CN104277969A - Preparation method of whole-cell catalytic transesterification reaction micro-reactor - Google Patents
Preparation method of whole-cell catalytic transesterification reaction micro-reactor Download PDFInfo
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- CN104277969A CN104277969A CN201310283691.6A CN201310283691A CN104277969A CN 104277969 A CN104277969 A CN 104277969A CN 201310283691 A CN201310283691 A CN 201310283691A CN 104277969 A CN104277969 A CN 104277969A
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Abstract
The invention relates to a preparation method of a whole-cell catalytic transesterification reaction micro-reactor and belongs to the technical field of biochemical engineering. The preparation method comprises that a chitosan powder (CTS)/polyvinylidene fluoride (PVDF) composite microcapsule is prepared from CTS powder, PVDF powder, sodium alginate, sodium citrate and calcium chloride as raw materials by curing balling, sol-gel membrane formation and liquefaction capsule-formation. The CTS/ PVDF composite microcapsule can be used as a whole-cell catalytic transesterification reaction micro-reactor. The CTS/ PVDF composite microcapsule has uniform particle sizes, good mechanical properties and good permselectivity, can be used for fixing lipase-producing bacterium whole-cell and greatly reduces an enzyme activity loss late.
Description
Technical field
The present invention relates to a kind of preparation method for whole-cell catalytic transesterification reaction microreactor.The invention belongs to technical field of biochemical industry.
Background technology
For optimizing biological respinse and transmitting performance, the microminiaturization of bio-reactor is one of new developing direction.Microreactor (also known as microcapsule), as the fixation support of cell, the selective penetrated property of outer polymeric membrane can ensure that the exchange of substance inside and outside microreactor, and interior spatial structure provides the surface of cell attachment, outside stirring can make that microcapsule are in suspended state and shearing force can not injure cytoactive, and be conducive to the object that cell mass tissue differentiation reaches volume increase, prior, the active substance that the inner cell of microcapsule produces can be reused, simplify purification procedures.Therefore, microcapsule earn widespread respect in cell large scale culturing and application aspect.
At present, material for the preparation of microcapsule comprises natural polymer (sodium alginate, polylysine, chitosan, starch etc.), semi-synthetic macromolecular material (contracting methylcellulose gum, methylcellulose gum, ethyl cellulose etc.) and complete synthesis macromolecular material (polyethylene, polystyrene, polyhutadiene, polypropylene, polyethers, polyureas, polyoxyethylene glycol, polyvinyl alcohol, polymeric amide, polyacrylamide, urethane, polymethylmethacrylate, polyvinylpyrrolidone, epoxy resin, polysiloxane etc.) three major types, be applied to Encapsulated Enzyme, medicine or cell etc.But above-mentioned microencapsulation material permeability is poor; though the environment of Cell protection can be provided; stop shearing force, phage etc. to the infringement of cell; but also make cell be in state of isolating relative to outside simultaneously; the exchange of substance of cell and environment is obstructed, and the input and the inner metabolism refuse that limit external nutrition export, and have both been unfavorable for that Growth of Cells was bred; also the output of its meta-bolites is unfavorable for, if cyst membrane intensity is more weak more easily mechanical damage occurs.Therefore the permeability of microcapsule membrane and physical strength affect its key factor applied.Chitosan is one of microencapsulation material comparatively commonly used, there is excellent film-forming properties, security, biocompatibility, but its mechanicalness is poor, greatly limit its application as catalyzed transesterification microreactor, the permeability of its film forming can not meet the requirement of catalyzed transesterification microreactor simultaneously.
Summary of the invention
The object of the invention is for above-mentioned existing problems, a kind of preparation method for whole-cell catalytic transesterification reaction microreactor is provided, namely the film-forming properties of chitosan and affinity, the nontoxicity to cell thereof is utilized, in conjunction with the film-forming properties of polyvinylidene difluoride (PVDF), porousness and excellent mechanicalness etc., obtain the whole-cell catalytic transesterification reaction microreactor of high-throughput, high strength.
The invention provides a kind of preparation method for whole-cell catalytic transesterification reaction microreactor, with Chitosan powder (CTS), polyvinylidene difluoride (PVDF) powder (PVDF), sodium alginate, Trisodium Citrate and calcium chloride for raw material, by solidification balling-up, sol-gel film forming, be liquefied as the operations such as capsule, prepare CTS/PVDF composite membrane microcapsule.
Preparation process comprises the steps:
1) in fermentation culture, add sodium alginate, form solution A; Solution A is dropwise instilled in calcium chloride solution, carries out calcification balling-up;
2) chitosan is dissolved in acetic acid, stirs and swelling, form solution B; Polyvinylidene difluoride (PVDF) is dissolved in N,N-DIMETHYLACETAMIDE, stirs and swelling, form solution C;
3) solution B is fully mixed according to a certain volume with solution C, form solution D;
4) by 1) in the calcification bead that formed be placed in solution D and flood certain hour, be then placed in 10% sodium hydroxide solution and solidify.
5) by 4) in bead after solidification be placed in sodium citrate solution and liquefy certain hour, obtain CTS/PVDF composite membrane microcapsule.
The mass concentration of described sodium alginate soln is 1-50%;
The mass concentration of described calcium chloride solution is 0.1-40%;
The mass concentration of described chitosan in acetic acid is 0.1-40%;
The mass concentration of described polyvinylidene difluoride (PVDF) in N,N-DIMETHYLACETAMIDE is 0.1-30%;
Described chitosan solution B and polyvinylidene difluoride (PVDF) solution C volume ratio are 50-1: 1;
Described calcification bead dipping time in solution D is 0.1-100h;
The mass concentration of described Trisodium Citrate is 0.01-50%;
After described solidification, bead liquefying time in sodium citrate solution is 0.1-80h.
CTS/PVDF composite membrane microcapsule can be used as whole-cell catalytic transesterification reaction microreactor.
Advantage of the present invention and effect: the Microcapsules Size obtained through the present invention is even, and mechanicalness is better, and selective permeability is good, for fixing yielding lipase bacterial whole cell, enzyme rate of loss alive reduces greatly.
Embodiment
Below in conjunction with embodiment, the present invention is further described, but they are not impose any restrictions the present invention.
Embodiment 1:
A kind of preparation method for whole-cell catalytic transesterification reaction microreactor, with Chitosan powder (CTS), polyvinylidene difluoride (PVDF) powder (PVDF), sodium alginate, Trisodium Citrate and calcium chloride for raw material, by solidification balling-up, sol-gel film forming, be liquefied as the operations such as capsule, prepare CTS/PVDF composite membrane microcapsule.
Preparation process comprises the steps:
1) fermentation culture 2 parts is prepared: peptone 1.0%, glucose 2.0%, ammonium sulfate 0.1%, yeast extract 0.5%, magnesium sulfate 0.05%, sweet oil 1.0%, dipotassium hydrogen phosphate 0.2%.PH is adjusted to be 8.5,1 × 10 before using
5pa sterilizing 20min.
2) in 1 part of fermentation culture, add sodium alginate, to stir and swelling, form the solution A containing 6% sodium alginate; Solution A dropwise being dripped to concentration is in the calcium chloride solution of 5%, calcification 24h, and obtain the gelled pill that particle diameter is about 3-4mm, physical strength is good;
3) chitosan is dissolved in acetic acid, stirs and swelling, form the solution B containing 2% chitosan; Polyvinylidene difluoride (PVDF) is dissolved in N,N-DIMETHYLACETAMIDE, stirs and swelling, form the solution C containing 2.25% polyvinylidene difluoride (PVDF);
4) ratio being 15: 1 by volume by solution B and solution C fully mixes, and forms solution D;
5) by 2) in the calcification bead that formed be placed in solution D and flood 10h, then take out and be placed in 10% sodium hydroxide solution and solidify 24h.
6) by 5) in bead after solidification be placed in 0.5% sodium citrate solution and liquefy 15h, obtain CTS/PVDF composite membrane microcapsule, diameter is 5-6mm.
7) in aseptic Bechtop, bacillus cloacae is inoculated into 6) the middle microcapsule inside formed, be placed in another portion of fermented liquid immediately to cultivate, temperature is 32 DEG C, rotating speed is 120rmp/min, get 1ml fermented liquid after 5 days and survey lipase enzymatic activity, result shows that enzyme rate of loss alive is only 20.7%.
Embodiment 2:
A kind of preparation method for whole-cell catalytic transesterification reaction microreactor, with Chitosan powder (CTS), polyvinylidene difluoride (PVDF) powder (PVDF), sodium alginate, Trisodium Citrate and calcium chloride for raw material, by solidification balling-up, sol-gel film forming, be liquefied as the operations such as capsule, prepare CTS/PVDF composite membrane microcapsule.
Preparation process comprises the steps:
1) fermentation culture 2 parts is prepared: peptone 1.0%, glucose 2.0%, ammonium sulfate 0.1%, yeast extract 0.5%, magnesium sulfate 0.05%, sweet oil 1.0%, dipotassium hydrogen phosphate 0.2%.PH is adjusted to be 8.5,1 × 10 before using
5pa sterilizing 20min.
2) in 1 part of fermentation culture, add sodium alginate, to stir and swelling, form the solution A containing 20% sodium alginate; Solution A dropwise being dripped to concentration is in the calcium chloride solution of 15%, calcification 24h, and obtain the gelled pill that particle diameter is about 3-4mm, physical strength is good;
3) chitosan is dissolved in acetic acid, stirs and swelling, form the solution B containing 20% chitosan; Polyvinylidene difluoride (PVDF) is dissolved in N,N-DIMETHYLACETAMIDE, stirs and swelling, form the solution C containing 22% polyvinylidene difluoride (PVDF);
4) ratio being 30: 1 by volume by solution B and solution C fully mixes, and forms solution D;
5) by 2) in the calcification bead that formed be placed in solution D and flood 20h, then take out and be placed in 10% sodium hydroxide solution and solidify 24h.
6) by 5) in bead after solidification be placed in 5% sodium citrate solution and liquefy 25h, obtain CTS/PVDF composite membrane microcapsule, diameter is 5-6mm.
7) in aseptic Bechtop, bacillus cloacae is inoculated into 6) the middle microcapsule inside formed, be placed in another portion of fermented liquid immediately to cultivate, temperature is 32 DEG C, rotating speed is 120rmp/min, get 1ml fermented liquid after 5 days and survey lipase enzymatic activity, result shows that enzyme rate of loss alive is only 35.5%.
Embodiment 3:
A kind of preparation method for whole-cell catalytic transesterification reaction microreactor, with Chitosan powder (CTS), polyvinylidene difluoride (PVDF) powder (PVDF), sodium alginate, Trisodium Citrate and calcium chloride for raw material, by solidification balling-up, sol-gel film forming, be liquefied as the operations such as capsule, prepare CTS/PVDF composite membrane microcapsule.
Preparation process comprises the steps:
1) fermentation culture 2 parts is prepared: peptone 1.0%, glucose 2.0%, ammonium sulfate 0.1%, yeast extract 0.5%, magnesium sulfate 0.05%, sweet oil 1.0%, dipotassium hydrogen phosphate 0.2%.PH is adjusted to be 8.5,1 × 10 before using
5pa sterilizing 20min.
2) in 1 part of fermentation culture, add sodium alginate, to stir and swelling, form the solution A containing 30% sodium alginate; Solution A dropwise being dripped to concentration is in the calcium chloride solution of 10%, calcification 24h, and obtain the gelled pill that particle diameter is about 3-4mm, physical strength is good;
3) chitosan is dissolved in acetic acid, stirs and swelling, form the solution B containing 6% chitosan; Polyvinylidene difluoride (PVDF) is dissolved in N,N-DIMETHYLACETAMIDE, stirs and swelling, form the solution C containing 8.5% polyvinylidene difluoride (PVDF);
4) ratio being 8: 1 by volume by solution B and solution C fully mixes, and forms solution D;
5) by 2) in the calcification bead that formed be placed in solution D and flood 35h, then take out and be placed in 10% sodium hydroxide solution and solidify 24h.
6) by 5) in bead after solidification be placed in 15% sodium citrate solution and liquefy 8h, obtain CTS/PVDF composite membrane microcapsule, diameter is 5-6mm.
7) in aseptic Bechtop, bacillus cloacae is inoculated into 6) the middle microcapsule inside formed, be placed in another portion of fermented liquid immediately to cultivate, temperature is 32 DEG C, rotating speed is 120rmp/min, get 1ml fermented liquid after 5 days and survey lipase enzymatic activity, result shows that enzyme rate of loss alive is only 23.4%.
The present invention discloses a kind of preparation method for whole-cell catalytic transesterification reaction microreactor, those skilled in the art are by using for reference present disclosure, the links such as appropriate change operational path realize, although method of the present invention is described by preferred embodiment, person skilled obviously can change method as herein described or reconfigure not departing from content of the present invention and scope, realizes net result.Special needs to be pointed out is, all similar replacements and change apparent to those skilled in the art, they are all deemed to be included in content of the present invention and scope.
Claims (10)
1. the preparation method for whole-cell catalytic transesterification reaction microreactor, with Chitosan powder (CTS), polyvinylidene difluoride (PVDF) powder (PVDF), sodium alginate, Trisodium Citrate and calcium chloride for raw material, by solidification balling-up, sol-gel film forming, be liquefied as the operations such as capsule, prepare CTS/PVDF composite membrane microcapsule.Preparation process comprises the steps:
1) in fermentation culture, add sodium alginate, form solution A; Solution A is dropwise instilled in calcium chloride solution, carries out calcification balling-up;
2) chitosan is dissolved in acetic acid, stirs and swelling, form solution B; Polyvinylidene difluoride (PVDF) is dissolved in N,N-DIMETHYLACETAMIDE, stirs and swelling, form solution C;
3) solution B is fully mixed according to a certain volume with solution C, form solution D;
4) by 1) in the calcification bead that formed be placed in solution D and flood certain hour, be then placed in 10% sodium hydroxide solution and solidify.
5) by 4) in after solidification bead be placed in sodium citrate solution and liquefy certain hour, obtain CTS/PVDF composite membrane microcapsule.
2. CTS/PVDF composite membrane microcapsule according to claim 1, it is characterized in that: CTS/PVDF composite membrane microcapsule can be used as whole-cell catalytic transesterification reaction microreactor, uniform particle sizes, mechanicalness is good, selective permeability is good, for fixing yielding lipase bacterial whole cell, enzyme rate of loss alive reduces greatly.
3. the preparation method for whole-cell catalytic transesterification reaction microreactor according to claim 1, is characterized in that: the mass concentration of described sodium alginate soln is 1-50%.
4. the preparation method for whole-cell catalytic transesterification reaction microreactor according to claim 1, is characterized in that: the mass concentration of described calcium chloride solution is 0.1-40%.
5. the preparation method for whole-cell catalytic transesterification reaction microreactor according to claim 1, is characterized in that: the mass concentration of described chitosan in acetic acid is 0.1-40%.
6. the preparation method for whole-cell catalytic transesterification reaction microreactor according to claim 1, is characterized in that: the mass concentration of described polyvinylidene difluoride (PVDF) in N,N-DIMETHYLACETAMIDE is 0.1-30%.
7. the preparation method for whole-cell catalytic transesterification reaction microreactor according to claim 1, is characterized in that: described chitosan solution B and polyvinylidene difluoride (PVDF) solution C volume ratio are 50-1: 1.
8. the preparation method for whole-cell catalytic transesterification reaction microreactor according to claim 1, is characterized in that: described calcification bead dipping time in solution D is 0.1-100h.
9. the preparation method for whole-cell catalytic transesterification reaction microreactor according to claim 1, is characterized in that: the mass concentration of described Trisodium Citrate is 0.01-50%.
10. the preparation method for whole-cell catalytic transesterification reaction microreactor according to claim 1, is characterized in that: after solidification, bead liquefying time in sodium citrate solution is 0.1-80h.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1546660A (en) * | 2003-12-01 | 2004-11-17 | 四川大学 | Microcapsule carrier with membrane hole for fixing microorganism and its preparation method |
| CN1616656A (en) * | 2003-11-10 | 2005-05-18 | 中国科学院大连化学物理研究所 | Method for preparing fixed yeast cell micro capsule |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1616656A (en) * | 2003-11-10 | 2005-05-18 | 中国科学院大连化学物理研究所 | Method for preparing fixed yeast cell micro capsule |
| CN1546660A (en) * | 2003-12-01 | 2004-11-17 | 四川大学 | Microcapsule carrier with membrane hole for fixing microorganism and its preparation method |
Non-Patent Citations (1)
| Title |
|---|
| 王家荣: "海藻酸钠-壳聚糖-海藻酸钠生物微胶囊的制备", 《宁波大学学报》, vol. 20, no. 4, 31 December 2007 (2007-12-31) * |
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