CN104293671A - Disruption method of recombinant escherichia coli - Google Patents
Disruption method of recombinant escherichia coli Download PDFInfo
- Publication number
- CN104293671A CN104293671A CN201310299971.6A CN201310299971A CN104293671A CN 104293671 A CN104293671 A CN 104293671A CN 201310299971 A CN201310299971 A CN 201310299971A CN 104293671 A CN104293671 A CN 104293671A
- Authority
- CN
- China
- Prior art keywords
- bacillus coli
- recombination bacillus
- breaking method
- diacetylmuramidase
- carry out
- 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
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/06—Lysis of microorganisms
- C12N1/066—Lysis of microorganisms by physical methods
Abstract
The invention discloses a disruption method of recombinant escherichia coli. The method includes following steps: performing concentration through a ceramic membrane or a centrifugal machine to a recombinant escherichia coli bacteria liquid obtained through fermentation; adding a lysozyme and Mg<2+> according to proportions; stirring the bacteria liquid for 0.5-1h at a low temperature; performing high-pressure homogenization disruption for 1-3 times through a homogenizer; and performing microscopic examination to the bacteria through a microscope until a standard is achieved. The method is a cell disruption method which is quick and high-efficient, is convenient to carry out and can be used in large-scale industrial production.
Description
Technical field
The present invention relates to the extensive extraction production field of intestinal bacteria recombinant protein, particularly a kind of breaking method of recombination bacillus coli.
Background technology
The bacterial cell disruption method that intestinal bacteria are conventional has: freeze-thaw method, sonioation method, lysozyme Method, chemical-agent technique etc. repeatedly, and freeze-thaw method is only limitted to short run and breaks bacterium repeatedly, and broken bacterium effect is poor, consuming time longer, and efficiency is low; Sonioation method crushing effect is better, but the laboratory scale of being only limitted to, exist and amplify bottleneck; N,O-Diacetylmuramidase crush method exists that broken not exclusively after broken, enzyme yield is lower, and the shortcoming such as costly; Chemical-agent technique is generally used for test in laboratory analysis, and as SDS etc., chemical reagent generally can cause protein denaturation, causes enzyme deactivation, and therefore the method major part is only limitted to the sex change detection analysis of protein.Clarifixator high-pressure homogenization crush method is widely used in laboratory and plant size, its crushing effect is good, and crushing efficiency is high, and is not easy to cause protein denaturation, but high-pressure homogenization broken due to operating pressure high, be generally 800 ~ 1000Bar, higher to equipment requirements, accessory is easily damaged, m of e costly, and cell concentration limits to some extent time broken, general wet thallus content is at 100g/L ~ 200g/L, and after cell concentration improves, crushing effect is had a greatly reduced quality.Time broken, the height of cell concentration directly affects the height of follow-up enzyme liquid concentration, and when lower enzyme liquid concentration causes doing enzymic catalytic reaction, enzyme liquid throwing amount is too high, brings difficulty to subsequent purification.
Summary of the invention
For the above various broken bacterium method Problems existing, the object of the invention is in order to provide a kind of rapidly and efficiently, run convenient and the method for cell disruption of large-scale commercial production can be used for.
For achieving the above object, the present invention takes following technical scheme: a kind of breaking method of recombination bacillus coli, is characterized in that: the recombination bacillus coli bacterium liquid that fermentation obtains, and after ceramic membrane or whizzer concentrate, proportionally adds N,O-Diacetylmuramidase and Mg
2+, and keep stirring 0.5 ~ 1h under cryogenic, then carry out high-pressure homogenization fragmentation 1 ~ 3 time through clarifixator, with sediments microscope inspection thalline to up to standard.
Further, described recombination bacillus coli bacterium liquid wet thallus concentration after ceramic membrane or whizzer concentrate is 200 ~ 400g/L.
Further, described N,O-Diacetylmuramidase add-on is 2 ~ 10mg/(g wet thallus), described Mg
2+add-on be final concentration 2 ~ 10 mM.Mg
2+n,O-Diacetylmuramidase can be impelled to keep high vigor state, thus decrease the usage quantity of N,O-Diacetylmuramidase, reduce production cost.
Further, describedly N,O-Diacetylmuramidase and Mg is added
2+after bacterium liquid keep at 4 ~ 8 DEG C 80 ~ 150rpm stir 0.5 ~ 1h.
Further, bacterium liquid carries out the high-pressure homogenization fragmentation 1 ~ 3 time of 300 ~ 400Bar through clarifixator, and it is completely broken to ensure to carry out microscopy with microscope.
Further, Mg is added
2+form be Mg
2+soluble inorganic salt and crystalline hydrate, such as include but not limited to MgCl
2, MgSO
4and crystalline hydrate.
The breaking method of recombination bacillus coli of the present invention, can effective broken high density thalline, and broken cell concentration reaches 200 ~ 400g/L, and percentage of damage reaches more than 99%, and simple high-pressure homogeneous fragmentation is only 100 ~ 200g/L.Meanwhile, effectively can reduce the pressure of clarifixator homogenate fragmentation, broken bacterium pressure of the present invention is 300 ~ 400Bar, and prior art is 800 ~ 1000Bar, therefore effectively extends the work-ing life of machine parts.And make N,O-Diacetylmuramidase maintain vigor, decrease the consumption of N,O-Diacetylmuramidase, reduce cost.
Embodiment
Embodiment 1
The recombination bacillus coli bacterium liquid 2000L that fermentation obtains, is concentrated into 500L through ceramic membrane or whizzer, cell concentration 300g/L, adds 300g N,O-Diacetylmuramidase and 246.47g MgSO after concentrated
47H
2o, under 8 DEG C of conditions, 100rpm stirs 1h, then utilizes clarifixator to carry out cytoclasis under the pressure of 400Bar, flow velocity 200L/h, and broken 1 time altogether, carry out microscopy with microscope, bacterial cell disruption rate is 80%.
Embodiment 2
The recombination bacillus coli bacterium liquid 2000L that fermentation obtains, is concentrated into 490L through ceramic membrane or whizzer, cell concentration 300g/L, adds 900g N,O-Diacetylmuramidase and 739.41g MgSO after concentrated
47H
2o, under 4 ~ 8 DEG C of conditions, 100rpm stirs 1h, then utilizes clarifixator to carry out cytoclasis under the pressure of 400Bar, flow velocity 200L/h, and broken 1 time altogether, carry out microscopy with microscope, bacterial cell disruption rate is 87%.
Embodiment 3
The recombination bacillus coli bacterium liquid 2000L that fermentation obtains, is concentrated into 500L through ceramic membrane or whizzer, cell concentration 300g/L, adds 300g N,O-Diacetylmuramidase and 246.47g MgSO after concentrated
47H
2o, under 4 ~ 8 DEG C of conditions, 100rpm stirs 1h, then utilizes clarifixator to carry out cytoclasis under the pressure of 400Bar, flow velocity 200L/h, and broken 2 times altogether, carry out microscopy with microscope, bacterial cell disruption rate is 93%.
Embodiment 4
The recombination bacillus coli bacterium liquid 2000L that fermentation obtains, is concentrated into 490L through ceramic membrane or whizzer, cell concentration 300g/L, adds 900g N,O-Diacetylmuramidase and 739.41g MgSO after concentrated
47H
2o, under 4 ~ 8 DEG C of conditions, 100rpm stirs 1h, then utilizes clarifixator to carry out cytoclasis under the pressure of 400Bar, flow velocity 200L/h, and broken 2 times altogether, carry out microscopy with microscope, bacterial cell disruption rate is 99%.
Embodiment 5
The recombination bacillus coli bacterium liquid 3000L that fermentation obtains, is concentrated into 500L through ceramic membrane or whizzer, cell concentration 400g/L, adds 2000g N,O-Diacetylmuramidase and 1232.35g MgSO after concentrated
47H
2o, under 4 DEG C of conditions, 80rpm stirs 1h, then utilizes clarifixator to carry out cytoclasis under the pressure of 300Bar, flow velocity 200L/h, and broken 3 times altogether, carry out microscopy with microscope, bacterial cell disruption rate is 99%.
Embodiment 6
The recombination bacillus coli bacterium liquid 1500L that fermentation obtains, is concentrated into 500L through ceramic membrane or whizzer, cell concentration 200g/L, adds 400g N,O-Diacetylmuramidase and 406g MgCl after concentrated
26H
2o, under 6 DEG C of conditions, 150rpm stirs 0.5h, then utilizes clarifixator to carry out cytoclasis under the pressure of 350Bar, flow velocity 200L/h, and broken 2 times altogether, carry out microscopy with microscope, bacterial cell disruption rate is 97%.
Embodiment 7
The recombination bacillus coli bacterium liquid 1500L that fermentation obtains, is concentrated into 500L through ceramic membrane or whizzer, cell concentration 200g/L, adds 400g N,O-Diacetylmuramidase and 481.66g MgSO after concentrated
4, under 6 DEG C of conditions, 150rpm stirs 0.5h, then utilizes clarifixator to carry out cytoclasis under the pressure of 350Bar, flow velocity 200L/h, and broken 2 times altogether, carry out microscopy with microscope, bacterial cell disruption rate is 99%.
Embodiment 8
The recombination bacillus coli bacterium liquid 1500L that fermentation obtains, is concentrated into 500L through ceramic membrane or whizzer, cell concentration 200g/L, adds 400g N,O-Diacetylmuramidase and 190.42g MgCl after concentrated
2, under 6 DEG C of conditions, 150rpm stirs 0.5h, then utilizes clarifixator to carry out cytoclasis under the pressure of 350Bar, flow velocity 200L/h, and broken 2 times altogether, carry out microscopy with microscope, bacterial cell disruption rate is 97%.
Embodiment 9
The recombination bacillus coli bacterium liquid 1500L that fermentation obtains, is concentrated into 500L through ceramic membrane or whizzer, cell concentration 200g/L, adds 400g N,O-Diacetylmuramidase and 769.23g Mg (NO after concentrated
3)
26H
2o, under 6 DEG C of conditions, 150rpm stirs 0.5h, then utilizes clarifixator to carry out cytoclasis under the pressure of 350Bar, flow velocity 200L/h, and broken 2 times altogether, carry out microscopy with microscope, bacterial cell disruption rate is 94%.
Claims (7)
1. a breaking method for recombination bacillus coli, is characterized in that: the recombination bacillus coli bacterium liquid that fermentation obtains, and after ceramic membrane or whizzer concentrate, proportionally adds N,O-Diacetylmuramidase and Mg
2+, and keep stirring 0.5 ~ 1h under cryogenic, then carry out high-pressure homogenization fragmentation 1 ~ 3 time through clarifixator, with sediments microscope inspection thalline to up to standard.
2. the breaking method of recombination bacillus coli according to claim 1, is characterized in that: described recombination bacillus coli bacterium liquid wet thallus concentration after ceramic membrane or whizzer concentrate is 200 ~ 400g/L.
3. the breaking method of recombination bacillus coli according to claim 1, is characterized in that: described N,O-Diacetylmuramidase add-on is 2 ~ 10mg/(g wet thallus), described Mg
2+add-on be final concentration 2 ~ 10 mM.
4. the breaking method of recombination bacillus coli according to claim 1, is characterized in that: described adds N,O-Diacetylmuramidase and Mg
2+after bacterium liquid keep at 4 ~ 8 DEG C 80 ~ 150rpm stir 0.5 ~ 1h.
5. the breaking method of recombination bacillus coli according to claim 1, is characterized in that: bacterium liquid carries out the high-pressure homogenization fragmentation 1 ~ 3 time of 300 ~ 400Bar through clarifixator, and it is completely broken to ensure to carry out microscopy with microscope.
6. the breaking method of the recombination bacillus coli according to any one of claim 1-5, is characterized in that: add Mg
2+form be Mg
2+soluble inorganic salt and crystalline hydrate.
7. the breaking method of recombination bacillus coli according to claim 6, is characterized in that: add Mg
2+form be MgCl
2or MgSO
4and crystalline hydrate.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310299971.6A CN104293671A (en) | 2013-07-17 | 2013-07-17 | Disruption method of recombinant escherichia coli |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310299971.6A CN104293671A (en) | 2013-07-17 | 2013-07-17 | Disruption method of recombinant escherichia coli |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN104293671A true CN104293671A (en) | 2015-01-21 |
Family
ID=52313629
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310299971.6A Pending CN104293671A (en) | 2013-07-17 | 2013-07-17 | Disruption method of recombinant escherichia coli |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104293671A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105441328A (en) * | 2015-12-28 | 2016-03-30 | 重庆医科大学 | Method for cracking colon bacillus cells at high flux |
| CN109439650A (en) * | 2018-10-10 | 2019-03-08 | 安徽欣伯玉生物科技有限公司 | A kind of extracting method of Agrobacterium plasmid DNA |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996014405A2 (en) * | 1994-11-04 | 1996-05-17 | Molecular Biology Resources, Inc. | Biologically active fragments of thermus flavus dna polymerase |
| CN101292027A (en) * | 2005-08-24 | 2008-10-22 | 先锋高级育种国际公司 | Rubisco activase with increased thermostability and methods of use thereof |
-
2013
- 2013-07-17 CN CN201310299971.6A patent/CN104293671A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1996014405A2 (en) * | 1994-11-04 | 1996-05-17 | Molecular Biology Resources, Inc. | Biologically active fragments of thermus flavus dna polymerase |
| CN101292027A (en) * | 2005-08-24 | 2008-10-22 | 先锋高级育种国际公司 | Rubisco activase with increased thermostability and methods of use thereof |
Non-Patent Citations (1)
| Title |
|---|
| 田文标,等: "重组大肠杆菌不耐热肠毒素B 亚单位的纯化及生物学活性鉴定", 《第三军医大学学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105441328A (en) * | 2015-12-28 | 2016-03-30 | 重庆医科大学 | Method for cracking colon bacillus cells at high flux |
| CN109439650A (en) * | 2018-10-10 | 2019-03-08 | 安徽欣伯玉生物科技有限公司 | A kind of extracting method of Agrobacterium plasmid DNA |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Pottkämper et al. | Applying metagenomics for the identification of bacterial cellulases that are stable in ionic liquids | |
| CN102191142B (en) | Surface yellow spot cleaning agent for neutral stones and stone cultural relics and preparation method thereof | |
| Mofrad et al. | Rapid formation of dry natural gas hydrate with high capacity and low decomposition rate using a new effective promoter | |
| CN102600987B (en) | Lithium oxide mineral separation collecting agent and application method thereof | |
| JP2017501705A5 (en) | ||
| CN107010853B (en) | A kind of method that microwave synergy is activated S95 grade slag powder | |
| CN104293671A (en) | Disruption method of recombinant escherichia coli | |
| CN101845187A (en) | Oil-based viscosity reducer for raw oil conveyed through pipeline | |
| CN104743952A (en) | Activation grinding aid used for grinding of high titanium slag powder and preparation method of activation grinding aid | |
| CN106398857B (en) | Method for extracting schizochytrium limacinum grease and application thereof | |
| Yang et al. | Comparative analysis of heterologous expression, biochemical characterization optimal production of an alkaline α‐amylase from alkaliphilic Alkalimonas amylolytica in Escherichia coli and Pichia pastoris | |
| CN106660845A (en) | Methods for enhancing the dewaterability of sludge with enzyme treatment | |
| CN106186368A (en) | A kind of preparation method of saprobia fermentation process agent | |
| CN105273831B (en) | A kind of broken wall of oil-containing micro-algae and the method for surfactant assisted extraction grease | |
| CN102965077A (en) | Powdered ore additive | |
| US20210237012A1 (en) | Electrical control system for slurry mixing | |
| CN102826622A (en) | Treatment method of wastewater containing rubber latex | |
| CN105435707B (en) | A kind of cationic Gemini surfactant and its construction method with similar cloud point phenomenon | |
| Ritter et al. | The influence of sorbitol on the production of cellulases and xylanases in an airlift bioreactor | |
| Suwannabun et al. | Pretreatment of rice straw by inorganic salts and 1-ethyl-3-methylimdazolium acetate for biofuel production | |
| CN105498511A (en) | Preparation method of magnesium hydroxide concentration slurry for flue gas desulfurization | |
| CN102503812A (en) | Method for increasing citric acid yield in extracting process of citric acid with calcium-salt method | |
| Li et al. | Expression, purification and characterization of a recombinant β-glucosidase from Volvariella volvacea | |
| CN102168174A (en) | Method for dephosphorizing high-phosphorus hematite | |
| CN104689924A (en) | Amphoteric combined collector for reverse flotation of hematite iron ore |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20150121 |
|
| RJ01 | Rejection of invention patent application after publication |