CN101899429A - Preparation of novel high-efficiency biocatalyst based on porous microcapsule technology - Google Patents
Preparation of novel high-efficiency biocatalyst based on porous microcapsule technology Download PDFInfo
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- CN101899429A CN101899429A CN200910103985XA CN200910103985A CN101899429A CN 101899429 A CN101899429 A CN 101899429A CN 200910103985X A CN200910103985X A CN 200910103985XA CN 200910103985 A CN200910103985 A CN 200910103985A CN 101899429 A CN101899429 A CN 101899429A
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Abstract
The invention relates to a new method for preparing a novel high-efficiency porous microcapsule biocatalyst by combining a coaxial double-layer technology and an in-situ erosion punching technology. A microcapsule with a diameter of 3-4mm is firstly prepared, wherein the inner core of the microcapsule comprises permeability beer or bread yeast cells, and an outer-layer shell of the microcapsule is prepared by polyvinyl alcohol/calcium alginate mixed gel; and finally, the mechanical strength of an outer layer of the microcapsule is increased by further solidifying through freezing and thawing repeatedly. Because cellular fluid is in the microcapsule, the microcapsule biocatalyst which is similar to a micro-bioreactor is formed; in addition, a phosphate solution or other calcium-ion complexing agents are used for carrying out chemical erosion to dissolve partial calcium alginate gel by the in-situ erosion punching technology in order to achieve an in-situ erosion punching purpose, thereby a microchannel is formed to promote the transmission of micromolecular materials and products in a gel coating layer, and moreover, the catalytic activity of cells in the porous microcapsule core is increased by the permeability treatment of the beer or bread yeast cells. Due to the adoption of the method, the novel high-efficiency porous microcapsule biocatalyst has high cell capacity, strong catalytic effect, low diffusional limitation and high mechanical strength and is used to prepare important chiral drugs, such as chiral alcohol, and the like and a medical and chemical intermediate.
Description
Technical field
The present invention relates to the new technology of a kind of " making up the high-performance bio catalyzer " based on novel porous microcapsule technology.
Background technology
Micro-capsule at biological field is enzyme, coenzyme, protein and other or microorganism, animal and plant cells to be enclosed in formed spherical micro-capsule in the hydrophilic semi-permeable membranes of one deck, biomacromolecule or cell are intercepted in the film of micro-capsule, oxygen, nutritive substance and other small-molecule substance then can free in and out cyst membrane and carry out material transfer, thereby reach catalysis, cultivation or immune isolated purpose.Biological micro-capsule is because it has the feature of liquid environment in semi-transparent microcapsule membrane of one deck and the film, so have the function that protective membrane endoenzyme molecule or cell and controlled substance transmit by cyst membrane.Thereby biological micro-capsule is just just as a small bio-reactor, for the catalysis of enzyme or the growth of cell provide a microenvironment.
Microcapsule technology is a kind of fixedly technique for packing, the selection of its effect and micro-capsule shell material is closely related, and the composition of capsule casing material has determined some performances of micro-capsule product, as solvability, slow-releasing, flowability etc., it also has certain influence to the encapsulation process method simultaneously, so the selection of capsule casing material is the key of carrying out microencapsulation.The condition that it must have: good flowability is arranged during high density, and guaranteeing has good operating performance in the microencapsulation process; Can also can form stable emulsification system by the emulsification core; Processing, can complete being embedded in its structure in the storage process with core; Easily dry and easy precipitation; Good solubility; Economy.J.K.Park etc. have systematically summarized the method for micro-capsule fixation of microbial cell.Above-mentioned particulate immobilized cell is compared with the micro-capsule immobilized cell, and the former occurs the cell seepage easily, and reaction substrate is limited with contacting of cell, to such an extent as to the productive rate of the work-ing life of this immobilized cell and product etc. all are a greater impact.And latter's cell carrying capacity height has been accelerated reaction process, has saved the reaction times, improves reaction yield, and has prolonged its use cell survival.
PVA can form good gel, is usually used in the cell embedding immobilization.Commonly used during it is used have a PVA cold method, the PVA chemical crosslink technique, and PVA radiation crosslinking method has a large amount of reports both at home and abroad in these areas.PVA chemical crosslink technique and PVA radiation crosslinking method or open connection easily, or pair cell is toxic and be subjected to certain limitation when practical application.The present invention is the physical method that PVA uses, it is the PVA cold method, gel forming utilizes PVA to form three-dimensional network at 0 ℃～-40 ℃ freezing reasonable time PVA interchain hydrogen bonds and crystallite district, thereby form the PVA hydrogel of physical crosslinking, and this gel has certain mechanical strength, only can swelling in water under the normal temperature and can not dissolve.
In outer gel, add simultaneously alginate calcium (CA) and be used for punching.Advantage widespread uses such as the sodium alginate immobilized cell is easy and simple to handle because of it, cytotoxicity is less.But it is unstable in phosphoric acid buffer reaches that its shortcoming is an alginate calcium, and decalcification makes loosely organized and caves in.The present invention utilizes the susceptibility of calcium alginate gel to phosphate solution or other calcium ion complexing agent chemical erosion just, be partly dissolved by the alginates that contains in the calcium alginate gel after chemical erosion immobilization, thereby in original outer gel, form the hole, reach the purpose of pore, the transmission efficiency of raw material and product when carrying out biocatalytic reaction to improve catalyzer.
With CTAB microorganism cells being carried out that permeability handles is cell catalysis effective methods when improving cell and being used for biocatalytic reaction.The present invention in immobilization before the cell, pair cell carries out permeability, has improved the activity of intracellular enzyme.And common cell fixation is not carry out permeability to handle.
Summary of the invention
The objective of the invention is to combine coaxial double-layer technology and in-situ chemical and corrode the new bio catalyzer of the novel method of punching technology in order to preparation permeability cerevisiae porous microcapsule.
To achieve these goals, main operational steps of the present invention comprises: at first, yeast cell is carried out permeability handle, to improve its catalytic activity; Adopting the coaxial double-layer technology to prepare kernel is the permeability cerevisiae, and softgel shell is the micro-capsule of PVA/CA.Its outer material is for mixing with the sodium alginate of suitable proportion and PVA, the permeability cell is carried out dressing after, splash in the calcium chloride solution of proper concn and form gel coat layer, and increase the mechanical property of coat layer through freeze thawing; Carry out chemical erosion with the phosphate solution of proper concn or other calcium ion complexing agent and make the dissolving of part calcium alginate gel, reach the purpose that original position corrodes punching, thereby form the microchannel to promote small molecules raw material and the transmission of product in gel coat layer.Compare with the micro-capsule before and after punching, the micro-capsule softgel shell that hydrophobic and hydrophilic molecules can see through after the punching easilier enter the capsule heart, and mass transfer coefficient is also apparently higher than the micro-capsule that does not punch.
Take pictures through scanning electron microscope, inverted microscope is observed, the blood cell counting counting, inspection such as methylene blue staining and enzymic activity, this new and effective biological catalyst have the physical strength height, desmo enzyme is active high, and storage stability is good, the cell carrying capacity is up to 80%, characteristics such as mass transfer effect is good.
Utilize the present invention can obtain the new bio catalyzer of porous microcapsule, cell carries the capacity height, and permeability is good, physical strength is high.At low temperatures can prolonged preservation, can prolonged and repeated use in biocatalytic reaction.This will help the widespread use of this new and effective biological catalyst in biocatalysis and bio-transformation industrial circle.
The concrete example of implementing
Example one:
With the purified water configuration concentration is that 15% PVA solution and concentration are 5% sodium alginate soln, and in high-pressure sterilizing pot with 121 ℃ of sterilization 20min; Get 15% PVA solution 70ml, and be prepared into capsule casing material behind 5% the sodium alginate soln 30ml thorough mixing and place the capsule casing material storage vessel of coaxial double-layer outer tube layer (coatings); In addition the cerevisiae of 20 grams being put into 0.2% CTAB solution carries out permeability and handled 20 minutes, cerevisiae after permeability handled is washed 3 times, centrifugal 5 minutes of 3000rmp then repeatedly with deionized water, with pack into the internal layer (core liquid) of coaxial double-layer pipe of the cerevisiae liquid after centrifugal, promptly in the core liquid storage vessel; Under certain pressure, the material in the coaxial double-layer pipe is squeezed out simultaneously, splash into 4%CaCl through sterilization
2Be cured moulding in the solution, the formation diameter is that the kernel of 3～4mm is a permeability cerevisiae liquid, and shell is the spherical micro-capsule of solid support material.Solidify after 5 minutes, after with sterilized water washing 2～3 times, be placed in-20 ℃ the refrigerator-freezer freezing the immobilization micro-capsule.After 18 hours, take out micro-capsule, in 0～4 ℃ of environment, thawed 2 hours, get final product for 2 times so repeatedly.It is 7.0 1mol/L phosphoric acid buffer that the micro-capsule that makes is placed the pH of 2～3 times of volumes, and be 30 minutes action time, takes out then and with purified water washing 2 times.Place purification of aqueous solutions after 12 hours the micro-capsule after the washing, promptly get novel high-efficiency multiple micro-capsule biological catalyst.
Example two:
With the purified water configuration concentration is that 10% PVA solution and concentration are 10% sodium alginate soln, and in high-pressure sterilizing pot with 121 ℃ of sterilization 20min; Get 10% PVA solution 85ml, and be prepared into capsule casing material behind 10% the sodium alginate soln 15ml thorough mixing and place the capsule casing material storage vessel of coaxial double-layer outer tube layer (coatings); In addition the bread leaven matricytes of 20 grams being put into 0.5% CTAB solution carries out permeability and handled 10 minutes, cerevisiae after permeability handled is washed 3 times, centrifugal 5 minutes of 3000rmp then repeatedly with deionized water, with pack into the internal layer (core liquid) of coaxial double-layer pipe of the cerevisiae liquid after centrifugal, promptly in the core liquid storage vessel; Under certain pressure, the material in the coaxial double-layer pipe is squeezed out simultaneously, splash into 2%CaCl through sterilization
2Be cured moulding in the solution, the formation diameter is that the kernel of 3～4mm is a permeability bread leaven matricyte liquid, and shell is the spherical micro-capsule of solid support material.Solidify after 5 minutes, after with sterilized water washing 2～3 times, be placed in-20 ℃ the refrigerator-freezer freezing the immobilization micro-capsule.After 12 hours, take out micro-capsule, in 0～4 ℃ of environment, thawed 2 hours, get final product for 2 times so repeatedly.It is 7.0 1mol/L phosphoric acid buffer that the micro-capsule that makes is placed the pH of 2～3 times of volumes, and be 20 minutes action time, takes out then and with purified water washing 2 times.Place purification of aqueous solutions after 12 hours the micro-capsule after the washing, promptly get novel high-efficiency multiple micro-capsule biological catalyst.
Claims (8)
1. make up the high-performance bio catalyzer based on novel porous microcapsule technology, its key step is:
A) cell permeability processing
Beer or bread leaven matricyte that will embedding be placed in cetyl trimethylammonium bromide solution (CTAB) or toluene or the alcohol mixed solvent, handle 2～120 minutes at 4～40 ℃, make cell permeabilityization, increase the enzymic activity of cell.
B) preparation of softgel shell mixing material
Dispose certain density PVA solution and sodium alginate soln with purified water, and in high-pressure sterilizing pot, sterilized 2～120 minutes with 80～150 ℃.Get two kinds of carrier solns by a certain percentage respectively, thorough mixing. prepare softgel shell coating material matrix.
C) preparation of micro-capsule (chemosetting)
Get a certain amount of polyvinyl alcohol and sodium alginate mixed carrier matrix places coaxial double-layer outer tube layer (coatings), promptly in the capsule casing material storage vessel; In addition cerevisiae liquid is packed into the internal layer (core liquid) of coaxial double-layer pipe promptly in the core liquid storage vessel, under certain pressure, squeezes out two kinds of materials in the coaxial double-layer pipe simultaneously, splashes into the CaC through the proper concn of sterilization
12Solidify for some time in the solution, the formation diameter is that the kernel of 3～4mm is a permeability cerevisiae liquid, and shell is the spherical micro-capsule of PVA/CA carrier plural gel.
D) physical solidification of micro-capsule
The micro-capsule that contains permeability yeast cell liquid of chemosetting moulding is washed several times with deionized water, the refrigerator and cooled that is placed on-60～-10 ℃ is then frozen, each freezing time is 1～24 hour, take out after freezing the finishing and be placed on 0～25 ℃ and thaw, thawed 1～5 hour, such frozen-thaw process carries out 1～10 time repeatedly at every turn.
E) preparation of porous microcapsule
The micro-capsule that makes is placed phosphoric acid salt or the citric acid or the edta buffer liquid of 1～5 times of volume, after the some time, take out, and with purified water washing 2 times.Place purification of aqueous solutions after 12 hours the micro-capsule after the washing, promptly get the porous microcapsule biological catalyst.
Liquid environment:
The described PVA aqueous solution is at 0.01%～15% (W/V);
Described sodium alginate concentration is at 0.01%～10% (W/V);
Described CaCl
2Strength of solution is at 0.1%～20% (W/V);
Described phosphoric acid salt or citric acid or EDTA concentration are 0.01～2mol/L;
It is 0.01% to 8% that permeability is handled used CTAB or toluene or alcohol mixed solvent solution; The permeability time is 1～60 minute.
2. in accordance with the method for claim 1, it is characterized in that carrying out permeability with CTAB or toluene or alcohol mixed solvent and handle described in the step a), thereby improve intracellular enzymic activity, thereby further improve the catalytic activity of biological catalyst.
3. in accordance with the method for claim 1, it is characterized in that in the mixing material of softgel shell described in the step b), the concentration of PVA is 1～15%, the concentration of alginate calcium be 0.1～10% with and mutual ratio.
4. in accordance with the method for claim 1, it is characterized in that the CaCl described in the step c)
2Solution is 0.1%～20% (W/V), and be 1～30 minute set time, and formed micro-capsule is that diameter is 3～4mm.
5. Freezing-Melting Condition is-60～-10 ℃, 1～10 time, and each 1～24 hour.
6. in accordance with the method for claim 1, it is characterized in that Freezing-Melting Condition is-60～-10 ℃ in the step d), 1～10 time, each 1～24 hour.
7. in accordance with the method for claim 1, it is characterized in that in the step e), in phosphoric acid salt or citric acid or EDTA solution, vibrate, gel coat punching 1～80 minute.Utilize the susceptibility of gel coat alginate calcium to phosphate solution or other calcium ion complexing agent chemical erosion, by chemical erosion the alginate in the softgel shell gel are partly dissolved, thereby the formation hole is to improve the transmission efficiency of raw material and product when carrying out biocatalytic reaction with biological catalyst.
8. in accordance with the method for claim 1, the porous microcapsule after it is characterized in that using, through removing the softgel shell coatings, clean cell after, can be again through requiring the step described in 1 from b), c), d) to e) described processing, repeated application.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109312294A (en) * | 2016-08-26 | 2019-02-05 | 株式会社钟化 | Freeze yeast cake formed body and its manufacturing method |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109312294A (en) * | 2016-08-26 | 2019-02-05 | 株式会社钟化 | Freeze yeast cake formed body and its manufacturing method |
| CN109312294B (en) * | 2016-08-26 | 2023-04-04 | 株式会社钟化 | Frozen fresh yeast molded body and method for producing same |
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Application publication date: 20101201 |