CN108893460A - A kind of method of the quick immobilized cell of magnetically fixed bed - Google Patents
A kind of method of the quick immobilized cell of magnetically fixed bed Download PDFInfo
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Abstract
The invention discloses a kind of methods of magnetically fixed quick immobilized cell of bed, with process, simple, less energy-consuming, time-consuming are short, equipment is simple, can operate continuously, is smaller on the influence of the cell activity of immobilization, facilitate the series of advantages such as loading and unloading cell, have a wide range of applications in fields such as biocatalysis.
Description
Technical field
The invention belongs to immobilized cell technique fields, especially by the method for the quick immobilization of cell in fermentation liquid,
More particularly to a kind of method of magnetically fixed quick immobilized cell of bed.
Background technique
Cell fixation is the important method that cell is reused in whole-cell catalytic and fermentation process, to the industrial mistake of raising
Production efficiency, the save the cost of journey have great importance.Common living cells process for fixation mainly has adsorption and entrapment,
The fixed cell density of absorption method is smaller, thus investment becomes most common living cells process for fixation.Investment it is basic
Process is：Firstly, the method by centrifugation, precipitating or filtering first collects cell from fermentation liquid, then cell is washed
To remove the Toxic Metabolites on surface, the method for being centrifuged, being precipitated or being filtered again collects cell；Secondly, by cell and height
Molecular material (for example sodium alginate, carragheen, PVA etc.) is vigorously stirred uniformly mixed；Again, above-mentioned mixed liquor is instilled and is agglomerated
It is formed in liquid and is embedded with the capsule of cell, having a size of several hundred microns between several millimeters；It is removed finally, capsule obtained is washed
The coagulation liquid on surface can be used for culture and bioconversion.The method of this traditional investment immobilized cell has a system
Lieque point：1, it is cumbersome that cell washing cell processes are collected；2, when being vigorously mixed with high molecular material, cell polar easy in inactivation；3, shape
At capsule size it is larger, often there is interior diffusion limitation；4, whole preparation process is cumbersome, and the time is longer, cost compared with
It is high.Therefore, it is significant to invent a kind of method that quick immobilized cell is used for biocatalysis.
Have some methods immobilized using magnetically fixed bed to cell at present, but traditional magnetically fixed bed is mostly with iron
Magnetisable material still has after removing magnetic field as magnetic conductivity medium, such as iron powder, iron wire, steel wire, steel ball, these magnetic conductive medias
Remanent magnetism, and then will lead to the Superparamagnetic particulates being attached in bed by magnetic field and be difficult to elute, seriously hinder magnetically fixed bed solid
Surely change the recycling in terms of cell.
Summary of the invention
It is an object of the invention to provide a kind of quick fixation cell of magnetically fixed bed in place of overcome the deficiencies in the prior art
The method of born of the same parents, this method are smaller on the influence of the cell activity of immobilization, and operating process is simple, have versatility, can be widely used for
Cell fixation during whole-cell catalytic.
The technical solution adopted by the present invention to solve the technical problems is：
A kind of method of the quick immobilized cell of magnetically fixed bed, including：
1) superparamagnetic fluid is prepared：By ferrous ion and ferric ion in molar ratio 1:1.5~2.5 ratio is mixed
It closes, is heated to 70~90 DEG C, every liter of reaction solution rapidly joins 110~130mL concentrated ammonia liquor, continues quickly 0.5~2h of stirring, reaction
Magneto separate after the completion removes supernatant, obtains superparamagnetic fluid；
2) superparamagnetic magnetic conductive media is prepared：Take 0.1~0.3mm of internal diameter, 0.05~0.2mm of wall thickness, 200~500mm of length
Hollow glass capillary, the superparamagnetic fluid for preparing step 1) in pipe inserts, removes under permanent magnet auxiliary super suitable
Dispersion liquid in magnetic fluid is fully filled with Superparamagnetic particulates in hollow glass capillary, after vacuum drying, closed hollow capillary
Glass tube both ends port forms the superparamagnetic magnetic conductive media being enclosed in capillary glass tube；
3) magnetically fixed bed reactor is prepared：It is filled in 10~50mm of diameter, the lucite tube of 200~500mm of length
The superparamagnetic magnetic conductive media of several step 2) preparations being enclosed in capillary glass tube, until by glass capillary in lucite tube
Pipe fills up, and particle, the liquid that the gap formed between capillary glass tube can facilitate size to be less than gap pass freely through；In organic glass
1~10 layer of copper wire is wound outside glass pipe, every layer of 500~1000 circle copper wire, form solenoid, obtain by 100~500 μm of brass wire diameter
Magnetically fixed bed reactor；Magnetic field, and the superparamagnetic magnetic conduction by being enclosed in capillary glass tube are generated after energization inside solenoid
Medium is by magnetic field surface peening；
4) magnetic fluid with cell interaction is prepared：By ferrous ion and ferric ion in molar ratio 1:1.5~
2.5 ratio mixing, is heated to 70~90 DEG C, and every liter of reaction solution rapidly joins 110~130mL concentrated ammonia liquor, reacts 8~12min
Afterwards, the polyacid or its salt that 10~50mL concentration is 4~6M is added dropwise in every liter of reaction system；Continue the temperature at 65~75 DEG C
Lower quickly 0.2~2h of stirring, Magneto separate, removes supernatant after the reaction was completed, and every liter of reaction solution remainder is scattered in 90 again
In~110mL deionized water, the electronegative magnetic fluid in surface to interact with cell is obtained；
5) cultured 0.2~2L of cell suspending liquid (cell concentration 10 is taken8~1010), be added step 4) preparation with
Cell interaction 5~20mL of magnetic fluid, adjust pH be 3~5 or additionally incorporate sodium chloride adjust salinity 0.2~
0.8mol/L obtains cell magnetic fluid mixed liquor after being stirred；Magnetically fixed bed reactor 0.5~2A of solenoid current is adjusted,
Cell magnetic fluid mixed liquor is passed through magnetically fixed bed reactor with the flow velocity of 5~20mL/min, cell is under the assistance of magnetic fluid
It is adsorbed in the superparamagnetic magnetic conductive media surface being enclosed in capillary glass tube in magnetically fixed bed reactor, it is solid to be secured to magnetic
Immobilized cell in the bed of fixed bed reactor.
Cell in magnetically fixed bed reactor is washed it is possible to further be passed through buffer, is just obtained after the completion of washing straight
The immobilized cell being fixed in bed is connect, can be used for subsequent bioconversion.
Further, it if you need to elute the immobilized cell in magnetically fixed bed reactor, can be reacted in magnetically fixed bed
Be passed through eluent in device and power off, the magnetic field of superparamagnetic magnetic conductive media completely disappears in magnetically fixed bed reactor, cell with it is super suitable
Magnetic conductance magnetic medium desorption flows out magnetic field with eluent.
Further, the concentration of the ferrous ion in the step 1) is 0.15~0.25mol/L, ferric ion
Concentration is 0.35~0.45mol/L.
Further, the concentration of the ferrous ion in the step 4) is 0.15~0.25mol/L, ferric ion
Concentration is 0.35~0.45mol/L.
Further, the polyacid in the step 4) or its salt are azelaic acid, citric acid, sodium citrate, ethanedioic acid etc.
At least one of.
Further, it in the step 5), when the mixing of cell magnetic fluid is passed through magnetically fixed bed reactor, is conveyed using pump
Cell magnetic fluid mixed liquor, lower section charging, top discharging；Magnetic field size can be adjusted by adjusting through solenoidal electric current；
If it is necessary, can pass through collet outside above-mentioned magnetically fixed bed reactor controls reaction temperature in magnetically fixed bed reactor；
Further, the cell suspending liquid in the step 5), predominantly single cell suspension, as Escherichia coli, yeast,
The suspension of lactic acid bacteria, bacillus etc..
Further, the salt used in the adjusting salinity in the step 5) includes such as sodium chloride, phosphate, lemon
The total concentration to the salt of cell fanout free region such as hydrochlorate.
Further, the buffer in the step 5) is physiological saline or the phosphate buffer of pH 7.3~7.5.
Compared with the background art, it has the following advantages that the technical program：
1, the present invention realizes the fast and convenient magnetic modification of cell, can the quick immobilization from suspension by cell in a few minutes
In magnetically fixed bed reactor, overcome that conventional cell process for fixation process is cumbersome, consuming time is long, cell activity loss is big
Defect.
2, method for immobilizing cell of the invention, cell are directly adsorbed on magnetic conductive media under magnetic fluid auxiliary, can
To control magnetic field strength by regulation electric current, realizes the absorption and desorption of cell, be conveniently replaceable immobilized cell.
3, the method for magnetically fixed bed reactor immobilized cell provided by the invention, easy to operate, equipment is simple, to cell
Activity influence it is smaller, it can be achieved that cell quick immobilization and recycling.
4, present invention improves over the magnetofluid making methods to interact with cell, with existing magnetofluid making method phase
Than greatly simplifying, can quickly be prepared.Meanwhile invention also improves the packing materials of magnetically fixed bed reactor, use
The packing material of superparamagnetic does not have remanence phenomenon generation.
In short, the method for the present invention process is simple, less energy-consuming, time-consuming is short, equipment is simple, can operate continuously, to the thin of separation
Cytoactive influence is smaller, has a wide range of applications in fields such as biocatalysis.
Detailed description of the invention
Present invention will be further explained below with reference to the attached drawings and examples.
Fig. 1 is the schematic diagram of magnetically fixed bed reactor of the invention.
Fig. 2 is to wash cell free growth curve comparison schematic diagram after free cell and immobilization in embodiment 7.
Specific embodiment
The contents of the present invention are illustrated below by embodiment：
Embodiment 1
1) superparamagnetic fluid is prepared：It is with 500mL concentration by the liquor ferri trichloridi that 500mL concentration is 0.4mol/L
The ferrous chloride solution of 0.2mol/L mixes in equal volume, is vigorously stirred, and is heated to 70 DEG C, every liter of reaction solution rapidly joins 120mL
Concentrated ammonia liquor (25~28%) continues quickly stirring 1h, after the reaction was completed Magneto separate, removes supernatant, obtain superparamagnetic fluid；
2) superparamagnetic magnetic conductive media is prepared：The hollow glass capillary of length 200mm, internal diameter 0.1mm, wall thickness 0.1mm are taken,
The superparamagnetic fluid for preparing step 1) in pipe is inserted, and the dispersion liquid in superparamagnetic fluid is removed under permanent magnet auxiliary,
Superparamagnetic particulates are fully filled in hollow glass capillary, after vacuum drying, both ends are sintered with alcolhol burner and are closed, and form closing
Superparamagnetic magnetic conductive media in capillary glass tube；
3) magnetically fixed bed reactor is prepared：The several steps 2) of filling in lucite tube (diameter 10mm, length 200mm)
The superparamagnetic magnetic conductive media of preparation being enclosed in capillary glass tube, until being filled out substantially in lucite tube by capillary glass tube
Full, particle, the liquid that the gap formed between capillary glass tube can facilitate size to be less than gap pass freely through；In lucite tube
2 layers of copper wire of outer winding, every layer of 600 circle copper wire (300 μm of brass wire diameter) form solenoid, obtain magnetically fixed bed reactor；It is powered
Generate magnetic field inside solenoid afterwards, and the superparamagnetic magnetic conductive media by being enclosed in capillary glass tube is by magnetic field surface peening；
4) magnetic fluid with cell interaction is prepared：By 500mL concentration be 0.4mol/L liquor ferri trichloridi with
500mL concentration is that the ferrous chloride solution of 0.2mol/L mixes in equal volume, is vigorously stirred, is heated to 70 DEG C, every liter of reaction solution is fast
120mL concentrated ammonia liquor is added in speed, after reacting 10min, is added dropwise the azelaic acid 20mL of 5M, continue 70 DEG C at a temperature of quickly stir
1h is mixed, after the reaction was completed Magneto separate, remove supernatant, remainder is scattered in again in 100mL deionized water, obtains and cell
The electronegative magnetic fluid in the surface of interaction, it is spare；
5) taking cultured 0.2L Escherichia coli (E.coli) suspension, (cell concentration is about 3.8 × 108), step is added
4) the magnetic fluid 10mL with cell interaction of preparation, adjusting pH is 3, and cell magnetic fluid mixed liquor is obtained after being stirred；
Magnetically fixed bed reactor 0.5~2A of solenoid current is adjusted, cell magnetic fluid mixed liquor is passed through magnetic with the flow velocity of 5mL/min and is consolidated
Fixed bed reactor, lower section charging, top discharging；Cell is adsorbed in the closing in magnetically fixed bed reactor under the assistance of magnetic fluid
Superparamagnetic magnetic conductive media surface in capillary glass tube, clear liquid outflow；
6) it is passed through physiological saline 1L, the cell in magnetically fixed bed reactor is washed, is just directly fixed after the completion of washing
Immobilized cell in bed can be used for subsequent bioconversion.
7) if you need to elute cell, eluent is passed through to magnetically fixed bed reactor and is powered off, it is super suitable in magnetically fixed bed reactor
The magnetic field of magnetic conductance magnetic medium completely disappears, and cell and superparamagnetic magnetic conductive media are desorbed, and flows out magnetic field with eluent.
Embodiment 2
1) superparamagnetic fluid is prepared：It is with 500mL concentration by the liquor ferri trichloridi that 500mL concentration is 0.4mol/L
The ferrous chloride solution of 0.2mol/L mixes in equal volume, is vigorously stirred, and is heated to 90 DEG C, every liter of reaction solution rapidly joins 120mL
Concentrated ammonia liquor continues quickly stirring 1h, after the reaction was completed Magneto separate, removes supernatant, obtain superparamagnetic fluid；
2) superparamagnetic magnetic conductive media is prepared：The hollow glass capillary of length 500mm, internal diameter 0.2mm, wall thickness 0.1mm are taken,
The superparamagnetic fluid for preparing step 1) in pipe is inserted, and the dispersion liquid in superparamagnetic fluid is removed under permanent magnet auxiliary,
Superparamagnetic particulates are fully filled in hollow glass capillary, after vacuum drying, both ends are sintered with alcolhol burner and are closed, and form closing
Superparamagnetic magnetic conductive media in capillary glass tube；
3) magnetically fixed bed reactor is prepared：The several steps 2) of filling in lucite tube (diameter 50mm, length 500mm)
The superparamagnetic magnetic conductive media of preparation being enclosed in capillary glass tube, until being filled out substantially in lucite tube by capillary glass tube
Full, particle, the liquid that the gap formed between capillary glass tube can facilitate size to be less than gap pass freely through；In lucite tube
5 layers of copper wire of outer winding, every layer of 1000 circle copper wire (500 μm of brass wire diameter) form solenoid, obtain magnetically fixed bed reactor；It is powered
Generate magnetic field inside solenoid afterwards, and the superparamagnetic magnetic conductive media by being enclosed in capillary glass tube is by magnetic field surface peening；
4) magnetic fluid with cell interaction is prepared：By 500mL concentration be 0.4mol/L liquor ferri trichloridi with
500mL concentration is that the ferrous chloride solution of 0.2mol/L mixes in equal volume, is vigorously stirred, is heated to 70 DEG C, every liter of reaction solution is fast
120mL concentrated ammonia liquor is added in speed, after reacting 10min, is added dropwise the azelaic acid 40mL of 5M, continue 70 DEG C at a temperature of quickly stir
1h is mixed, after the reaction was completed Magneto separate, remove supernatant, remainder is scattered in again in 100mL deionized water, obtains and cell
The electronegative magnetic fluid in the surface of interaction, it is spare；
5) taking cultured E. coli suspension 2L, (cell concentration is about 1.2 × 109), be added step 4) preparation with
The magnetic fluid 20mL of cell interaction, adjusting pH is 3, and cell magnetic fluid mixed liquor is obtained after being stirred；It adjusts magnetically fixed
Bed reactor solenoid current 1A, is passed through magnetically fixed bed reactor for cell magnetic fluid mixed liquor with the flow velocity of 20mL/min, under
Side's charging, top discharging；Cell is adsorbed in magnetically fixed bed reactor under the assistance of magnetic fluid and is enclosed in capillary glass tube
In superparamagnetic magnetic conductive media surface, clear liquid outflow；
6) it is passed through physiological saline 1L, washs the cell in magnetically fixed bed reactor, Escherichia coli are successfully fixed on magnetically fixed
In bed.
Embodiment 3
1) superparamagnetic fluid is prepared：It is with 500mL concentration by the liquor ferri trichloridi that 500mL concentration is 0.4mol/L
The ferrous chloride solution of 0.2mol/L mixes in equal volume, is vigorously stirred, and is heated to 70 DEG C, every liter of reaction solution rapidly joins 120mL
Concentrated ammonia liquor continues quickly stirring 1h, after the reaction was completed Magneto separate, removes supernatant, obtain superparamagnetic fluid；
2) superparamagnetic magnetic conductive media is prepared：The hollow glass capillary of length 300mm, internal diameter 0.1mm, wall thickness 0.1mm are taken,
The superparamagnetic fluid for preparing step 1) in pipe is inserted, and the dispersion liquid in superparamagnetic fluid is removed under permanent magnet auxiliary,
Superparamagnetic particulates are fully filled in hollow glass capillary, after vacuum drying, both ends are blocked with 502 glue curings, form envelope
Close the superparamagnetic magnetic conductive media in capillary glass tube；
3) magnetically fixed bed reactor is prepared：The several steps 2) of filling in lucite tube (diameter 20mm, length 300mm)
The superparamagnetic magnetic conductive media of preparation being enclosed in capillary glass tube, until being filled out substantially in lucite tube by capillary glass tube
Full, particle, the liquid that the gap formed between capillary glass tube can facilitate size to be less than gap pass freely through；In lucite tube
8 layers of copper wire of outer winding, every layer of 700 circle copper wire (400 μm of brass wire diameter) form solenoid, obtain magnetically fixed bed reactor；It is powered
Generate magnetic field inside solenoid afterwards, and the superparamagnetic magnetic conductive media by being enclosed in capillary glass tube is by magnetic field surface peening；
4) magnetic fluid with cell interaction is prepared：By 500mL concentration be 0.4mol/L liquor ferri trichloridi with
500mL concentration is that the ferrous chloride solution of 0.2mol/L mixes in equal volume, is vigorously stirred, is heated to 70 DEG C, every liter of reaction solution is fast
120mL concentrated ammonia liquor is added in speed, and after reacting 10min, the citric acid 20mL of 5M is added dropwise；Continue 70 DEG C at a temperature of quickly stir
1h is mixed, after the reaction was completed Magneto separate, remove supernatant, remainder is scattered in again in 100mL deionized water, obtains and cell
The electronegative magnetic fluid in the surface of interaction, it is spare；
5) taking cultured E. coli suspension 2L, (cell concentration is about 6.9 × 108), be added step 4) preparation with
The magnetic fluid 10mL of cell interaction, adjusting pH is 5, and cell magnetic fluid mixed liquor is obtained after being stirred；It adjusts magnetically fixed
Bed reactor solenoid current 0.5A, is passed through magnetically fixed bed reactor for cell magnetic fluid mixed liquor with the flow velocity of 10mL/min,
Lower section charging, top discharging；Cell is adsorbed in magnetically fixed bed reactor under the assistance of magnetic fluid and is enclosed in glass capillary
Superparamagnetic magnetic conductive media surface in pipe, clear liquid outflow；
6) it is passed through 7.4 phosphate buffer 1L of pH, washs the cell in magnetically fixed bed reactor, Escherichia coli are successfully fixed
In magnetically fixed bed.
Embodiment 4
1) superparamagnetic fluid is prepared：It is with 500mL concentration by the liquor ferri trichloridi that 500mL concentration is 0.4mol/L
The ferrous chloride solution of 0.2mol/L mixes in equal volume, is vigorously stirred, and is heated to 70 DEG C, every liter of reaction solution rapidly joins 120mL
Concentrated ammonia liquor continues quickly stirring 1h, after the reaction was completed Magneto separate, removes supernatant, obtain superparamagnetic fluid；
2) superparamagnetic magnetic conductive media is prepared：The hollow glass capillary of length 200mm, internal diameter 0.1mm, wall thickness 0.1mm are taken,
The superparamagnetic fluid for preparing step 1) in pipe is inserted, and the dispersion liquid in superparamagnetic fluid is removed under permanent magnet auxiliary,
Superparamagnetic particulates are fully filled in hollow glass capillary, after vacuum drying, both ends are blocked with 502 glue curings, form envelope
Close the superparamagnetic magnetic conductive media in capillary glass tube；
3) magnetically fixed bed reactor is prepared：The several steps 2) of filling in lucite tube (diameter 20mm, length 200mm)
The superparamagnetic magnetic conductive media of preparation being enclosed in capillary glass tube, until being filled out substantially in lucite tube by capillary glass tube
Full, particle, the liquid that the gap formed between capillary glass tube can facilitate size to be less than gap pass freely through；In lucite tube
6 layers of copper wire of outer winding, every layer of 1000 circle copper wire (200 μm of brass wire diameter) form solenoid, obtain magnetically fixed bed reactor；It is powered
Generate magnetic field inside solenoid afterwards, and the superparamagnetic magnetic conductive media by being enclosed in capillary glass tube is by magnetic field surface peening；
4) magnetic fluid with cell interaction is prepared：By 500mL concentration be 0.4mol/L liquor ferri trichloridi with
500mL concentration is that the ferrous chloride solution of 0.2mol/L mixes in equal volume, is vigorously stirred, is heated to 70 DEG C, every liter of reaction solution is fast
120mL concentrated ammonia liquor is added in speed, and after reacting 10min, the citric acid 40mL of 5M is added dropwise；Continue 70 DEG C at a temperature of quickly stir
1h is mixed, after the reaction was completed Magneto separate, remove supernatant, remainder is scattered in again in 100mL deionized water, obtains and cell
The electronegative magnetic fluid in the surface of interaction, it is spare；
5) taking cultured saccharomycete, (cell concentration is about 3.2 × 108) suspension 2L, be added step 4) preparation with it is thin
The magnetic fluid 20mL of cell phase interaction, adjusting pH is 5, and cell magnetic fluid mixed liquor is obtained after being stirred；Adjust magnetically fixed bed
Cell magnetic fluid mixed liquor is passed through magnetically fixed bed reactor, lower section with the flow velocity of 10mL/min by reactor solenoid current 1A
Charging, top discharging；Cell is adsorbed in being enclosed in capillary glass tube in magnetically fixed bed reactor under the assistance of magnetic fluid
Superparamagnetic magnetic conductive media surface, clear liquid outflow；
6) it is passed through 7.4 phosphate buffer 1L of pH, washs the cell in magnetically fixed bed reactor, saccharomycete is successfully fixed on
In magnetically fixed bed.
Embodiment 5
1) superparamagnetic fluid is prepared：It is with 500mL concentration by the liquor ferri trichloridi that 500mL concentration is 0.4mol/L
The ferrous chloride solution of 0.2mol/L mixes in equal volume, is vigorously stirred, and is heated to 80 DEG C, every liter of reaction solution rapidly joins 120mL
Concentrated ammonia liquor continues quickly stirring 1h, after the reaction was completed Magneto separate, removes supernatant, obtain superparamagnetic fluid；
2) superparamagnetic magnetic conductive media is prepared：The hollow glass capillary of length 200mm, internal diameter 0.2mm, wall thickness 0.1mm are taken,
The superparamagnetic fluid for preparing step 1) in pipe is inserted, and the dispersion liquid in superparamagnetic fluid is removed under permanent magnet auxiliary,
Superparamagnetic particulates are fully filled in hollow glass capillary, after vacuum drying, both ends are blocked with 502 glue curings, form envelope
Close the superparamagnetic magnetic conductive media in capillary glass tube；
3) magnetically fixed bed reactor is prepared：The several steps 2) of filling in lucite tube (diameter 40mm, length 200mm)
The superparamagnetic magnetic conductive media of preparation being enclosed in capillary glass tube, until being filled out substantially in lucite tube by capillary glass tube
Full, particle, the liquid that the gap formed between capillary glass tube can facilitate size to be less than gap pass freely through；In lucite tube
10 layers of copper wire of outer winding, every layer of 600 circle copper wire (300 μm of brass wire diameter) form solenoid, obtain magnetically fixed bed reactor；It is powered
Generate magnetic field inside solenoid afterwards, and the superparamagnetic magnetic conductive media by being enclosed in capillary glass tube is by magnetic field surface peening；
4) magnetic fluid with cell interaction is prepared：By 500mL concentration be 0.4mol/L liquor ferri trichloridi with
500mL concentration is that the ferrous chloride solution of 0.2mol/L mixes in equal volume, is vigorously stirred, is heated to 70 DEG C, every liter of reaction solution is fast
120mL concentrated ammonia liquor is added in speed, and after reacting 10min, the citric acid 10mL of 5M is added dropwise；Continue 70 DEG C at a temperature of quickly stir
1h is mixed, after the reaction was completed Magneto separate, remove supernatant, remainder is scattered in again in 100mL deionized water, obtains and cell
The electronegative magnetic fluid in the surface of interaction, it is spare；
5) taking cultured saccharomycete, (cell concentration is about 3.2 × 108) suspension 2L, be added step 4) preparation with it is thin
The magnetic fluid 20mL of cell phase interaction, adjusting pH is 7, and cell magnetic fluid mixed liquor is obtained after being stirred；Adjust magnetically fixed bed
Cell magnetic fluid mixed liquor is passed through magnetically fixed bed reactor with the flow velocity of 5mL/min by reactor solenoid current 1A, lower section into
Material, top discharging；Cell is adsorbed in being enclosed in capillary glass tube in magnetically fixed bed reactor under the assistance of magnetic fluid
Superparamagnetic magnetic conductive media surface, clear liquid outflow；
6) it is passed through physiological saline 1L, washs the cell in magnetically fixed bed reactor, saccharomycete is successfully fixed on magnetically fixed bed
In.
Embodiment 6
1) yeast cells of the immobilization for obtaining embodiment 5 in the reactor removes magnetic in the case where closing electric current
The magnetic field of fixed bed reactors is passed through physiological saline 1L with 5mL/min, and the yeast that elution unloads in magnetically fixed bed reactor is thin
Born of the same parents, until trickle is not celliferous clear liquid；
2) taking cultured saccharomycete, (cell concentration is about 3.2 × 108) suspension 2L, be added step 4) preparation with it is thin
The magnetic fluid 20mL of cell phase interaction, adjusting pH is 7, and cell magnetic fluid mixed liquor is obtained after being stirred；Regulating step 1) it looks for
Magnetically fixed bed reactor solenoid current 1A after that elution cell, by cell magnetic fluid mixed liquor with the flow velocity of 5mL/min
It is passed through magnetically fixed bed reactor, lower section charging, top discharging；Cell is adsorbed in magnetically fixed bed reactor under the assistance of magnetic fluid
The interior superparamagnetic magnetic conductive media surface being enclosed in capillary glass tube, clear liquid outflow；
3) it is passed through 7.4 phosphate buffer 1L of pH, washs the cell in magnetically fixed bed reactor, saccharomycete is successfully solid again
It is scheduled in magnetically fixed bed.
The recyclable operation of the process, can fast implement the immobilization and unloading of cell.Compared to traditional immobilized cell method
Entrapment media could be replaced by needing to dismantle reactor, and this method can realize the online immobilization unloading for completing cell, have bright
Aobvious superiority；Furthermore this method can substantially reduce the microbiological contamination risk in immobilization process.
Embodiment 7
1) Bacillus coli cells of the immobilization for obtaining embodiment 1 in the reactor are gone in the case where closing electric current
Except the magnetic field of magnetically fixed bed reactor, 7.4 phosphate buffer 0.2L of pH is passed through with 20mL/min, it is anti-that elution unloads magnetically fixed bed
Answer the Bacillus coli cells in device.Dilution-plate method measurement viable count is 2.7 × 10 step by step8A/mL, with initial cell concentration
3.8×108A/mL is compared and is closer to, and shows that the immobilization process is smaller to the Active lesions of cell；
2) the elution cell 5mL in step 1 is taken, the LB culture medium of 200mL is inoculated into, measures growth curve.By embodiment 1
5mL is taken out in the 0.2L Bacillus coli cells suspension of middle step 5) and is not passed through magnetically fixed bed reactor, is directly inoculated into 200mL
LB culture medium, measure growth curve.Compare immobilization before and after cell growth curve as shown in Fig. 2, the two growth curve compared with
Be it is close, show immobilization on cell activity influence it is smaller.
The above is only the preferred embodiment of the present invention, the range implemented of the present invention that therefore, it cannot be limited according to, i.e., according to
Equivalent changes and modifications made by the invention patent range and description, should still be within the scope of the present invention.
Claims (10)
1. a kind of method of the quick immobilized cell of magnetically fixed bed, it is characterised in that：Including：
1) superparamagnetic fluid is prepared：By ferrous ion and ferric ion in molar ratio 1:1.5~2.5 ratio mixing, adds
For heat to 70~90 DEG C, 110~130mL concentrated ammonia liquor is added in every liter of reaction solution, continues 0.5~2h of stirring, after the reaction was completed Magneto separate,
Supernatant is removed, superparamagnetic fluid is obtained；
2) superparamagnetic magnetic conductive media is prepared：Take the sky of 0.1~0.3mm of internal diameter, 0.05~0.2mm of wall thickness, 200~500mm of length
Heart glass capillary, the superparamagnetic fluid for preparing step 1) in pipe are inserted, and the dispersion liquid in superparamagnetic fluid is removed,
Superparamagnetic particulates are fully filled in hollow glass capillary, dry rear enclosed hollow glass capillary both ends port forms closing
Superparamagnetic magnetic conductive media in capillary glass tube；
3) magnetically fixed bed reactor is prepared：It is filled in 10~50mm of diameter, the lucite tube of 200~500mm of length several
The superparamagnetic magnetic conductive media of step 2) preparation being enclosed in capillary glass tube, until being filled out in lucite tube by capillary glass tube
It is full；1~10 layer of copper wire is wound outside lucite tube, every layer of 500~1000 circle copper wire, form by 100~500 μm of brass wire diameter
Solenoid obtains magnetically fixed bed reactor；
4) magnetic fluid with cell interaction is prepared：By ferrous ion and ferric ion in molar ratio 1:1.5~2.5
Ratio mixing is heated to 70~90 DEG C, and 110~130mL concentrated ammonia liquor is added in every liter of reaction solution, and after reacting 8~12min, every liter anti-
Answer system that the polyacid or its salt that 10~50mL concentration is 4~6M is added dropwise；Continue 65~75 DEG C at a temperature of stir 0.2
~2h, Magneto separate, removes supernatant after the reaction was completed, and every liter of reaction solution remainder is scattered in 90~110mL water again, obtains
To the electronegative magnetic fluid in surface to interact with cell；
5) taking cell concentration is 108~10100.2~2L of cell suspending liquid, be added step 4) preparation with cell interact
5~20mL of magnetic fluid, adjust pH be 3~5 or adjust salinity be 0.2~0.8mol/L, obtain Cell magnetic after being stirred
Fluid mixed liquor；Magnetically fixed bed reactor 0.5~2A of solenoid current is adjusted, by cell magnetic fluid mixed liquor with 5~20mL/
The flow velocity of min is passed through magnetically fixed bed reactor, and cell is adsorbed in the closing in magnetically fixed bed reactor under the assistance of magnetic fluid
Superparamagnetic magnetic conductive media surface in capillary glass tube, the fixation cell being secured in the bed of magnetically fixed bed reactor
Born of the same parents.
2. the method for the quick immobilized cell of magnetically fixed bed according to claim 1, it is characterised in that：Further include：6) lead to
Enter buffer and wash cell in magnetically fixed bed reactor, is secured in the bed of magnetically fixed bed reactor after the completion of washing
Immobilized cell.
3. the method for the quick immobilized cell of magnetically fixed bed according to claim 1, it is characterised in that：Further include：7) exist
Eluent is passed through in magnetically fixed bed reactor and is powered off, and the immobilized cell in magnetically fixed bed reactor is eluted.
4. the method for the quick immobilized cell of magnetically fixed bed according to claim 1, it is characterised in that：In the step 1)
The concentration of ferrous ion be 0.15~0.25mol/L, the concentration of ferric ion is 0.35~0.45mol/L.
5. the method for the quick immobilized cell of magnetically fixed bed according to claim 1, it is characterised in that：In the step 4)
The concentration of ferrous ion be 0.15~0.25mol/L, the concentration of ferric ion is 0.35~0.45mol/L.
6. the method for the quick immobilized cell of magnetically fixed bed according to claim 1, it is characterised in that：In the step 4)
Polyacid or its salt be at least one of azelaic acid, citric acid, sodium citrate, ethanedioic acid.
7. the method for the quick immobilized cell of magnetically fixed bed according to claim 1, it is characterised in that：The step 5)
In, cell magnetic fluid is mixed when being passed through magnetically fixed bed reactor by the way of lower section charging, top discharging；It is logical by adjusting
Solenoidal electric current is crossed, magnetic field size is adjusted；Pass through collet outside the magnetically fixed bed reactor and controls magnetically fixed bed reactor
Interior reaction temperature.
8. the method for the quick immobilized cell of magnetically fixed bed according to claim 1, it is characterised in that：In the step 5)
Cell suspending liquid be Escherichia coli, yeast, lactic acid bacteria, bacillus suspension.
9. the method for the quick immobilized cell of magnetically fixed bed according to claim 1, it is characterised in that：In the step 5)
Adjusting salinity in the salt that uses for sodium chloride, phosphate, citrate.
10. the method for the quick immobilized cell of magnetically fixed bed according to claim 2, it is characterised in that：The step 5)
In buffer be physiological saline or pH 7.3~7.5 phosphate buffer.
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101458985A (en) * | 2007-12-13 | 2009-06-17 | 比亚迪股份有限公司 | Preparation for magnetic fluid and chitosan magnetic micro-sphere |
| CN101475930A (en) * | 2008-12-26 | 2009-07-08 | 中国科学院过程工程研究所 | Superparamagnetic nano particle in situ adsorptive separation-immobilized Rhodococcus biological desulphurization process |
| CN101763930A (en) * | 2010-01-21 | 2010-06-30 | 上海大学 | Improved ferroferric oxide magnetic fluid and method for preparing same |
-
2018
- 2018-06-29 CN CN201810701521.8A patent/CN108893460B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101458985A (en) * | 2007-12-13 | 2009-06-17 | 比亚迪股份有限公司 | Preparation for magnetic fluid and chitosan magnetic micro-sphere |
| CN101475930A (en) * | 2008-12-26 | 2009-07-08 | 中国科学院过程工程研究所 | Superparamagnetic nano particle in situ adsorptive separation-immobilized Rhodococcus biological desulphurization process |
| CN101763930A (en) * | 2010-01-21 | 2010-06-30 | 上海大学 | Improved ferroferric oxide magnetic fluid and method for preparing same |
Non-Patent Citations (5)
| Title |
|---|
| SEYED MORTAZA ROBATJAZI等: "Continuous biodegradation of parathion by immobilized Sphingomonas sp. in magnetically fixed-bed bioreactors and evaluation of the enzyme stability of immobilized bacteria", 《BIOTECHNOL LETT》 * |
| 徐炎: "固定化细胞技术的优势与应用领域", 《科技展望》 * |
| 林檬: "细胞的磁修饰及其用于罗伊氏乳杆菌转化甘油的研究", 《中国优秀硕士学位论文全文数据库基础科学辑》 * |
| 林檬等: "磁辅助生物反应器研究进展", 《化工进展》 * |
| 陈佳璐等: "羧基修饰的超顺磁纳米粒子直接固定化罗伊氏乳杆菌", 《化工学报》 * |
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