CN105316232A - Device for preparation/reaction of immobilized enzyme and application thereof - Google Patents
Device for preparation/reaction of immobilized enzyme and application thereof Download PDFInfo
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- CN105316232A CN105316232A CN201410313414.XA CN201410313414A CN105316232A CN 105316232 A CN105316232 A CN 105316232A CN 201410313414 A CN201410313414 A CN 201410313414A CN 105316232 A CN105316232 A CN 105316232A
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Abstract
The invention discloses a device for preparation/reaction of immobilized enzyme and an application thereof. The device comprises: a pressure-resistant tank body, a flange cover, a material feeding inlet and a sight glass arranged on the upper part of the flange cover, a compressed gas inlet arranged on the upper part of the pressure-resistant tank body and a material discharge outlet disposed on the bottom of the pressure-resistant tank body, a stirring device and a heater. An O-shaped sealing ring is disposed between the pressure-resistant tank body and the flange cover. A distributor is connected to a pipeline on the material discharge outlet through a flange and is composed of a distribution cover and a distribution plate in which a plurality of circular through holes are uniformly formed. A valve A is disposed on a material discharge pipe above the distributor. A branched material discharge pipe is disposed on the material discharge pipe above the valve A. A valve B is disposed on the branched material discharge pipe. The device not only can be used as a preparation device of the immobilized enzyme but also can be used as an enzymolysis reaction device of the immobilized enzyme. The device is not only increased in utilization rate but also satisfies industrial production requirements.
Description
Technical field
The present invention relates to a kind of device for immobilized enzyme preparation/reaction and application thereof, specifically, relate to a kind of preparation that both can be used for immobilized enzyme, also can be used for device and the embody rule thereof of the enzyme digestion reaction of immobilized enzyme.
Background technology
Enzyme is a kind of biological catalyst produced by the viable cell in organism.Enzyme catalysis have efficient, single-minded, reaction conditions is gentle, the feature such as pollution-free, can under the mild conditions in body, the various biochemical reaction of catalysis expeditiously, promotes the metabolism of organism.Because most of enzyme is protein, very responsive to environment during unbound state, poor at environment stability inferiors such as high temperature, strong acid, highly basic, high ion concentration and part organic solvents, easy sex change, thus its catalytic activity is even lost in reduction, and enzyme can not be reused, after carrying out enzymic catalytic reaction, have residual in product, not easily purifying.Not enough for overcoming these, enzyme immobilization technology arises at the historic moment the sixties in 20th century and progressively grows up.
Body endoenzyme is many to combine with film class material and carries out distinctive catalyzed reaction.Enzyme immobilization technology is the mode of action of analogue body endoenzyme, by means that are chemical or physics, with carrier by enzyme constraint or be limited in certain region, enzyme molecule is made to carry out peculiar and active katalysis in this region, and a kind of technology that is recyclable and that repeatedly use.At present, the Theory and applications research of immobilized enzyme has achieved many important achievement, and is used widely in fields such as food, medicine, chemical industry.Immobilized enzyme is compared with resolvase, both the catalysis characteristics of enzyme had been remained, overcome again the deficiency of resolvase, have that stability strengthens, environmental sensibility reduces, can repeat or use continuously, easily and product separation reduce enzyme auto-degradation, be applicable to the advantages such as multienzymatic reaction.
Crosslinking and entrapping method are enzyme immobilization methods the most conventional at present, two kinds of methods can be used alone, also can conbined usage, improve the immobilization efficiency of immobilized enzyme and the object of Reusability rate to reach, meet actual production or scientific research to the requirement of immobilized enzyme.
Crosslinking refers to by poly functional reagent, the amino-acid residue of resolvase and bifunctional or multi-group crosslink agent be reacted and crosslinked process for fixation occurs.Conventional bifunctional reagent has glutaraldehyde, bis-diazotized benzidine-2-2 '-disulfonic acid etc., and conventional poly functional reagent is the different sulphur cyanogen of Toluene-2,4-diisocyanate-isocyanide-4-, and wherein most widely used is glutaraldehyde.Glutaraldehyde has two aldehyde radicals, all can react with the free amine group of enzyme or protein, forms Schiff, thus makes enzyme molecule crosslinked, make immobilized enzyme.
Entrapping method enzyme is embedded in porous support the method making enzyme fixing, and its preparation technology is easy and condition is comparatively gentle.Carrier does not react with zymoprotein amino-acid residue, seldom changes the higher structure of enzyme, can obtain higher enzymatic activity recovery.Therefore, comparatively speaking, the immobilized enzyme adopting entrapping method to prepare has larger advantage.But because entrapping method prepares the conventional carrier of immobilized enzyme as gelatin, agar, agarose, chitosan, sodium alginate etc., all have full-bodied characteristic, shaping therefore in preparation process has just become a very large technical problem.In prior art, the preparation of immobilized enzyme gel particle generally adopts glass syringe method, that is: draw immobilized enzyme coagulant liquid with syringe, dropwise instill method shaping in forming agent.Actual preparation too much becomes the bottleneck affecting production efficiency because moulding process is consuming time often, so that cannot realize large-scale industrial production.
Due to the plurality of advantages of enzymic catalytic reaction, in recent years, the research of this respect obtains the features such as the enzyme digestion reaction of deep carrying out, particularly diphasic system is fast with speed of reaction, productive rate is high, by product is few and receives much concern.In the enzyme-catalyzed reaction that diphasic system carries out, while reaction, product is enriched to organic phase from aqueous phase, eliminates the restraining effect of product to catalyzed reaction, reduces the generation of by product simultaneously.But due to enzyme easy in inactivation in organic solvent, so often react with the form of immobilized enzyme.Enzyme digestion reaction need operate with special sealed reaction device, and market existing enzyme digestion reaction device is expensive, and can only carry out single enzyme digestion reaction operation, and can not be used for the production of immobilized enzyme, plant factor is not high.
Summary of the invention
The object of this invention is to provide a kind of device for immobilized enzyme preparation/reaction and application thereof, the technical barriers such as the difficult forming caused because carrier viscosity is large existing when adopting entrapping method or crosslinked entrapping method to prepare immobilized enzyme to solve, production efficiency are low, can not accomplish scale production; Realize this device can the enzyme digestion reaction of single, double phase system of being fixed enzyme simultaneously, improves the utilization ratio of device.
For achieving the above object, the present invention adopts following technical scheme:
A kind of device for immobilized enzyme preparation/reaction, comprise: the discharge port of withstand voltage tank body, flange lid, the opening for feed being arranged on flange top of closure and visor, the pressurized gas entrance being arranged on withstand voltage tank body top and bottom, whipping appts and well heater, be provided with O-ring seals between described withstand voltage tank body and flange lid; It is characterized in that: on the pipeline of bottom discharge mouth, have sparger by Flange joint, described sparger is made up of point sheet and the grid distributor being evenly distributed with some manholes; Discharge pipe above sparger is provided with valve A, the discharge pipe above valve A is provided with discharging arm, this discharging arm is provided with valve B.
Preferably, the diameter of described manhole is 2.0 ~ 5.0mm.
Preferably, muff is provided with in the outside of described withstand voltage tank body.
Preferably, described opening for feed and pressurized gas entrance are equipped with sealable locking closure.
Device for immobilized enzyme preparation/reaction of the present invention not only can be used for adopting entrapping method or crosslinked entrapping method to prepare immobilized enzyme, also can be used for the enzyme digestion reaction of the single, double phase system of immobilized enzyme.
Utilizing said apparatus of the present invention to carry out entrapping method, to prepare the operation of immobilized enzyme as follows:
1) by opening for feed, the solid support material and water of preparing immobilized enzyme are dropped in withstand voltage tank body by proportioning, open whipping appts and well heater, solid support material is dissolved completely under stirring heating, the being fixed enzyme carrier aqueous solution;
2) after the to be fixedization enzyme carrier aqueous solution is cooled to room temperature, add enzyme powder or the enzyme solution of proportional quantity, closed-type feed mouth, stir, prepare immobilized enzyme coagulant liquid;
3) open valve A, and select whether pass into pressurized gas according to immobilized enzyme coagulant liquid viscosity size, immobilized enzyme coagulant liquid is flowed out from sparger, instillation is placed on is equipped with in the container of salts solution immediately below sparger;
4) immobilized enzyme particle that formed in salts solution of collecting by filtration, washing, anhydrates, obtains immobilized enzyme particle.
Utilizing said apparatus of the present invention to carry out crosslinked entrapping method, to prepare the operation of immobilized enzyme as follows:
1) by opening for feed, the solid support material and water of preparing immobilized enzyme are dropped in withstand voltage tank body by proportioning, open whipping appts and well heater, solid support material is dissolved completely under stirring heating, the being fixed enzyme carrier aqueous solution;
2) after the to be fixedization enzyme carrier aqueous solution is cooled to room temperature, add the enzyme powder of proportional quantity or the linking agent of enzyme solution and proportional quantity, closed-type feed mouth, stir and make mixing, leave standstill crosslinked, prepare immobilized enzyme coagulant liquid;
3) open valve A, and select whether pass into pressurized gas according to immobilized enzyme coagulant liquid viscosity size, immobilized enzyme coagulant liquid is flowed out from sparger, instillation is placed on is equipped with in the container of salts solution immediately below sparger;
4) immobilized enzyme particle that formed in salts solution of collecting by filtration, washing, anhydrates, obtains immobilized enzyme particle.
Preferably, the container that salts solution is housed is placed on range distribution device 20 ~ 40cm place.
Utilize the operation of being fixed of said apparatus enzyme enzyme digestion reaction of the present invention as follows:
A) by opening for feed, immobilized enzyme, damping fluid or immobilized enzyme, damping fluid and organic solvent are dropped in withstand voltage tank body by proportioning, confined reaction system, open whipping appts and well heater, make stirring heating, carry out enzyme digestion reaction;
B) question response terminates, and opens opening for feed and valve B, releases reaction solution.
Preferably, the bottom in withstand voltage tank body is placed with screening plant.
Compared with prior art, the present invention has following significance progress:
1, solve that the difficult forming caused because carrier viscosity is large existing in immobilized enzyme preparation, production efficiency are low, the technical barrier such as can not to accomplish scale production, not only achieve and adopt entrapping method or crosslinked entrapping method mass-producing to prepare immobilized enzyme, and ratio of briquetting and preparation efficiency all very high, there is significant practical value;
2, apparatus of the present invention also can be used for the enzyme digestion reaction of the single-phase of immobilized enzyme or diphasic system, improve utilization ratio of device, decrease the input cost of equipment, have multiple using value;
3, apparatus of the present invention structure is simple, easy to operate, meets industrialization production requirements.
Accompanying drawing explanation
Fig. 1 is the structural representation of a kind of device for immobilized enzyme preparation/reaction provided by the invention;
Fig. 2 is a kind of structural representation of sparger of the present invention;
Fig. 3 is the structural representation of the grid distributor of sparger of the present invention.
In figure: 1, withstand voltage tank body; 2, flange lid; 3, opening for feed; 4, visor; 5, pressurized gas entrance; 6, discharge port; 7, whipping appts; 8, flange; 9, sparger; 91, sheet is divided; 92, grid distributor; 93, manhole; 10, valve A; 11, discharging arm; 12 valve B; 13, muff.
Embodiment
Below in conjunction with drawings and Examples, technical scheme of the present invention is further elaborated.
As shown in Figure 1 to Figure 3: a kind of device for immobilized enzyme preparation/reaction provided by the invention, comprise: the discharge port 6 of withstand voltage tank body 1, flange lid 2, the opening for feed 3 being arranged on flange lid 2 top and visor 4, the pressurized gas entrance 5 being arranged on withstand voltage tank body 1 top and bottom, whipping appts 7 and well heater (not shown), be provided with O-ring seals (not shown) between described withstand voltage tank body 1 and flange lid 2; The pipeline of bottom discharge mouth 6 is connected with sparger 9 by flange 8, and described sparger 9 is made up of point sheet 91 and the grid distributor 92 being evenly distributed with some manholes 93; Discharge pipe above sparger 9 is provided with valve A10, the discharge pipe above valve A10 is provided with discharging arm 11, this discharging arm 11 is provided with valve B12.
The diameter of described manhole 93 is preferably 2.0 ~ 5.0mm, and to meet immobilized enzyme particle size requirements, concrete aperture can be selected according to the immobilized enzyme particle size of required preparation.
Be provided with muff 13 in the outside of described withstand voltage tank body 1, be beneficial to insulation.
Opening for feed 3 and pressurized gas entrance 5 are equipped with sealable locking closure, to ensure that enzyme digestion reaction carries out in confined conditions, reduce the volatilization of solvent.
Embodiment 1: application apparatus of the present invention adopt entrapping method to prepare immobilized enzyme
1) by opening for feed 3, sodium alginate and water are dropped in withstand voltage tank body 1 by proportioning, open whipping appts 7 and well heater, at 50 ~ 60 DEG C, be stirred to sodium alginate dissolve completely, obtain sodium alginate aqueous solution (solution final concentration is 1wt%);
2) be cooled to after room temperature until sodium alginate aqueous solution, add the beta-glucosidase powder (5U/mL sodium alginate aqueous solution) of proportional quantity, closed-type feed mouth, stir, obtained beta-glucosidase coagulant liquid;
3) opening opening for feed makes tank interior be in atmospheric pressure state, open valve A10, beta-glucosidase coagulant liquid is flowed out from sparger 9, instillation is placed on 20 ~ 40cm place immediately below sparger 9 and is equipped with in the container of (1 ~ 3) wt% calcium chloride water, to form smooth gelled pill; On the grid distributor 92 of described sparger 9, the diameter of the manhole 93 of distribution is 4.0mm;
4) until room temperature vulcanizing after 2 hours, the gelled pill that collecting by filtration is formed in calcium chloride water, with deionized water wash, blots surface-moisture, obtains immobilized enzyme beta-glucoside enzyme granulate.
Adopt apparatus of the present invention that immobilized enzyme molding rate can be made to reach 100mL/ minute, and under similarity condition, the molding rate adopting syringe to prepare immobilized enzyme is only 1.0mL/ minute, visible, the molding rate that apparatus of the present invention can make entrapping method prepare immobilized enzyme is 100 times of prior art, has significance progress and industrial applications value.
Embodiment 2: application apparatus of the present invention adopt crosslinked entrapping method to prepare immobilized enzyme
1) by opening for feed 3, sodium alginate and water are dropped in withstand voltage tank body 1 by proportioning, open whipping appts 7 and well heater, at 50 ~ 60 DEG C, be stirred to sodium alginate dissolve completely, obtain sodium alginate aqueous solution (solution final concentration is 3wt%);
2) be cooled to after room temperature until sodium alginate aqueous solution, add beta-glucosidase powder (5U/mL sodium alginate aqueous solution) and the glutaraldehyde (making its final concentration be 0.5vol%) of proportional quantity, closed-type feed mouth, stir, normal temperature crosslinked reaction 2 hours, obtained beta-glucosidase coagulant liquid;
3) valve A10 is opened, from pressurized gas entrance, pass into proper amount of gas simultaneously, beta-glucosidase coagulant liquid is flowed out fast from sparger 9, instillation is placed on 30cm place immediately below sparger 9 and is equipped with in the container of (0.5 ~ 3) wt% calcium chloride water, to form smooth gelled pill; On the grid distributor 92 of described sparger 9, the diameter of the manhole 93 of distribution is 2.0mm;
4) until room temperature vulcanizing after 2 hours, the gelled pill that collecting by filtration is formed in calcium chloride water, with deionized water wash, blots surface-moisture, obtains immobilized enzyme beta-glucoside enzyme granulate.
Adopt apparatus of the present invention that immobilized enzyme molding rate can be made to reach 150mL/ minute, and under similarity condition, the molding rate adopting syringe to prepare immobilized enzyme is only 0.5mL/ minute, visible, the molding rate that apparatus of the present invention can make crosslinked entrapping method prepare immobilized enzyme is 300 times of prior art, has significance progress and industrial applications value.
Embodiment 3: application apparatus of the present invention carry out the reaction of single_phase system immobilized enzyme hydrolysis
A) by opening for feed 3, the Acetic acid-sodium acetate damping fluid of immobilized enzyme, baicalin, pH=4.0 ~ 6.0 is pressed proportioning (20: 1: 500, weight ratio) drop in withstand voltage tank body 1, confined reaction system, open whipping appts 7 and well heater, stir at 40 ~ 60 DEG C and make to carry out enzyme digestion reaction;
B) question response terminates, and opens opening for feed and makes reaction system be in atmospheric pressure state, then open valve B12, releases reaction solution.
Preferably, the bottom in withstand voltage tank body 1 is placed with screening plant (such as screen cloth), to sieve out immobilized enzyme particle, after question response liquid is released completely, can open flange lid 2, be taken out by screen cloth, obtain collected immobilized enzyme.
Embodiment 4: application apparatus of the present invention carry out the reaction of diphasic system immobilized enzyme hydrolysis
A) by opening for feed 3, the Acetic acid-sodium acetate damping fluid of immobilized enzyme, jasminoidin, pH=3.5 ~ 5.5 and ethyl acetate are pressed proportioning (10: 1: 100: 100, weight ratio) drop in withstand voltage tank body 1, confined reaction system, open whipping appts 7 and well heater, stir at 45 ~ 65 DEG C and make to carry out enzyme digestion reaction;
B) question response terminates, and opens opening for feed and makes reaction system be in atmospheric pressure state, then open valve B12, releases reaction solution.
Preferably, the bottom in withstand voltage tank body 1 is placed with screening plant (such as screen cloth), to sieve out immobilized enzyme particle, after question response liquid is released completely, can open flange lid 2, be taken out by screen cloth, obtain collected immobilized enzyme.
Embodiment 5: application apparatus of the present invention carry out the reaction of diphasic system immobilized enzyme hydrolysis
A) by opening for feed 3, the Acetic acid-sodium acetate damping fluid of immobilized enzyme, baicalin, pH=4.0 ~ 6.0 and chloroform are pressed proportioning (20: 1: 500: 500, weight ratio) drop in withstand voltage tank body 1, confined reaction system, open whipping appts 7 and well heater, stir at 40 ~ 60 DEG C and make to carry out enzyme digestion reaction;
B) question response terminates, and opens opening for feed and makes reaction system be in atmospheric pressure state, then open valve B12, releases reaction solution.
Preferably, the bottom in withstand voltage tank body 1 is placed with screening plant (such as screen cloth), to sieve out immobilized enzyme particle, after question response liquid is released completely, can open flange lid 2, be taken out by screen cloth, obtain collected immobilized enzyme.
Finally be necessary described herein:
The further description that above-described embodiment is only done for the technical scheme proposed the present invention, only understanding technical scheme of the present invention for helping, can not be interpreted as limiting the scope of the invention.The non-intrinsically safe that those skilled in the art's foregoing according to the present invention is made improves and adjustment all belongs to the scope of protection of present invention.
Claims (8)
1. the device for immobilized enzyme preparation/reaction, comprise: the discharge port of withstand voltage tank body, flange lid, the opening for feed being arranged on flange top of closure and visor, the pressurized gas entrance being arranged on withstand voltage tank body top and bottom, whipping appts and well heater, be provided with O-ring seals between described withstand voltage tank body and flange lid; It is characterized in that: on the pipeline of bottom discharge mouth, have sparger by Flange joint, described sparger is made up of point sheet and the grid distributor being evenly distributed with some manholes; Discharge pipe above sparger is provided with valve A, the discharge pipe above valve A is provided with discharging arm, this discharging arm is provided with valve B.
2. device as claimed in claim 1, is characterized in that: the diameter of described manhole is 2.0 ~ 5.0mm.
3. device as claimed in claim 1, is characterized in that: be provided with muff in the outside of described withstand voltage tank body.
4. device as claimed in claim 1, is characterized in that: described opening for feed and pressurized gas entrance are equipped with sealable locking closure.
5. the application of device described in any one in Claims 1-4, is characterized in that: as the preparation facilities of immobilized enzyme.
6. apply as claimed in claim 5, it is characterized in that: described preparation facilities is used for entrapping method or crosslinked entrapping method prepares immobilized enzyme.
7. the application of device described in any one in Claims 1-4, is characterized in that: as the enzyme digestion reaction device of immobilized enzyme.
8. apply as claimed in claim 7, it is characterized in that: described enzyme digestion reaction device is used for the enzyme digestion reaction of the single-phase or diphasic system of immobilized enzyme.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201410313414.XA CN105316232B (en) | 2014-07-02 | 2014-07-02 | A kind of device and its application for immobilised enzymes preparation/reaction |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201410313414.XA CN105316232B (en) | 2014-07-02 | 2014-07-02 | A kind of device and its application for immobilised enzymes preparation/reaction |
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| CN105316232A true CN105316232A (en) | 2016-02-10 |
| CN105316232B CN105316232B (en) | 2018-04-06 |
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN86105304A (en) * | 1985-08-06 | 1987-02-04 | 三井东压化学株式会社 | The method and the employed device that prepare immobilized enzyme |
| CN2132749Y (en) * | 1992-07-21 | 1993-05-12 | 南京化工学院 | Made-up device of fixed biocatalyst |
| CN101402910A (en) * | 2008-03-04 | 2009-04-08 | 周剑平 | Reaction still for producing high-viscosity cell immobilization carrier |
| CN201424470Y (en) * | 2009-04-01 | 2010-03-17 | 湖北五瑞生物工程有限公司 | Zymohydrolysis pot |
| CN201525847U (en) * | 2009-07-21 | 2010-07-14 | 南京工业大学 | Immobilized enzyme carrier preparation device |
| CN202482327U (en) * | 2012-03-16 | 2012-10-10 | 湖北五瑞生物工程有限公司 | Complete stainless steel enzymolysis tank with external heating plate |
| CN203923205U (en) * | 2014-07-02 | 2014-11-05 | 上海中医药大学 | A kind of device for immobilized enzyme preparation/reaction |
-
2014
- 2014-07-02 CN CN201410313414.XA patent/CN105316232B/en active Active
Patent Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN86105304A (en) * | 1985-08-06 | 1987-02-04 | 三井东压化学株式会社 | The method and the employed device that prepare immobilized enzyme |
| CN2132749Y (en) * | 1992-07-21 | 1993-05-12 | 南京化工学院 | Made-up device of fixed biocatalyst |
| CN101402910A (en) * | 2008-03-04 | 2009-04-08 | 周剑平 | Reaction still for producing high-viscosity cell immobilization carrier |
| CN201424470Y (en) * | 2009-04-01 | 2010-03-17 | 湖北五瑞生物工程有限公司 | Zymohydrolysis pot |
| CN201525847U (en) * | 2009-07-21 | 2010-07-14 | 南京工业大学 | Immobilized enzyme carrier preparation device |
| CN202482327U (en) * | 2012-03-16 | 2012-10-10 | 湖北五瑞生物工程有限公司 | Complete stainless steel enzymolysis tank with external heating plate |
| CN203923205U (en) * | 2014-07-02 | 2014-11-05 | 上海中医药大学 | A kind of device for immobilized enzyme preparation/reaction |
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