CN101235368A - Method for preparing immobilized enzyme biological catalyst - Google Patents
Method for preparing immobilized enzyme biological catalyst Download PDFInfo
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- CN101235368A CN101235368A CNA2008100341049A CN200810034104A CN101235368A CN 101235368 A CN101235368 A CN 101235368A CN A2008100341049 A CNA2008100341049 A CN A2008100341049A CN 200810034104 A CN200810034104 A CN 200810034104A CN 101235368 A CN101235368 A CN 101235368A
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Abstract
The invention designs a process for preparation, which utilizes silica-based mesoporous molecular sieves and calcium alginate to fix enzyme biocatalyst, and belongs to the technical field of fixing enzyme biocatalyst preparation technique. The process for preparation comprises utilizing silica-based mesoporous molecular sieves MCM-41 to be carrying agent, fixing biocatalyst molecule in meso-porous pore path of silicon oxide carrier, secondarily fixing immobilized enzyme biocatalyst which is prepared in calcium alginate gel. The process for preparation of the invention adopts a physical adsorption embedding method and a collosol-gel method. The fix enzyme biocatalyst of the invention has better enzyme dispersibility and higher enzymes loading, and has higher enzymatic activity recovery ratio and repeated utilization ratio. The invention can fix various enzymes such as horse radish peroxidase, proteinase, lipase and the like. The fix enzyme biocatalyst of the invention can be used to process waste water which contains hydroxybenzene and decolorize, which has higher catalytic activity.
Description
Technical field
The present invention designs a kind of preparation method who uses silicon-based mesoporous molecular sieve and alginate calcium immobilized enzyme biological catalyst, belongs to immobilized enzyme biological catalyst fabricating technology field.
Background technology
The synthetic of ordered mesoporous molecular sieve just began as far back as the seventies in 20th century, and just the report of the mesopore molecular sieves such as MCM-41 of Mobil company in 1992 just causes people's extensive attention, and was considered to the ordered mesoporous molecular sieve synthetic and really begins.They use tensio-active agent as template, have synthesized M41S series mesopore molecular sieve.Have big specific surface area, the duct is regularly arranged and the synthetic of adjustable ordered mesoporous molecular sieve series M41S is called a milestone on the molecular sieve development history by people.There are various synthetic systems and route of synthesis to come out afterwards again, many just researchs of these ordered mesoporous molecular sieves and Application Areas are needed, it is current class recruit sieve with wide application prospect, send at the catalyzer, semi-conductor, optics, computer, senser element, the medicine that separate purification, biomolecules sieve, chemosynthesis and conversion, many research fields such as gas and liquid adsorption, acoustics, Superlight molecular sieve have the potential purposes, at chemical industry, numerous areas such as biotechnology, environmental energy have important use and are worth.The aperture of mesopore molecular sieve is in very large range adjustable, so can not be replaced by zeolite molecular sieve, therefore, becomes one of popular research field.Mesopore molecular sieve is because the adjustable aperture of its homogeneous, high specific surface area and regular pore passage structure and be easy to advantages such as surface-functionalized, what be widely used in zymoprotein at present fixedly serves as the biocatalysis agent carrier, solved natural enzyme poor stability, easy inactivation, can not reuse, and sneak into product after the reaction, purification difficult makes problems such as its application is restricted.Not as before the fixation support of enzyme, the silica-based inorganic molecule sieve of sol-gel processing synthetic once was used to the fixing or embedding of bioactive moleculess such as enzyme widely at mesopore molecular sieve, and it is few to it is advantageous that enzyme leaks.But the activity of enzyme is subjected to colloidal sol-preparing gel condition effect, and mass transfer has been limited in narrow and small grid duct; Simultaneously, sol-gel processing is difficult to the distribution in control aperture, and is very little to the selectivity of fixation particle diameter; And enzyme still coagulable in this class matrix, and the carrier that fixed orifices distributes will help preventing condensing of enzyme.In addition, because the easy inactivation of water-soluble enzyme, less stable, and can only use and once cause the processing cost height.In order to improve enzymic activity, stability and to reduce cost, the immobilization technology of development enzyme is the main direction of studying in this field.
Summary of the invention
The preparation method who the purpose of this invention is to provide a kind of immobilized enzyme biocatalyst.The inventive method is to be carrier with silicon-based mesoporous molecular sieve MCM-41, and fixed biologically enzyme molecule in the mesopore orbit of silica supports is fixed in the immobilized enzyme biocatalyst secondary for preparing in the calcium alginate gel body again.
The present invention is a kind of preparation method of immobilized enzyme biocatalyst, it is characterized in that having following preparation process and step:
A. the preparation of silicon-based mesoporous molecular sieve carrier:
Zhi Bei silicon-based mesoporous molecular sieve MCM-41 carrier at first: with the hexadecyl brometo de amonio is template, with tetraethoxy as the silicon source.The hexadecyl brometo de amonio is dissolved in the deionized water, adds proper ammonia, dehydrated alcohol and expanding agent TMB again in above-mentioned solution.Stirred solution adds tetraethoxy then, at room temperature stirs 1-2 hour, then sample solution is carried out suction filtration, washing, oven dry processing, and the white powder that obtains is the former MCM-41 that has template, is that former MCM-41 is calcined at last.Calcining temperature is 550 ℃, and keeps 6 hours under this temperature, then cooling naturally.Powder after calcining promptly is the silica-based molecular sieve MCM-41 that has mesopore orbit.
B. upload enzyme or immobilized enzyme at silicon-based mesoporous molecular sieve:
The silicon-based mesoporous molecular sieve MCM-41 of preparation is joined in the phosphate buffer soln, and place on the magnetic stirring apparatus and to stir 1-2 hour, it is disperseed in solution, add enzyme then, it is fixed, and used enzyme is any of horseradish peroxidase, proteolytic enzyme or lipase.Continue to stir 3-4 hour after adding enzyme, separate with high-speed centrifuge then, and with the seasoning at room temperature of separating obtained product, dried year enzyme mesopore molecular sieve joined in the sodium alginate soln, after stirring, splash in the calcium chloride solution with syringe, make its reaction 1-2 hour.Control injection volume and speed, finally make the gel beads of different-grain diameter, be immobilized enzyme biocatalyst.
Adopted physical adsorption entrapping method and sol-gel processing in the inventive method, the immobilized enzyme biocatalyst that makes has the dispersed and higher enzyme amount of carrying of good enzyme, and higher the enzymic activity rate of recovery and repeating utilization factor are arranged during use.In addition, its technology is simple, and is easy to operate, and product stock is stable.
Mesopore molecular sieve is compared with other carriers as enzyme immobilization carrier, and the former has certain superiority: the aperture of mesopore molecular sieve is adjustable, applicable to the immobilization of plurality of enzymes; In addition, the slightly acidic hydroxyl is rich on the surface, helps the immobilization of zymoprotein molecule.Adopt alginate calcium embedding once more, have better mechanical stability and thermostability, reduce the spillage of enzyme in immobilized enzyme biocatalyst, thereby increase the repeated use number of times of immobilized enzyme biocatalyst.
Description of drawings
Fig. 1 is an immobilized enzyme biocatalyst process flow sheet of the present invention.
Fig. 2 removes the recycling number of times and the phenol clearance of phenol in the water for immobilized enzyme biocatalyst of the present invention.
Embodiment
Now a specific embodiment of the present invention is given unnecessary details in after.
Process flow sheet referring to Fig. 1.At first under normal temperature condition, prepare silicon-based mesoporous molecular sieve MCM-41 carrier.With the hexadecyl brometo de amonio is template, and tetraethoxy is as the silicon source.Earlier the CTAB with 0.75g is dissolved in the deionized water of 15g, take by weighing then 4.50g ammoniacal liquor (25wt%, 0.066mol), the dehydrated alcohol (C of 23ml
2H
5OH) and 0.6g expanding agent TMB join in the surfactant soln.Stirred solution 15min, the tetraethoxy (TEOS) that adds 1.4g then, at room temperature stir 2h after, sample solution is carried out suction filtration, washing, oven dry handles, the white powder of gained is the former MCM-41 that has template, is that former MCM-41 is calcined at last.Calcining temperature is 550 ℃, and keeps 6 hours under this temperature, then cooling naturally.Powder after the calcining promptly is the MCM-41 that has the duct.It is in 8 the 10ml phosphate buffer soln that 0.25gMCM-41 is joined pH, places and stirs 1h on the magnetic stirring apparatus, and it is uniformly dispersed in solution.Stirring finishes, and horseradish peroxidase is added in the solution, makes the enzyme concn in the solution reach 50u/ml, continues to stir 4 hours.Stirring finishes, at room temperature with the centrifugal 20min of 8000rpm speed, and with products therefrom seasoning at room temperature.Dried year enzyme mesopore molecular sieve joined in 3% the sodium alginate soln of 5ml, after stirring, splash in 2% calcium chloride solution reaction 1h with syringe.The control injection speed makes the gel beads that particle diameter is 4mm, is immobilized enzyme biocatalyst.
With above-mentioned embedding the immobilized biocatalyst of horseradish peroxidase handle the phenol solution that total phenol content is 100mg/L, test shows, after 10 recyclings, immobilized enzyme biocatalyst still has advantages of high catalytic activity, and the clearance of the phenol in the aqueous solution still can reach more than 80%.Referring to Fig. 2, the recycling number of times of this horseradish peroxidase biological catalyst phenol in removing water and the empirical curve of corresponding phenol clearance have been shown among Fig. 2.
Claims (1)
1. the preparation method of an immobilized enzyme biocatalyst is characterized in that having following preparation process and step:
A. the preparation of silicon-based mesoporous molecular sieve MCM-41 carrier: with the hexadecyl brometo de amonio is template, with tetraethoxy as the silicon source.The hexadecyl brometo de amonio is dissolved in the deionized water, adds proper ammonia, dehydrated alcohol and expanding agent TMB again in above-mentioned solution.Stirred solution adds tetraethoxy then, at room temperature stirs 1-2 hour, then sample solution is carried out suction filtration, washing, oven dry processing, and the white powder that obtains is the former MCM-41 that has template, is that former MCM-41 is calcined at last.Calcining temperature is 550 ℃, and keeps 6 hours under this temperature, then cooling naturally.Powder after calcining promptly is the silica-based molecular sieve MCM-41 that has mesopore orbit.
B. upload enzyme or immobilized enzyme at silicon-based mesoporous molecular sieve: the silicon-based mesoporous molecular sieve MCM-41 carrier of step a preparation is joined in the phosphate buffer soln, and place on the magnetic stirring apparatus and to stir 1-2 hour, it is disperseed in solution, add enzyme then, it is fixed, and used enzyme is any of horseradish peroxidase, proteolytic enzyme or lipase.Continue to stir 3-4 hour after adding enzyme, separate with high-speed centrifuge then, and with the seasoning at room temperature of separating obtained product, dried year enzyme mesopore molecular sieve joined in the sodium alginate soln, after stirring, splash in the calcium chloride solution with syringe, make its reaction 1-2 hour.Control injection volume and speed, finally make the gel beads of different-grain diameter, be immobilized enzyme biocatalyst.
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101514338B (en) * | 2008-12-04 | 2011-11-16 | 浙江大学 | Method for preparing immobilized lipase by adopting methacryloxypropyl gels |
| CN101575135B (en) * | 2009-06-05 | 2011-12-21 | 上海大学 | Method for separating out phenols substances in water by catalytic oxidation with enzyme-carrying silicon-based mesoporous molecular sieve SBA-15 |
| CN102304505A (en) * | 2011-08-19 | 2012-01-04 | 无锡市寰创环境科技发展有限公司 | Synthesis method for peroxidase-magnetic iron nanoparticle functional material |
| CN102533913A (en) * | 2011-12-23 | 2012-07-04 | 南昌大学 | Method for preparing antioxidant active peptide by hydrolyzing fish scale collagen with co-immobilized double enzymes |
| CN103013976A (en) * | 2013-01-21 | 2013-04-03 | 天津工业大学 | Method for preparing organic-inorganic composite hydrogel membrane and grafting material containing immobilized biological macromolecules |
| CN108383083A (en) * | 2018-03-16 | 2018-08-10 | 三峡大学 | A kind of support type hydrochloric acid decolorising agent and preparation method thereof and decoloration process |
| CN108926745A (en) * | 2018-08-01 | 2018-12-04 | 北京大学 | A kind of preparation method and its compound system of nanoporous micro rack |
| CN109097353A (en) * | 2018-08-22 | 2018-12-28 | 江苏邦臣生物科技股份有限公司 | A kind of chemically composited catalyst of biological enzyme-and preparation method thereof |
| CN109750025A (en) * | 2018-12-29 | 2019-05-14 | 浙江医药股份有限公司昌海生物分公司 | A kind of hydrogel cladding dendroid silica immobilization CPO enzyme reactor and its preparation method and application |
| CN113025607A (en) * | 2021-03-24 | 2021-06-25 | 江南大学 | Interface immobilized enzyme based on amphiphilic mesoporous nano silicon spheres and preparation method thereof |
| CN113636892A (en) * | 2021-09-23 | 2021-11-12 | 山东爱果者生物技术有限公司 | Preparation method of seaweed extract and seaweed fertilizer |
| CN115703640A (en) * | 2021-08-13 | 2023-02-17 | 同济大学 | Molecular sieve microsphere material, preparation method and loudspeaker |
| CN116180268A (en) * | 2023-04-24 | 2023-05-30 | 山东天纤新材料有限公司 | Diclofenac sodium modified terylene large biological fiber and preparation method thereof |
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2008
- 2008-02-29 CN CNA2008100341049A patent/CN101235368A/en active Pending
Cited By (14)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101514338B (en) * | 2008-12-04 | 2011-11-16 | 浙江大学 | Method for preparing immobilized lipase by adopting methacryloxypropyl gels |
| CN101575135B (en) * | 2009-06-05 | 2011-12-21 | 上海大学 | Method for separating out phenols substances in water by catalytic oxidation with enzyme-carrying silicon-based mesoporous molecular sieve SBA-15 |
| CN102304505A (en) * | 2011-08-19 | 2012-01-04 | 无锡市寰创环境科技发展有限公司 | Synthesis method for peroxidase-magnetic iron nanoparticle functional material |
| CN102533913A (en) * | 2011-12-23 | 2012-07-04 | 南昌大学 | Method for preparing antioxidant active peptide by hydrolyzing fish scale collagen with co-immobilized double enzymes |
| CN102533913B (en) * | 2011-12-23 | 2013-07-31 | 南昌大学 | Method for preparing antioxidant active peptide by hydrolyzing fish scale collagen with co-immobilized double enzymes |
| CN103013976A (en) * | 2013-01-21 | 2013-04-03 | 天津工业大学 | Method for preparing organic-inorganic composite hydrogel membrane and grafting material containing immobilized biological macromolecules |
| CN108383083A (en) * | 2018-03-16 | 2018-08-10 | 三峡大学 | A kind of support type hydrochloric acid decolorising agent and preparation method thereof and decoloration process |
| CN108926745A (en) * | 2018-08-01 | 2018-12-04 | 北京大学 | A kind of preparation method and its compound system of nanoporous micro rack |
| CN109097353A (en) * | 2018-08-22 | 2018-12-28 | 江苏邦臣生物科技股份有限公司 | A kind of chemically composited catalyst of biological enzyme-and preparation method thereof |
| CN109750025A (en) * | 2018-12-29 | 2019-05-14 | 浙江医药股份有限公司昌海生物分公司 | A kind of hydrogel cladding dendroid silica immobilization CPO enzyme reactor and its preparation method and application |
| CN113025607A (en) * | 2021-03-24 | 2021-06-25 | 江南大学 | Interface immobilized enzyme based on amphiphilic mesoporous nano silicon spheres and preparation method thereof |
| CN115703640A (en) * | 2021-08-13 | 2023-02-17 | 同济大学 | Molecular sieve microsphere material, preparation method and loudspeaker |
| CN113636892A (en) * | 2021-09-23 | 2021-11-12 | 山东爱果者生物技术有限公司 | Preparation method of seaweed extract and seaweed fertilizer |
| CN116180268A (en) * | 2023-04-24 | 2023-05-30 | 山东天纤新材料有限公司 | Diclofenac sodium modified terylene large biological fiber and preparation method thereof |
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