, its seed culture fluid is characterised in that: with the yeast powder 480g of technical grade, peptone 900g; 1M phosphoric acid buffer (pH=6.8) 6L; Vitamin H 24mg, glucose 600g drop into respectively in the 100L seeding tank, with the tap water constant volume to 60L; Stir; Directly sterilize with steam then, 115 degree insulations 30 minutes are cooled to 37 ℃ again, insert 600mL first order seed bacterium liquid; In air quantity 0.6: 1,37 ± 1 ℃ of temperature, cultivated 6 hours under the condition of pressure tank 0.03～0.05MPa, the intestinal bacteria secondary seed solution of enlarged culturing.
4. according to claim 1, a kind of method of escherichia coli high-level expression recombination human source superoxide-dismutase is characterized in that: with the glucose 5kg of technical grade, the KH of 6.65kg
2PO
4, (NH4) 2HPO4 of 2kg, the MgSO47H2O of 6kg, Hydrocerol A 1.5kg, ironic citrate (III) 0.05kg drop into respectively in the 1000L fermentor tank,, stir to 500L with the tap water constant volume; Directly sterilize with steam then, 115 degree insulations 30 minutes are cooled to 37 degree again, and the sterilization back is by 28%NH
4OH adjusts pH to 7.0, adds the liquid microelement of 0.8L again; The CuSO45H of 100g
2The ZnSO of O, 50g
4, insert 60L first order seed nutrient solution; Under the condition of air quantity 1: 0.6～0.8,37 ± 1 ℃ of temperature, pressure tank 0.03～0.05MPa, cultivate, mend glucose after for some time, to OD
600Reach about 40 and begin to induce with IPTG, induce 2 hours after, stream adds trace element, CuSO in fermentor tank
45H
2O and ZnSO
4, coinduction 10 hours obtains fermented liquid, utilizes tubular-bowl centrifuge that fermented liquid is carried out solid-liquid separation, obtains thalline and fermented liquid clear liquid, after ultrasonication, records the enzyme 21723U/L alive of anti-oxidant dismutase.
5. the method for a kind of escherichia coli high-level expression recombination human source superoxide-dismutase according to claim 4 is characterized in that: the trace element of 2.5mg/L is the MnCl of KI, 1.5mg/L
24H
2The CuCl of O, 1.5mg/L
22H
2The H of O, 3.0mg/L
3BO
3, 2.5mg/L Na
2MoO
42H
2Zn (the CH of O, 1.3mg/L
3COO)
22H
2O, hydrochloric acid sulphur ammonium 4.5mg/L.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012100425958A CN102533687A (en) | 2012-02-24 | 2012-02-24 | Method for efficiently expressing recombinant human superoxide dismutase by escherichia coli |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2012100425958A CN102533687A (en) | 2012-02-24 | 2012-02-24 | Method for efficiently expressing recombinant human superoxide dismutase by escherichia coli |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102533687A true CN102533687A (en) | 2012-07-04 |
Family
ID=46341721
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2012100425958A Pending CN102533687A (en) | 2012-02-24 | 2012-02-24 | Method for efficiently expressing recombinant human superoxide dismutase by escherichia coli |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102533687A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104313108A (en) * | 2014-09-23 | 2015-01-28 | 青岛康和食品有限公司 | Lactose bile salt fermentation culture medium and application thereof |
| CN109628533A (en) * | 2019-01-18 | 2019-04-16 | 重庆派金生物科技有限公司 | The soluble high efficiency recombinant expressed method of fibroblast growth factor albumen |
-
2012
- 2012-02-24 CN CN2012100425958A patent/CN102533687A/en active Pending
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104313108A (en) * | 2014-09-23 | 2015-01-28 | 青岛康和食品有限公司 | Lactose bile salt fermentation culture medium and application thereof |
| CN109628533A (en) * | 2019-01-18 | 2019-04-16 | 重庆派金生物科技有限公司 | The soluble high efficiency recombinant expressed method of fibroblast growth factor albumen |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Ammar | Cultivation of Microalgae Chlorella vulgaris in Airlift photobioreactor for Biomass Production using commercial NPK Nutrients | |
| KR20090055497A (en) | Novel culture process for a heterotrophic microalga | |
| CN106929422A (en) | A kind of method that chlorella and yeast co-culture purification yeast wastewater | |
| CN110627213B (en) | Method for efficiently treating high-ammonia-nitrogen wastewater by microalgae photo-fermentation method | |
| CN105647825B (en) | Method that is a kind of while improving spiral algal biomass and polysaccharide yield | |
| Nguyen et al. | Effect of Tris-(hydroxymethyl)-amino methane on microalgae biomass growth in a photobioreactor | |
| CN105506048A (en) | Fermentation method for preparing beta-carotin by using Blakeslea trispora | |
| CN105441525A (en) | Method for increasing yield of haematococcaceae astaxanthin with saccharose as carbon source through co-culture | |
| He et al. | Production of coenzyme Q10 by purple non-sulfur bacteria: current development and future prospect | |
| CN103444981A (en) | Method for Aspergillus oryzae to degrade edible and medicinal fungus dregs to produce protein feed | |
| CN103881923A (en) | Method for culturing microalgae by using coking wastewater | |
| CN102492752A (en) | Method for co-producing pullulan polysaccharide and melanin by Aureobasidium pullulan | |
| WO2020134688A1 (en) | Method for preparing high-purity hericium erinaceus polysaccharide by fermenting hericium erinaceus, and fermentation medium thereof | |
| CN102533687A (en) | Method for efficiently expressing recombinant human superoxide dismutase by escherichia coli | |
| CN105483171B (en) | A kind of raising ubiquinone10The production method of industrial output | |
| CN102181509A (en) | Method for producing vernine | |
| CN101988075A (en) | Method for preparing hydrogen by fermentation through using special anaerobic clostridium pasteurianum | |
| CN104946536A (en) | Isochrysis zhangjiangensis culture method | |
| KR101810567B1 (en) | Method for producing dry yeast containing selenium using fermentation | |
| CN111500475B (en) | Rhodotorula mucilaginosa ZOR1 and method for producing mannan and carotenoid | |
| CN103352061A (en) | Method for improving output of chondroitin sulfate analoge prepared by fermentation method | |
| CN102321683B (en) | Process for preparing fumaric acid fermentation liquid by fermentation method and for separating and extracting pure fumaric acid from fumaric acid fermentation liquid | |
| Leca et al. | Innovative and sustainable cultivation strategy for the production of Spirulina platensis using anaerobic digestates diluted with residual geothermal water | |
| CN105969709A (en) | Recombinant escherichia coli strain and method for preparing lycopene by adopting same | |
| CN102559641A (en) | Method for producing beta-1,3-1,4-glucanase through submerged fermentation of recombinant Pichia pastoris liquid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20120704 |