CN101492665B - Solid phase support crosslinking enzyme aggregation and method for preparing the same - Google Patents
Solid phase support crosslinking enzyme aggregation and method for preparing the same Download PDFInfo
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- CN101492665B CN101492665B CN2008100565942A CN200810056594A CN101492665B CN 101492665 B CN101492665 B CN 101492665B CN 2008100565942 A CN2008100565942 A CN 2008100565942A CN 200810056594 A CN200810056594 A CN 200810056594A CN 101492665 B CN101492665 B CN 101492665B
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Abstract
The invention discloses immobilized crosslinked enzyme aggregates (CLEAs) and a preparation method thereof. The crosslinked enzyme aggregates are fixed on a porous microsphere; the preparation method thereof comprises: dispersing detached enzyme needing to be immobilized into the porous microsphere; carrying out depositing and crosslinking on zymoprotein in the porous microsphere in sequence, and finally rinsing the porous microsphere, thus preparing the immobilized crosslinked enzyme aggregates. The method of the immobilized enzyme can be used for immobilizing the enzyme of various types or sources; the immobilized method is simple and easy to be carried out, is easy to be operated, does not need special devices and has low production cost; the prepared crosslinked enzyme aggregates have high stability and strong capacity for resisting trypsinase, has a certain physical form and a stable structure, can be recycled and repeatedly used and can be used for continuous industrial production.
Description
Technical field
The present invention relates to immobilized enzyme, relate in particular to and be fixed in cross-linked enzyme aggregate on the solid phase carrier and preparation method thereof, belong to the immobilization field of enzyme.
Background technology
Begun the sixties from eighties of last century since the immobilization research of enzyme, people inquire into and study the immobilization technology of enzyme in all its bearings, successfully used the method for physics and chemistry that enzyme has been carried out immobilization, wherein many technology and product have obtained application in industrial production.But the fixed object all is resolvase basically, because resolvase is higher to environmental requirement, and relatively more fragile soluble protein, generally speaking, all unstable to heat, strong acid, highly basic.In process for fixation commonly used, because stronger chemical reaction, usually proteinic character is caused great influence, because enzyme has certain senior space structure again, very easily be subjected to the influence of factors such as tensio-active agent, carrier surface physical behavior, chemical graft, irreversible destruction is caused in space structure or active centre to enzyme, makes enzyme activity descend or inactivation, has had a strong impact on the effect and the use of immobilized enzyme.
Sheldon group of Dutch Delft university in 2002 proposes to precipitate zymoprotein with protein precipitant, obtain having the enzyme aggregate of stable state structure, use glutaraldehyde cross-linking again, prepared cross-linked enzyme aggregate (Cross Linked EnzymeyAggregates, CLEAs).It is crosslinked that seven kinds of business-like lipase have been carried out aggregate, the carrier-free immobilization enzyme (P.Lopez-Serrano, L.Cao, the F.van Rantwijk ﹠amp that have obtained having better enzymic activity; R.A.Sheldon etal.Cross-linked enzyme aggregates with enhanced activity:application tolipases.Biotechnology letters, 2002,24:1379-1383).Dong Xiao in 2003 is firm, and that urase has been carried out aggregate is crosslinked, obtained to have better stability cross-linked urease congeries (preparation of Dong Xiaoyi, Xia Shiwen cross-linked urease congeries and Preliminary Applications [J]. biotechnology journal, 2003,19 (3): 332 336).
Aggregate crosslinking enzyme (CLEAs) is a kind of immobilized enzyme that grows up behind crosslinked lytic enzyme (CLEs), crystal crosslinking enzyme (CLECs), belongs to the scope of carrier-free immobilization.Because the enzyme molecule is in the stable state structure in the aggregate crosslinking enzyme, so characteristics such as the immobilized enzyme that forms has the thermostability height, and the antisteapsin ability is strong.But because the aggregate crosslinking enzyme is a carrier-free immobilization, has the dispersiveness of height, so lost the topmost characteristics of immobilized enzyme: be difficult to after reaction finishes recycle; Do not have the certain physical form, in suitability for industrialized production, can not be used for continuity production.
Summary of the invention
The present invention's technical problem at first to be solved is to overcome the existing shortcoming that can not recycling of aggregate crosslinking enzyme, a kind of cross-linked enzyme aggregate that is fixed on the carrier is provided, this cross-linked enzyme aggregate removes has the thermostability height, outside the advantages such as the antisteapsin ability is strong, also have certain physical form and rock steady structure, can recycle, can be applied successively to produce.
The present invention's technical problem at first to be solved is achieved through the following technical solutions:
A kind of cross-linked enzyme aggregate that is fixed on the solid phase carrier, wherein, described solid phase carrier is a porous microsphere.
Described porous microsphere can be made by various materials, for example, can be made by organic materials (for example organic polymer porous material etc.), inorganic materials materials such as (sintered glass, porous ceramicss etc.); With the immobilization carrier of described porous microsphere, need not it is carried out special processing, as long as its main hole is greater than treating 1～2 times of fixed protein molecule size as cross-linked enzyme aggregate.Preferably, the mean pore size of porous microsphere of the present invention is between 10nm～300nm, more preferably 30nm.
But the scope that is applicable to immobilized enzyme of the present invention is very extensive, so long as can (be adopted existing aggregate crosslinking technological it can be prepared into the aggregate crosslinking enzyme in other words by the sedimentary enzyme of protein precipitant, the enzyme of this class is all applicable to the present invention) all can be used as the object of immobilized enzyme of the present invention basically, for example, can be organic phosphorus degrading enzyme, Sumylact L, zytase, lipase, glucose oxidase or phytase etc., be preferably organic phosphorus degrading enzyme, Sumylact L.
Another technical problem to be solved by this invention provides a kind of method for preparing the cross-linked enzyme aggregate of said fixing on solid phase carrier.
Another technical problem to be solved by this invention is achieved through the following technical solutions:
The method of the cross-linked enzyme aggregate on the solid phase carrier is fixed in a kind of preparation, comprises the precipitation, crosslinked of zymoprotein, and it is characterized in that: at first allowing needs fixed free state enzyme to be scattered in the porous microsphere; In porous microsphere, carry out the precipitation, crosslinked of zymoprotein more successively, last rinsing porous microsphere, promptly.
Preferably, described dispersing method comprises: the free state enzyme is dissolved in adding porous microsphere in back in the damping fluid, slowly stirs, allow the free state enzyme be scattered in the porous microsphere; Preferred, the free state enzyme is dissolved in the phosphate buffered saline buffer, be mixed with the enzyme solution that concentration is 0.1-1.5mg/ml (more preferably being mixed with the enzyme solution of 0.4-0.5mg/ml), under 4～25 ℃ of temperature condition, slowly stirred 1～3 hour, allow the free state enzyme be scattered in the porous microsphere.
The precipitation of the precipitation of described zymoprotein, method such as crosslinked zymoprotein during with the existing preparation cross-linked enzyme aggregate, crosslinked method are identical; Preferably, the precipitation of described zymoprotein is carried out in accordance with the following methods: the protein precipitant of adding slowly stirred 3～5 hours under 4～25 ℃ temperature condition; The protein precipitant that is added includes but not limited to: ammonium sulfate, the trimethyl carbinol or ethanol etc.; The concentration range that is added is at 33%～80% (v/v).
Described crosslinkedly preferably carry out in accordance with the following methods: add bi-functional cross-linking agent and under 4～25 ℃ of temperature condition, slowly stirred 15～24 hours; The bi-functional cross-linking agent that is added includes but not limited to: glutaraldehyde, phenyl two different sulphur cyanogen or bis-diazotized benzidine-2 etc.; The concentration range that is added is between 0.1%～2.5% (v/v).
The inflexible porous microsphere is because self properties of materials has good chemical stability and certain mechanical strength.The present invention rationally utilizes the hole characteristic of porous microsphere, the aggregate crosslinking enzyme is fixed in the hole of carrier in the process that forms, irregular hole is retained in the carrier stable state solid enzyme of formation, reached the purpose of immobilized enzyme, and just become the mass transfer of substrate in the chemical reaction system, product and the passage of convection current less than the hole of protein molecule.
The invention solves the existing shortcoming that can not recycling of aggregate crosslinking enzyme, aggregate crosslinking enzyme certain physical form and rock steady structure have been given, the advantage that has kept the aggregate crosslinking enzyme again, be adapted to industrialization batch and serialization production, opened up the prospect of aggregate crosslinking enzyme industrial applications.
Immobilized enzyme method of the present invention is simple, simple and easy to do, and reaction conditions is easy to optimizing, need not specific installation.The immobilized enzyme stability height that the inventive method is prepared, the antitrypsin ability is strong, can use repeatedly, can fill the device of different shape, has higher flow rate of liquid, is easy to use in suitability for industrialized production.
Description of drawings
Fig. 1 p-nitrophenol typical curve;
Fig. 2 o-nitrophenol (ONP) typical curve;
The thermostability of immobilized crosslinked organic phosphorus degrading enzyme aggregate of Fig. 3 and liquid organic phosphorus degrading enzyme relatively;
The firmness test of the immobilized crosslinked organic phosphorus degrading enzyme aggregate of Fig. 4 (rotating speed of vibration shaking table is 200 rev/mins);
Batch method successive reaction test of the immobilized crosslinked organic phosphorus degrading enzyme aggregate of Fig. 5;
The firmness test of the immobilized crosslinked Sumylact L aggregate of Fig. 6 (rotating speed of vibration shaking table is 200 rev/mins);
Batch method successive reaction test of the immobilized crosslinked Sumylact L aggregate of Fig. 7.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage of the present invention and characteristics will be more clear along with description.But these embodiment only are exemplary, scope of the present invention are not constituted any restriction.It will be understood by those skilled in the art that and down can make amendment or replace without departing from the spirit and scope of the present invention, but these modifications and replacing all fall within the scope of protection of the present invention the details of technical solution of the present invention and form.
The preparation of embodiment 1 immobilized crosslinked organic phosphorus degrading enzyme aggregate
Take by weighing organic phosphorus degrading enzyme and be dissolved in the phosphate buffered saline buffer, be mixed with the enzyme solution that protein concentration is 0.4mg/ml, (polystyrene type nonpolar adsorption resin, model are ADS-5 to add porous microsphere; Mean pore size is 30nm, and particle diameter is greater than 100 μ m; Performance: protein, carbohydrate, mineral acid, alkali, salt, small molecules hydrophilic organics hydrophilicity are not all adsorbed; Under conditions such as heat, acid, alkali, organic solvent, very high stability is arranged all.Tianjin Nankai with become scientific and technological limited formula production), stirred 3 hours down slowly at 4 ℃; Adding ammonium sulfate to saturation ratio again is 67.5%, slowly stirs 5 hours down at 4 ℃; Adding glutaraldehyde (25%) solution to ultimate density is 2.2%, slowly stirs 18 hours down at 4 ℃; Filter and take out porous microsphere, rinsing to solution is clarified repeatedly.
Take by weighing Sumylact L and be dissolved in the phosphate buffered saline buffer, be mixed with the enzyme solution that protein concentration is 0.5mg/ml, (polystyrene type nonpolar adsorption resin, model are ADS-5 to add porous microsphere.Mean pore size is 30nm, and particle diameter is greater than 100 μ m; Performance: protein, carbohydrate, mineral acid, alkali, salt, small molecules hydrophilic organics hydrophilicity are not all adsorbed; Under conditions such as heat, acid, alkali, organic solvent, very high stability is arranged all.Tianjin Nankai with become scientific and technological limited formula production), stirred 3 hours down slowly at 4 ℃.Adding ammonium sulfate to saturation ratio is 67.5%, slowly stirs 5 hours down at 4 ℃.Adding glutaraldehyde (25%) solution to ultimate density is 2.2%, 4 ℃ of slow down stirring 18 hours.Filter and take out porous microsphere, rinsing to solution is clarified repeatedly.
The performance test of the immobilized cross-linked enzyme aggregate of test example 1 the present invention
One, test materials
The immobilized crosslinked organic phosphorus degrading enzyme aggregate that given the test agent 1: embodiment 1 is prepared;
The immobilized crosslinked Sumylact L aggregate that given the test agent 2: embodiment 2 is prepared;
Two, test event
1. organic phosphorus degrading enzyme enzyme activity determination method
The drafting of p-nitrophenol typical curve:
Get the 0.08346g p-nitrophenol, with a small amount of 95% dissolve with ethanol, water is settled to 100mL (6mmol/L) then earlier.In test tube, add the p-nitrophenol solution and 1000,997.5,995,992.5,990,985,980,970,960 of 0,2.5,5,7.5,10,15,20,30,40,50 μ L, 50mmol/LTris-Cl (pH8.0) damping fluid of 950 μ L respectively, cumulative volume is 1mL, every then pipe adds the 1mL10% trichoroacetic acid(TCA), adds 1mL10%Na again
2CO
3Solution, cumulative volume are 3mL, and 410nm measures absorbance value.Simultaneously, draw p-nitrophenol typical curve (Fig. 1).
The vitality test of the immobilized crosslinked organic phosphorus degrading enzyme aggregate that resolvase and embodiment 1 are prepared:
The mensuration of resolvase: in test tube, add 5 μ L (10mg/mL) parathion-methyl solution, pH8.0,900 μ L (50mmol/L) Tris-HCl damping fluids and 100 μ L are through the enzyme liquid of appropriateness dilution, 37 ℃ are incubated 10 minutes, add 1mL 10% trichoroacetic acid(TCA) termination reaction, add 1mL 10%Na again
2CO
3The solution colour developing, 410nm measures absorbance value.Calculate enzyme activity according to the following equation:
N is the extension rate of enzyme liquid;
A unit of enzyme activity (U) is defined as: at 37 ℃, per minute discharges the required enzyme amount of 1 μ mol p-nitrophenol.
The mensuration of immobilized enzyme: in test tube, add 5 μ L (10mg/mL) parathion-methyl solution, the given the test agent 1 of pH8.0,995 μ L (50mmol/L) Tris-HCl damping fluids and 60mg, 37 ℃ are incubated 10 minutes, add 1mL 10% trichoroacetic acid(TCA) termination reaction, add 1mL 10%Na again
2CO
3The solution colour developing, 410nm measures absorbance value.Calculate enzyme activity according to above-mentioned formula.
2. Sumylact L enzyme activity determination method
The drafting of o-nitrophenol (ONP) typical curve:
Take by weighing 0.25g ONP dissolving, constant volume in 100ml 0.1M HAc-NaAc damping fluid.In test tube, add the ONPG solution and 1000,950,900,850,800,750 of 0,50,100,150,200,250,300 μ L, 0.1mmol/L HAc-NaAc (pH5.2) damping fluid of 700 μ L respectively, cumulative volume is 1mL, every then pipe adds the 1mL10% trichoroacetic acid(TCA), adds 2mL 1M Na again
2CO
3Solution, cumulative volume are 4mL, and 420nm measures absorbance value.Simultaneously, draw o-nitrophenol (ONP) typical curve (Fig. 2).
The vitality test of the immobilized crosslinked Sumylact L aggregate that resolvase and embodiment 2 are prepared
The mensuration of resolvase: the 0.25% o-nitrophenol-β-D galactoside (ONPG) that in test tube, adds 800 μ L, the test tube that adds above substrate is put into 60 ℃ of waters preheating 2min, the enzyme liquid that adds 200 μ L through the appropriateness dilution, 60 ℃ are incubated 15 minutes, add 1mL 10% trichoroacetic acid(TCA) termination reaction, add 2mL1M Na again
2CO
3The solution colour developing, 420nm measures absorbance value.Calculate enzyme activity according to the following equation:
N is the extension rate of enzyme liquid
A unit of enzyme activity (U/ml) is defined as: at 60 ℃, per minute decomposes o-NP-β-D galactoside (ONPG) and generates the required enzyme amount of 1 μ mol o-nitrophenol ONP.
The mensuration of immobilized enzyme: in test tube, add the 0.2%ONPG solution of 1000 μ L and the given the test agent of 10mg and be incubated 15 minutes for 1,60 ℃, add 1mL 10% trichoroacetic acid(TCA) termination reaction, add 2mL 1M Na again
2CO
3The solution colour developing, 420nm measures absorbance value.
3. heat stability test
Get the prepared immobilized crosslinked organic phosphorus degrading enzyme aggregate of an amount of organic phosphorus degrading enzyme liquid enzymes and embodiment 1 respectively, after in 70 degree water-baths, being incubated 0,30,60,90,120,150,180,210 minute respectively, measuring enzyme according to the enzyme activity determination method and live.
4. firmness test
In triangular flask, add the prepared immobilized crosslinked Sumylact L aggregate of the prepared immobilized crosslinked organic phosphorus degrading enzyme aggregate of 10 gram embodiment 1, embodiment 2 respectively, and the phosphoric acid buffer of 200ml pH7.5, place 37 ℃ of shaking tables, rotating speed is 200r.p.m.Vibrate after 2 hours, take out about 0.5 gram immobilization porous microsphere, put 4 ℃ standby.Drain the damping fluid of triangular flask, add new damping fluid, vibrated once more 2 hours, repeat the above-mentioned steps sampling.Immobilized enzyme is total to continuous oscillation 14 hours in shaking table.After vibration finishes, measure enzyme respectively according to said determination enzyme method alive and live.
5. successive reaction batch test
(1) mensuration of the prepared immobilized crosslinked organic phosphorus degrading enzyme aggregate of embodiment 1:
In 7 milliliters of cillin bottles, add the prepared immobilized crosslinked organic phosphorus degrading enzyme aggregate of 60 milligrams of embodiment 1,995 μ L (50mmol/L, pH8.0) Tris-HCl damping fluid, 5 μ L (10mg/mL) parathion-methyl solution, 37 ℃ of insulations 10 minutes.After reaction finished, careful sucking-off reaction solution placed test tube, added 1mL 10% trichoroacetic acid(TCA) termination reaction, added 1mL 10%Na again
2CO
3The solution colour developing, 410nm measures absorbance value.
(5 μ L (10mg/mL) parathion-methyl solution carry out the reaction of a new round for 50mmol/L, pH8.0) Tris-HCl damping fluid, and reaction repeated is 50 times altogether to add 995 μ L in remaining with the cillin bottle of immobilized enzyme again.
(2) mensuration of the prepared immobilized crosslinked Sumylact L aggregate of embodiment 2:
In 7 milliliters of cillin bottles, add the prepared immobilized crosslinked Sumylact L aggregate of 10 milligrams of embodiment 2, add the 0.2%ONPG solution of 1000 μ L, 60 ℃ of insulations 15 minutes.After reaction finished, careful sucking-off reaction solution placed test tube, added 1mL 10% trichoroacetic acid(TCA) termination reaction, added 2mL 1M Na again
2CO
3The solution colour developing, 420nm measures absorbance value.
Add the 0.2%ONPG solution of 1000 μ L again in remaining with the cillin bottle of immobilized enzyme, carry out the reaction of a new round, reaction repeated is 40 times altogether.
Three, test result
1, heat stability test
Measure the thermostability of the liquid enzymes of prepared immobilized crosslinked organic phosphorus degrading enzyme aggregate of present embodiment 1 and organic phosphorus degrading enzyme respectively, measurement result shows, the thermostability height of the immobilized crosslinked organic phosphorus degrading enzyme aggregate that present embodiment is prepared will be far superior to the thermostability (Fig. 3) of the liquid enzymes of organic phosphorus degrading enzyme.
2, firmness test
Adopt vibration shaking table (rotating speed of vibration shaking table is 200 rev/mins) to measure the firmness of the immobilized enzyme of given the test agent 1 and given the test agent 2 respectively, the measurement result explanation, immobilized crosslinked organic phosphorus degrading enzyme aggregate that the present invention is prepared and crosslinked Sumylact L aggregate firmness good (Fig. 4, Fig. 6).
3, successive reaction test
By the test of batch method successive reaction, show that given the test agent 1 and given the test agent 2 can use repeatedly, be adapted to industrialization batch and serialization production (Fig. 5, Fig. 7).
Claims (1)
1. cross-linked enzyme aggregate, it is characterized in that: described cross-linked enzyme aggregate is fixed on the porous microsphere carrier; Its preparation method comprises: at first the free state enzyme is dissolved in the phosphate buffered saline buffer, be mixed with the enzyme solution that concentration is 0.1～1.5mg/ml, add the porous microsphere carrier, under 4～25 ℃ of temperature condition, slowly stirred 1～3 hour, allow the free state enzyme be scattered in the porous microsphere carrier; In the porous microsphere carrier, carry out the precipitation, crosslinked of zymoprotein more successively, last rinsing porous microsphere carrier, promptly;
Wherein, described free state enzyme is selected from organic phosphorus degrading enzyme or Sumylact L; Described porous microsphere carrier is the polystyrene type nonpolar adsorption resin, and model is ADS-5, and mean pore size is 30nm, and particle diameter is greater than 100 μ m; Described intermediate processing is: adding ammonium sulfate to saturation ratio is 67.5%, slowly stirs 5 hours down at 4 ℃; Described cross-linking method is: glutaraldehyde solution to the ultimate density of adding 25% is 2.2%, slowly stirs 18 hours down at 4 ℃.
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| CN102154256A (en) * | 2010-12-23 | 2011-08-17 | 深圳大学 | Carrier-free immobilized lipase and preparation method thereof |
| CN102304498A (en) * | 2011-09-08 | 2012-01-04 | 天津市林业果树研究所 | Cross-linked enzyme aggregate prepared in water-in-oil emulsion and preparation method thereof |
| CN102505008B (en) * | 2011-11-09 | 2014-06-11 | 华南理工大学 | Magnetic immobilized cross-linked lipase aggregate and preparation method and application thereof |
| IN2014KN01233A (en) * | 2011-11-11 | 2015-10-16 | Augustine A Dinovo | |
| CN103583832B (en) * | 2013-07-18 | 2015-03-11 | 上海交通大学 | Preparation method of nano zeolite assembly immobilized phytase compound feed additive |
| CN103805589A (en) * | 2014-02-28 | 2014-05-21 | 湖北工业大学 | Method for immobilizing cellulose crosslinking crystal on silica gel plate |
| CN104046609A (en) * | 2014-06-24 | 2014-09-17 | 东北农业大学 | Preparation method for efficient immobilized lipase |
| CN104480096B (en) * | 2014-11-27 | 2018-07-10 | 陕西师范大学 | The method of the immobilized beta-glucosidase of cross-linked polymeric |
| CN107922897A (en) * | 2015-08-28 | 2018-04-17 | 荷兰联合利华有限公司 | The liquid detergent compositions of enzyme comprising protease and non-protein enzyme |
| CN107012136A (en) * | 2017-06-12 | 2017-08-04 | 浙江工业大学 | A kind of method of immobilization Thermomyces lanuginosus lipase |
| CN109994163A (en) * | 2019-03-22 | 2019-07-09 | 陕西省生物农业研究所 | A kind of simulation natural polymers design a model |
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Non-Patent Citations (2)
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| Linqiu Cao et al.Immobilised enzymes: science or art?.《Current Opinion in Chemical Biology》.2005,第9卷第218页右栏第2段，图1. * |
| Moon Il Kim et al.Crosslinked Enzyme Aggregates in Hierarchically-Ordered Mesoporous Silica:A Simple and Effective Method for Enzyme Stabilization..《Biotechnology and Bioengineering》.2007,第96卷(第2期),摘要,第212页左栏第2段. * |
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