CN106867990A - Preparation method of immobilized beta-glucosidase - Google Patents
Preparation method of immobilized beta-glucosidase Download PDFInfo
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- CN106867990A CN106867990A CN201510920113.8A CN201510920113A CN106867990A CN 106867990 A CN106867990 A CN 106867990A CN 201510920113 A CN201510920113 A CN 201510920113A CN 106867990 A CN106867990 A CN 106867990A
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- glucosidase
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- beta
- preparation
- gelation
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- 108010047754 beta-Glucosidase Proteins 0.000 title claims abstract description 78
- 102000006995 beta-Glucosidase Human genes 0.000 title claims abstract description 78
- 238000002360 preparation method Methods 0.000 title claims abstract description 22
- 229930091371 Fructose Natural products 0.000 claims abstract description 38
- 239000005715 Fructose Substances 0.000 claims abstract description 38
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 claims abstract description 38
- BOTDANWDWHJENH-UHFFFAOYSA-N Tetraethyl orthosilicate Chemical compound CCO[Si](OCC)(OCC)OCC BOTDANWDWHJENH-UHFFFAOYSA-N 0.000 claims abstract description 22
- 238000006460 hydrolysis reaction Methods 0.000 claims abstract description 9
- 230000007062 hydrolysis Effects 0.000 claims abstract description 8
- 238000006243 chemical reaction Methods 0.000 claims abstract description 5
- 239000004088 foaming agent Substances 0.000 claims abstract description 4
- 238000003980 solgel method Methods 0.000 claims abstract description 4
- 238000001879 gelation Methods 0.000 claims description 31
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 22
- 239000007864 aqueous solution Substances 0.000 claims description 17
- 239000000243 solution Substances 0.000 claims description 13
- 239000008351 acetate buffer Substances 0.000 claims description 10
- 238000003756 stirring Methods 0.000 claims description 9
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 9
- 239000000843 powder Substances 0.000 claims description 8
- 239000007853 buffer solution Substances 0.000 claims description 7
- 239000000499 gel Substances 0.000 claims description 6
- 239000011541 reaction mixture Substances 0.000 claims description 6
- 238000007171 acid catalysis Methods 0.000 claims description 2
- 239000002245 particle Substances 0.000 claims description 2
- 238000000034 method Methods 0.000 abstract description 13
- 239000001913 cellulose Substances 0.000 abstract description 8
- 229920002678 cellulose Polymers 0.000 abstract description 8
- 230000008569 process Effects 0.000 abstract description 3
- 230000008901 benefit Effects 0.000 abstract description 2
- 238000004064 recycling Methods 0.000 abstract description 2
- 238000001179 sorption measurement Methods 0.000 abstract description 2
- 108010093096 Immobilized Enzymes Proteins 0.000 abstract 1
- 229940079919 digestives enzyme preparation Drugs 0.000 abstract 1
- 102000004190 Enzymes Human genes 0.000 description 35
- 108090000790 Enzymes Proteins 0.000 description 35
- 229940088598 enzyme Drugs 0.000 description 34
- 239000007788 liquid Substances 0.000 description 15
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 14
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 9
- 108010059892 Cellulase Proteins 0.000 description 8
- 230000000694 effects Effects 0.000 description 8
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 7
- 238000013019 agitation Methods 0.000 description 7
- 230000008859 change Effects 0.000 description 7
- 239000008103 glucose Substances 0.000 description 7
- 238000000227 grinding Methods 0.000 description 7
- 238000003760 magnetic stirring Methods 0.000 description 7
- 229910052757 nitrogen Inorganic materials 0.000 description 7
- 239000012299 nitrogen atmosphere Substances 0.000 description 7
- 239000003755 preservative agent Substances 0.000 description 7
- 230000002335 preservative effect Effects 0.000 description 7
- 238000002604 ultrasonography Methods 0.000 description 7
- 238000001291 vacuum drying Methods 0.000 description 7
- 230000009286 beneficial effect Effects 0.000 description 5
- 229940106157 cellulase Drugs 0.000 description 5
- 238000005516 engineering process Methods 0.000 description 5
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 4
- 239000000463 material Substances 0.000 description 4
- 239000002699 waste material Substances 0.000 description 4
- GUBGYTABKSRVRQ-CUHNMECISA-N D-Cellobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-CUHNMECISA-N 0.000 description 3
- 101710112457 Exoglucanase Proteins 0.000 description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 3
- 229930006000 Sucrose Natural products 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 239000005720 sucrose Substances 0.000 description 3
- 108010008885 Cellulose 1,4-beta-Cellobiosidase Proteins 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 239000000017 hydrogel Substances 0.000 description 2
- 230000009466 transformation Effects 0.000 description 2
- 241000609240 Ambelania acida Species 0.000 description 1
- 102000005744 Glycoside Hydrolases Human genes 0.000 description 1
- 108010031186 Glycoside Hydrolases Proteins 0.000 description 1
- 229920001410 Microfiber Polymers 0.000 description 1
- 240000000111 Saccharum officinarum Species 0.000 description 1
- 235000007201 Saccharum officinarum Nutrition 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000010905 bagasse Substances 0.000 description 1
- 150000001720 carbohydrates Chemical class 0.000 description 1
- 235000014633 carbohydrates Nutrition 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 235000008504 concentrate Nutrition 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 238000007599 discharging Methods 0.000 description 1
- 238000011038 discontinuous diafiltration by volume reduction Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 239000011261 inert gas Substances 0.000 description 1
- 229920005610 lignin Polymers 0.000 description 1
- 208000020442 loss of weight Diseases 0.000 description 1
- 239000003658 microfiber Substances 0.000 description 1
- 238000002156 mixing Methods 0.000 description 1
- 239000000203 mixture Substances 0.000 description 1
- 235000013379 molasses Nutrition 0.000 description 1
- 239000010413 mother solution Substances 0.000 description 1
- 229920001542 oligosaccharide Polymers 0.000 description 1
- 150000002482 oligosaccharides Chemical class 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 230000001172 regenerating effect Effects 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 230000006641 stabilisation Effects 0.000 description 1
- 238000011105 stabilization Methods 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 239000006188 syrup Substances 0.000 description 1
- 235000020357 syrup Nutrition 0.000 description 1
- 150000004043 trisaccharides Chemical class 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
Landscapes
- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention discloses a preparation method of immobilized beta-glucosidase, belonging to the field of enzyme preparations. The method adopts fructose as a pore-foaming agent and prepares the immobilized beta-glucosidase by a sol-gel method of tetraethoxysilane hydrolysis reaction. Different immobilized beta-glucosidase samples were prepared by adding different concentrations of aqueous fructose solution and different doses of beta-glucosidase. The obtained immobilized beta-glucosidase can avoid ineffective adsorption in the cellulose enzymolysis process, the cellulose enzymolysis conversion efficiency is obviously improved, the immobilized enzyme has the advantages of good mechanical strength and more recycling times, and the cellulose hydrolysis saccharification cost can be reduced.
Description
Technical field
The invention belongs to enzymic preparation field, more particularly to a kind of preparation method of immobilized β-glucosidase.
Background technology
Cellulose is the maximum polymeric carbohydrate of reserves on the earth, for regenerative resource and Material Field provide abundant raw material.The bioconversion of cellulose is an important transformation technology of cellulosic material, and the preparation that isolates and purifies of cellulase is key technique therein.Cellulase is the compound enzyme system of multicomponent of glycoside hydrolase, and 3 class components are divided into according to catalysis：Endoglucanase；Exoglucanase, i.e. cellobiohydrolase and beta-glucosidase [12,19~21].
Beta-glucosidase is to mainly act on β-(Isosorbide-5-Nitrae) glycosidic bond, also acts on β-(1,1), (1,2), (1,3), (1,6) glycosidic bond.It can hydrolyze the β-D-Glucose glycosidic bond for being incorporated into end irreducibility, while discharging β-D-Glucose and corresponding aglucon.Cellulase is acted on the noncrystalline domain of microfibre in catalyzing hydrolysis cellulose by endoglucanase first, it is set to expose many ends for circumscribed enzyme effect, exoglucanase (cellobiohydrolase) is decomposed successively from non-reducing end, produce cellobiose, the cellulose of Partial digestion is further acted synergistically by the poly- restriction endonuclease in Portugal and excision enzyme, the oligosaccharide such as generation cellobiose, trisaccharide are decomposed, finally by beta-glucosidase action breaks into glucose.In this process, beta-glucosidase plays a key effect, and beta-glucosidase content is few in cellulose components, vigor is low, constrain endoglucanase and the maximum effect of exoglucanase performance in enzyme system, so as to cellobiose is accumulated in causing hydrolysis sugar liquid, the available sugar amount of subsequent fermentation is reduced, the bottleneck as cellulase hydrolysis often needs additionally to add beta-glucosidase during cellulase hydrolysis.
Enzyme immobilization technology is to improve an important technology of enzyme stability, utilization rate and reduces cost, mainly including investment；Absorption method；Covalent method, four kinds of cross-linking method.These four methods cut both ways, and are mainly reflected in the load capacity and enzyme activity of immobilised enzymes.Because enzyme is bioactivator, to temperature, the conditional parameter such as pH value is very sensitive, so enzyme immobilization method has specificity for different enzymes for it.The present invention is directed to specific one-component beta-glucosidase, and template (pore-foaming agent) is done using fructose, and immobilized β-glucosidase is prepared by tetraethyl orthosilicate (TEOS) hydrolysis sol-gel process.By adding the fructose aqueous solution of various concentrations and the beta-glucosidase of various dose to prepare different immobilized β-glucosidase samples.
The content of the invention
It is high it is an object of the invention to be directed to beta-glucosidase production and use cost, it is easy to the problem inactivated by the lignin ineffective adsorption in lignocellulosic substrate, immobilized β-glucosidase is prepared with sol-gel investment.The high concentration syrup for obtaining will be for the first time squeezed directly to evaporate, concentrate, crystallizing and obtain sucrose, crystalline mother solution (molasses) mix with bagasse after solid fermentation production ethanol.Sugarcane Application way of the invention, efficiently make use of sucrose therein, reduce the cost of sugar industry, alleviate the pollution problem in sucrose and ethanol production process.
The present invention provides a kind of preparation method of immobilized β-glucosidase, comprises the following steps：
Pore-foaming agent is done using fructose, reacts sol-gel process by teos hydrolysis to prepare immobilized β-glucosidase.
The preparation method of immobilized β-glucosidase of the present invention, it is preferred that comprise the following steps：
(1) a certain amount of tetraethyl orthosilicate mixes with water, adds hydrochloric acid, under hydrochloric acid catalysis, is stirred in inert gas, and flowed back 1-2h when temperature then being risen into 55-65 DEG C, and room temperature is cooled to afterwards, obtains reaction mixture；
(2) reaction mixture is vacuumized, until weightless more than 50%；
(3) the quantitative fructose aqueous solution is added in the reaction solution after being vacuumized to step (2), is stirred, add the beta-glucosidase for being intended to immobilization, stirring, gelation under stand at low temperature obtains the immobilized β-glucosidase of gelation；
(4) the immobilized β-glucosidase sample of the gelation is dried under vacuum to constant weight, particle of the mechanical crushing into 20 mesh.
The preparation method of immobilized β-glucosidase of the present invention, wherein, the beta-glucosidase for being intended to immobilization is preferably the beta-glucosidase of one-component, and is diluted addition with buffer solution when adding.
The preparation method of immobilized β-glucosidase of the present invention, wherein, the buffer solution is acetate buffer solution, and pH value is preferably 4.0-5.0.
The preparation method of immobilized β-glucosidase of the present invention, wherein, in step (3), the mass fraction of fructose is preferably in 0-70% in the fructose aqueous solution.
The preparation method of immobilized β-glucosidase of the present invention, wherein, in step (1) described reaction mixture, tetraethyl orthosilicate is preferably 1 with the mol ratio of water:2.
The preparation method of immobilized β-glucosidase of the present invention, wherein, in the immobilized β-glucosidase of step (3) described gelation, the consumption of beta-glucosidase is preferably in 500-2000IU/g gels.
The preparation method of immobilized β-glucosidase of the present invention, wherein, in step (3), the gelation time is preferably 1-4 days.
Beta-glucosidase enzyme immobilization method of the invention, the advantage is that：
(1) beta-glucosidase enzyme load capacity is high, reaches more than 90%；
(2) enzyme activity stabilization, continuous to use 30 days, enzyme activity only declines within 5%；
(3) template molecule is cheap, and WATER-WASHING METHOD can just be removed, and minimizing technology is simply pollution-free.
The present invention is remarkably improved the enzyme activity stability of beta-glucosidase by immobilized β-glucosidase, increases recycling number of times, and for reducing lignocellulosic enzymolysis conversion cost, improving enzymolysis transformation efficiency has significantly value.
Brief description of the drawings
Fig. 1 is the scanning electron microscope (SEM) photograph on the surface of the 50% fructose and 1.155mL enzyme immobilizatios β-sweet enzyme of glucose observed with SEM；
Fig. 2 is the scanning electron microscope (SEM) photograph on the surface of the 50% fructose and 2.31mL enzyme immobilizatios β-sweet enzyme of glucose observed with SEM；
Fig. 3 is the scanning electron microscope (SEM) photograph on the surface of the 60% fructose and 1.155mL enzyme immobilizatios β-sweet enzyme of glucose observed with SEM；
Fig. 4 is the scanning electron microscope (SEM) photograph on the surface of the 60% fructose and 2.31mL enzyme immobilizatios β-sweet enzyme of glucose observed with SEM；
Fig. 5 is the scanning electron microscope (SEM) photograph on the surface of the 70% fructose and 0.5mL enzyme immobilizatios β-sweet enzyme of glucose observed with SEM；
Fig. 6 is the scanning electron microscope (SEM) photograph on the surface of the 70% fructose and 4.61mL enzyme immobilizatios β-sweet enzyme of glucose observed with SEM.
Specific embodiment
Embodiments of the invention are elaborated below：The present embodiment is implemented under premised on technical solution of the present invention; give detailed implementation method and process; but protection scope of the present invention is not limited to following embodiments, the experimental technique of unreceipted actual conditions in the following example, generally according to normal condition.
Buffer solution：
In the present invention, buffer solution is not particularly limited, the usual buffer solution is acetate buffer solution, and pH value is 4.0-5.0；
If pH value is less than 4.0, because pH value is too small, hydrogel is caused to solidify rapidly, it is impossible to realize uniform mixing, and pH value is more than 5.0, because pH value is excessive, causes enzyme enzyme to inactivate, and has no other beneficial effects.
Step
(3)
In, the mass fraction of fructose in the fructose aqueous solution：
In the present invention, in step (3), the mass fraction of fructose is not particularly limited in the fructose aqueous solution, and in usual step (3), the mass fraction of fructose is in 40-70% in the fructose aqueous solution；
If in step (3), the mass fraction of fructose is less than 40% in the fructose aqueous solution, because the mass fraction of fructose is too small, cause the effective duct volume reduction of material, and in step (3), in the fructose aqueous solution, the mass fraction of fructose is more than 70%, because the mass fraction of fructose is too high, cause fructose to waste, have no other beneficial effects.
The mol ratio of tetraethyl orthosilicate and water：
In the present invention, tetraethyl orthosilicate is not particularly limited with the mol ratio of water, in usual step (1) reaction mixture, tetraethyl orthosilicate is 1 with the mol ratio of water:2；
If tetraethyl orthosilicate is less than 1 with the mol ratio of water:2, because the consumption of tetraethyl orthosilicate is too small, cause follow-up vacuum step to predict loss of weight quality, lead to not to form hydrogel, and tetraethyl orthosilicate is more than 1 with the mol ratio of water:2, because tetraethyl orthosilicate consumption is excessive, cause to waste, and the reaction of tetraethyl orthosilicate is insufficient, has no other beneficial effects.
β
-
The consumption of glucuroide：
In the present invention, the consumption to beta-glucosidase is not particularly limited, and in the immobilized β-glucosidase of usual step (3) gelation, the consumption of beta-glucosidase is in 500-2000IU/g gels；
If the consumption of beta-glucosidase is less than 500IU/g, because the consumption of beta-glucosidase is too small, cause the effective enzyme activity of unit too low, and the consumption of gel beta-glucosidase is more than 2000IU/g, because the consumption of gel beta-glucosidase is excessive, cause to waste, and be susceptible to embedding not exclusively, have no other beneficial effects.
Gelation time：
In the present invention, gelation time is not particularly limited, in usual step (3), the gelation time is 1-4 days；
If gelation time is less than 1 day, because the time is too short, cause gelation not thorough, the strength of materials is poor；And gelation time is, more than 4 days, due to overlong time, to cause the time to waste, other beneficial effects are had no.
Embodiment 1
The addition 32g tetraethyl orthosilicates in the three neck round bottom flask of 500mL, the hydrochloric acid of 7.5mL 0.034mol, in a nitrogen atmosphere, magnetic agitation (1500rpm) 15min, then raises the temperature of water-bath, and flow back 1h when being raised to 60 DEG C, room temperature is subsequently cooled to, nitrogen is removed.
Vacuumize under magnetic stirring, until weightless 50% or so.The 50% fructose aqueous solution is added, is stirred, then ultrasound 5min, to remove bubble.The liquid that will be obtained averagely is assigned in 3 small beakers of 100mL, add enzyme liquid (beta-glucosidase and 50mM, the mixed liquor of the acetate buffer solution of pH5.0) 1.155mL, stirring 2min, sealed with preservative film, and 30~40 holes are pricked on film, and 48h in 4 DEG C of refrigerator is put into, make its gelation.
After 48h, the immobilized β-glucosidase sample of gelation is put into vacuum drying chamber and is vacuum dried, to weight not in change, then ground sample, and the sample of grinding is crossed into 20 mesh sieves, yield less than the powder equal to 20 mesh.
The surface of immobilized β-glucosidase is observed with SEM (SEM), Fig. 1, Fig. 2, Fig. 3, Fig. 4, Fig. 5, Fig. 6 is referred to.
Embodiment 2
The addition 32g tetraethyl orthosilicates in the three neck round bottom flask of 500mL, the hydrochloric acid of 7.5mL 0.034mol, in a nitrogen atmosphere, magnetic agitation (1500rpm) 15min, then raises the temperature of water-bath, and flow back 2h when being raised to 55 DEG C, room temperature is subsequently cooled to, nitrogen is removed.
Vacuumize under magnetic stirring, until weightless 50% or so.The 50% fructose aqueous solution is added, is stirred, then ultrasound 5min, to remove bubble.The liquid that will be obtained averagely is assigned in 3 small beakers of 100mL, add enzyme liquid (beta-glucosidase and 50mM, the mixed liquor of the acetate buffer solution of pH5.0) 2.31mL, stirring 2min, sealed with preservative film, and 30~40 holes are pricked on film, and 96h in 4 DEG C of refrigerator is put into, make its gelation.
After 96h, the immobilized β-glucosidase sample of gelation is put into vacuum drying chamber and is vacuum dried, to weight not in change, then ground sample, and the sample of grinding is crossed into 20 mesh sieves, yield less than the powder equal to 20 mesh.
Embodiment 3
The addition 32g tetraethyl orthosilicates in the three neck round bottom flask of 500mL, the hydrochloric acid of 7.5mL 0.034mol, in a nitrogen atmosphere, magnetic agitation (1500rpm) 15min, then raises the temperature of water-bath, and flow back 1h when being raised to 65 DEG C, room temperature is subsequently cooled to, nitrogen is removed.
Vacuumize under magnetic stirring, until weightless 50% or so.The 60% fructose aqueous solution is added, is stirred, then ultrasound 5min, to remove bubble.The liquid that will be obtained averagely is assigned in 3 small beakers of 100mL, add enzyme liquid (beta-glucosidase and 50mM, the mixed liquor of the acetate buffer solution of pH5.0) 1.155mL, stirring 2min, sealed with preservative film, and 30~40 holes are pricked on film, and 48h in 4 DEG C of refrigerator is put into, make its gelation.
After 48h, the immobilized β-glucosidase sample of gelation is put into vacuum drying chamber and is vacuum dried, to weight not in change, then ground sample, and the sample of grinding is crossed into 20 mesh sieves, yield less than the powder equal to 20 mesh.
Embodiment 4
The addition 32g tetraethyl orthosilicates in the three neck round bottom flask of 500mL, the hydrochloric acid of 7.5mL 0.034mol, in a nitrogen atmosphere, magnetic agitation (1500rpm) 15min, then raises the temperature of water-bath, and flow back 1.5h when being raised to 60 DEG C, room temperature is subsequently cooled to, nitrogen is removed.
Vacuumize under magnetic stirring, until weightless 50% or so.The 60% fructose aqueous solution is added, is stirred, then ultrasound 5min, to remove bubble.The liquid that will be obtained averagely is assigned in 3 small beakers of 100mL, add enzyme liquid (beta-glucosidase and 50mM, the mixed liquor of the acetate buffer solution of pH5.5) 2.31mL, stirring 4min, sealed with preservative film, and 30~40 holes are pricked on film, and 96h in 4 DEG C of refrigerator is put into, make its gelation.
After 96h, the immobilized β-glucosidase sample of gelation is put into vacuum drying chamber and is vacuum dried, to weight not in change, then ground sample, and the sample of grinding is crossed into 20 mesh sieves, yield less than the powder equal to 20 mesh.
Embodiment 5
The addition 32g tetraethyl orthosilicates in the three neck round bottom flask of 500mL, the hydrochloric acid of 7.5mL 0.034mol, in a nitrogen atmosphere, magnetic agitation (1500rpm) 15min, then raises the temperature of water-bath, and flow back 1h when being raised to 60 DEG C, room temperature is subsequently cooled to, nitrogen is removed.
Vacuumize under magnetic stirring, until weightless 50% or so.The 70% fructose aqueous solution is added, is stirred, then ultrasound 5min, to remove bubble.The liquid that will be obtained averagely is assigned in 3 small beakers of 100mL, add enzyme liquid (beta-glucosidase and 50mM, the mixed liquor of the acetate buffer solution of pH5.0) 0.5mL, stirring 2min, sealed with preservative film, and 30~40 holes are pricked on film, and 48h in 4 DEG C of refrigerator is put into, make its gelation.
After 48h, the immobilized β-glucosidase sample of gelation is put into vacuum drying chamber and is vacuum dried, to weight not in change, then ground sample, and the sample of grinding is crossed into 20 mesh sieves, yield less than the powder equal to 20 mesh.
Embodiment 6
The addition 32g tetraethyl orthosilicates in the three neck round bottom flask of 500mL, the hydrochloric acid of 7.5mL 0.034mol, in a nitrogen atmosphere, magnetic agitation (1500rpm) 15min, then raises the temperature of water-bath, and flow back 1h when being raised to 60 DEG C, room temperature is subsequently cooled to, nitrogen is removed.
Vacuumize under magnetic stirring, until weightless 50% or so.The 50% fructose aqueous solution is added, is stirred, then ultrasound 5min, to remove bubble.The liquid that will be obtained averagely is assigned in 3 small beakers of 100mL, add enzyme liquid (beta-glucosidase and 50mM, the mixed liquor of the acetate buffer solution of pH4.0) 2.31mL, stirring 2min, sealed with preservative film, and 30~40 holes are pricked on film, and 48h in 4 DEG C of refrigerator is put into, make its gelation.
After 48h, the immobilized β-glucosidase sample of gelation is put into vacuum drying chamber and is vacuum dried, to weight not in change, then ground sample, and the sample of grinding is crossed into 20 mesh sieves, yield less than the powder equal to 20 mesh.
Embodiment 7
The addition 32g tetraethyl orthosilicates in the three neck round bottom flask of 500mL, the hydrochloric acid of 7.5mL 0.034mol, in a nitrogen atmosphere, magnetic agitation (1500rpm) 15min, then raises the temperature of water-bath, and flow back 1h when being raised to 60 DEG C, room temperature is subsequently cooled to, nitrogen is removed.
Vacuumize under magnetic stirring, until weightless 50% or so.The 70% fructose aqueous solution is added, is stirred, then ultrasound 5min, to remove bubble.The liquid that will be obtained averagely is assigned in 3 small beakers of 100mL, add enzyme liquid (beta-glucosidase and 50mM, the mixed liquor of the acetate buffer solution of pH5.0) 4.61mL, stirring 2min, sealed with preservative film, and 30~40 holes are pricked on film, and 96h in 4 DEG C of refrigerator is put into, make its gelation.
After 96h, the immobilized β-glucosidase sample of gelation is put into vacuum drying chamber and is vacuum dried, to weight not in change, then ground sample, and the sample of grinding is crossed into 20 mesh sieves, yield less than the powder equal to 20 mesh.
Claims (8)
1. a kind of preparation method of immobilized β-glucosidase, comprises the following steps：
Pore-foaming agent is done using fructose, reacts sol-gel process by teos hydrolysis to prepare immobilization
Beta-glucosidase.
2. the preparation method of immobilized β-glucosidase according to claim 1, including following step
Suddenly：
(1) a certain amount of tetraethyl orthosilicate mixes with water, adds hydrochloric acid, under hydrochloric acid catalysis, indifferent gas
Stirred in body, flowed back 1-2h when temperature then being risen into 55-65 DEG C, and room temperature is cooled to afterwards, must be reacted
Mixed liquor；
(2) reaction mixture is vacuumized, until weightless more than 50%；
(3) the quantitative fructose aqueous solution is added in the reaction solution after being vacuumized to step (2), is stirred,
The beta-glucosidase for being intended to immobilization is added, stirring, gelation under stand at low temperature obtains the immobilization of gelation
Beta-glucosidase；
(4) the immobilized β-glucosidase sample of the gelation is dried under vacuum to constant weight, mechanical powder
It is broken into the particle of 20 mesh.
3. the preparation method of immobilized β-glucosidase according to claim 2, it is characterised in that：
It is described be intended to immobilization beta-glucosidase for one-component beta-glucosidase, and add when buffer solution
It is diluted addition.
4. the preparation method of immobilized β-glucosidase according to claim 3, it is characterised in that：
The buffer solution is acetate buffer solution, and pH value is 4.0-5.0.
5. the preparation method of the immobilized β-glucosidase according to any one of claim 2~4, its
It is characterised by：In step (3), the mass fraction of fructose is in 40-70% in the fructose aqueous solution.
6. the preparation method of the immobilized β-glucosidase according to any one of claim 2~4, its
It is characterised by：In step (1) described reaction mixture, tetraethyl orthosilicate is 1 with the mol ratio of water:2.
7. the preparation method of the immobilized β-glucosidase according to any one of claim 2~4, its
It is characterised by：In the immobilized β-glucosidase of step (3) described gelation, beta-glucosidase
Consumption in 500-2000IU/g gels.
8. the preparation method of the immobilized β-glucosidase according to any one of claim 2~4, its
It is characterised by：In step (3), the gelation time is 1-4 days.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510920113.8A CN106867990B (en) | 2015-12-10 | 2015-12-10 | Preparation method of immobilized beta-glucosidase |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510920113.8A CN106867990B (en) | 2015-12-10 | 2015-12-10 | Preparation method of immobilized beta-glucosidase |
Publications (2)
| Publication Number | Publication Date |
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| CN106867990A true CN106867990A (en) | 2017-06-20 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108660129A (en) * | 2018-04-28 | 2018-10-16 | 江南大学 | The method that sol-gal process fixes saccharase and the double enzymes of glucose oxidase |
| CN109082420A (en) * | 2018-08-21 | 2018-12-25 | 江苏大学 | Metal-organic framework material immobilized β-glucosidase and its preparation method and application |
| CN111004792A (en) * | 2020-01-06 | 2020-04-14 | 陈炜山 | Preparation method and application of high-tolerance β -glucosidase |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102533717A (en) * | 2012-03-02 | 2012-07-04 | 江南大学 | Method for immobilizing beta-glucosidase and hydrolyzing straw cellulose by cooperating beta-glucosidase with cellulase |
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| CN102533717A (en) * | 2012-03-02 | 2012-07-04 | 江南大学 | Method for immobilizing beta-glucosidase and hydrolyzing straw cellulose by cooperating beta-glucosidase with cellulase |
Non-Patent Citations (1)
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| 高春光等: "溶胶- 凝胶法固定化α-淀粉酶的研究", 《化学与生物工程》 * |
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108660129A (en) * | 2018-04-28 | 2018-10-16 | 江南大学 | The method that sol-gal process fixes saccharase and the double enzymes of glucose oxidase |
| CN108660129B (en) * | 2018-04-28 | 2021-11-30 | 江南大学 | Method for immobilizing beta-fructofuranosidase and glucose oxidase double enzymes by sol-gel method |
| CN109082420A (en) * | 2018-08-21 | 2018-12-25 | 江苏大学 | Metal-organic framework material immobilized β-glucosidase and its preparation method and application |
| CN109082420B (en) * | 2018-08-21 | 2021-08-03 | 江苏大学 | Metal organic framework material immobilized beta-glucosidase and preparation method and application thereof |
| CN111004792A (en) * | 2020-01-06 | 2020-04-14 | 陈炜山 | Preparation method and application of high-tolerance β -glucosidase |
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