CN105316232B - A kind of device and its application for immobilised enzymes preparation/reaction - Google Patents
A kind of device and its application for immobilised enzymes preparation/reaction Download PDFInfo
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- CN105316232B CN105316232B CN201410313414.XA CN201410313414A CN105316232B CN 105316232 B CN105316232 B CN 105316232B CN 201410313414 A CN201410313414 A CN 201410313414A CN 105316232 B CN105316232 B CN 105316232B
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- immobilised enzymes
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- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention discloses a kind of device for immobilised enzymes preparation/reaction and its application.Described device includes：Discharging opening, agitating device and the heater of pressure-resistant tank body, flange lid, the charging aperture for being arranged on flange top of closure and visor, the compressed gas entrance for being arranged on pressure-resistant tank body top and bottom, O-ring seals are provided between described pressure-resistant tank body and flange lid；It is characterized in that：Distributor is connected with by flange on the pipeline of bottom discharge mouth, the distribution grid that the distributor is covered by distribution and be evenly distributed with some manholes forms；Valve A is provided with the discharge pipe above distributor, discharging branch pipe is provided with the discharge pipe above valve A, valve B is provided with the discharging branch pipe.Apparatus of the present invention both can be used as the preparation facilities of immobilised enzymes, it is also possible to makees the enzyme digestion reaction device of immobilised enzymes, not only increases utilization ratio of device, and meet industrialization production requirements.
Description
Technical field
The present invention relates to a kind of device for immobilised enzymes preparation/reaction and its application, is to be related to one kind both specifically
Preparation available for immobilised enzymes, it can also be used to the device and its concrete application of the enzyme digestion reaction of immobilised enzymes.
Background technology
Enzyme is a kind of biocatalyst as caused by the living cells in organism.Enzyme catalysis have efficient, single-minded, anti-
Answer mild condition, it is pollution-free the features such as, can expeditiously be catalyzed various biochemical reactions under the temperate condition in body,
Promote the metabolism of organism.It is very sensitive to environment during free state because most of enzymes are protein, in high temperature, strong
The environment stability inferiors such as acid, highly basic, high ion concentration and part organic solvent are poor, easily denaturation, so as to reduce or even lose it
Catalytic activity, and enzyme can not be reused, and after carrying out enzymic catalytic reaction, residual be had in product, not easy purification.To overcome these
Deficiency, enzyme immobilization technology arise at the historic moment the 1960s and progressively grown up.
Internal enzyme is more to be combined with film class material and carries out distinctive catalytic reaction.Enzyme immobilization technology is enzyme in analogue body
The mode of action, by means chemically or physically, enzyme is fettered or is limited in certain region with carrier, enzyme molecule is existed
This region carries out peculiar and active catalytic action, and a kind of technology that is recyclable and repeatedly using.At present, immobilised enzymes
Theory and application study achieved many important achievements, and be used widely in fields such as food, medicine, chemical industry.Gu
Surely change enzyme compared with resolvase, both remain the catalysis characteristics of enzyme, overcome the deficiency of resolvase again, there is stability to strengthen,
Environmental sensibility reduces, repeats or be used continuously, easily separated with product and reduce enzyme auto-degradation, suitable for multienzymatic reaction etc.
Advantage.
Cross-linking method and investment are presently the most conventional enzyme immobilization method, and two methods can be used alone, and can also join
Close and use, improve the immobilization efficiency of immobilised enzymes and the purpose of Reusability rate to reach, meet actual production or scientific research pair
The requirement of immobilised enzymes.
Cross-linking method refers to amino acid residue and difunctional or the multi-group crosslink agent for making resolvase by poly functional reagent
The process for fixation for reacting and crosslinking.Conventional bifunctional reagent has glutaraldehyde, bis-diazotized benzidine -2-2 '-disulfonic acid
Deng conventional poly functional reagent is the different sulphur cyanogen of Toluene-2,4-diisocyanate-isocyanide -4-, wherein most widely used is glutaraldehyde.Glutaraldehyde has
It two aldehyde radicals, can be reacted with the free amine group of enzyme or protein, form Schiff, so that enzyme molecule is crosslinked, be made solid
Surely enzyme is changed.
Investment is that enzyme is embedded in the method for fixing enzyme in porous carrier, and its preparation technology is easy and condition is more warm
With.Carrier does not react with zymoprotein amino acid residue, seldom changes the higher structure of enzyme, can obtain higher enzyme activity recovery
Rate.Therefore, comparatively, the immobilised enzymes prepared using investment has bigger advantage.However, because investment is prepared admittedly
Surely enzyme conventional carrier such as gelatin, agar, agarose, chitosan, sodium alginate etc. are changed, all with highly viscous characteristic, therefore
Shaping in preparation process is just into a very big technical problem.In the prior art, the preparation one of immobilised enzymes gel particle
As use glass syringe method, i.e.,：Immobilised enzymes coagulant liquid is drawn with syringe, instills in forming agent the method being molded dropwise.It is actual
Prepare turns into the bottleneck for influenceing production efficiency because forming process is time-consuming excessive often, so that large-scale industry metaplasia can not be realized
Production.
Due to the plurality of advantages of enzymic catalytic reaction, in recent years, the research of this respect obtains deep development, particularly two-phase
The enzyme digestion reaction of system is received much concern with the features such as reaction rate is fast, yield is high, accessory substance is few.In the enzyme that diphasic system is carried out
In catalytic reaction process, product is enriched to organic phase from aqueous phase while reaction, eliminates suppression of the product to catalytic reaction
Make and use, while reduce the generation of accessory substance.But due to enzyme easy in inactivation in organic solvent, so often with the shape of immobilised enzymes
Formula is reacted.Enzyme digestion reaction need to be operated with special sealing reaction unit, the existing enzyme digestion reaction device price in market
Costliness, and single enzyme digestion reaction operation can only be carried out, it is impossible to be used in the production of immobilised enzymes, utilization rate of equipment and installations be not high.
The content of the invention
It is an object of the invention to provide a kind of device for immobilised enzymes preparation/reaction and its application, to solve to use
Investment or crosslinking investment prepare the existing difficult forming caused by carrier viscosity is big during immobilised enzymes, production effect
The technical barriers such as rate is low, can not accomplish scale production；Simultaneously realize the device being fixed enzyme single, double phase system
Enzyme digestion reaction, improve the utilization rate of device.
To achieve the above object, the present invention adopts the following technical scheme that：
A kind of device for immobilised enzymes preparation/reaction, including：Pressure-resistant tank body, flange lid, it is arranged on flange lid
The charging aperture and visor on top, the discharging opening of the compressed gas entrance for being arranged on pressure-resistant tank body top and bottom, agitating device and
Heater, O-ring seals are provided between described pressure-resistant tank body and flange lid；It is characterized in that：In the pipe of bottom discharge mouth
Distributor is connected with by flange on road, the distributor is covered by distribution and be evenly distributed with the distribution grid group of some manholes
Into；Valve A is provided with the discharge pipe above distributor, discharging branch pipe is provided with the discharge pipe above valve A, at this
Discharging branch pipe is provided with valve B.
Preferably, a diameter of 2.0~5.0mm of the manhole.
Preferably, it is provided with muff in the outside of the pressure-resistant tank body.
Preferably, the charging aperture and compressed gas entrance are equipped with sealable locking closure.
The device for immobilised enzymes preparation/reaction of the present invention cannot be only used for using investment or crosslinking embedding legal system
Standby immobilised enzymes, it can also be used to the enzyme digestion reaction of the single, double phase system of immobilised enzymes.
The operation that immobilised enzymes is prepared using the said apparatus progress investment of the present invention is as follows：
1) carrier material for preparing immobilised enzymes is put into pressure-resistant tank body with water by proportioning by charging aperture, opens stirring
Device and heater, carrier material is set to be completely dissolved under agitating and heating, the being fixed zymophore aqueous solution；
2) after the to be fixedization zymophore aqueous solution is cooled to room temperature, the enzyme powder or enzyme solutions of proportional quantity, closed-type feed are added
Mouthful, stirring, prepare immobilised enzymes coagulant liquid；
3) valve A is opened, and chooses whether to be passed through compressed gas according to immobilised enzymes gel fluid viscosity size, makes immobilization
Enzyme coagulant liquid is flowed out from distributor, and instillation is placed in the container equipped with salting liquid immediately below distributor；
4) immobilized enzyme particle formed in salting liquid is collected by filtration, washes, removes water, produce immobilized enzyme particle.
Using the present invention said apparatus carry out be crosslinked investment prepare immobilised enzymes operation it is as follows：
1) carrier material for preparing immobilised enzymes is put into pressure-resistant tank body with water by proportioning by charging aperture, opens stirring
Device and heater, carrier material is set to be completely dissolved under agitating and heating, the being fixed zymophore aqueous solution；
2) after the to be fixedization zymophore aqueous solution is cooled to room temperature, the enzyme powder or enzyme solutions and proportional quantity of proportional quantity are added
Crosslinking agent, closed-type feed mouth, stirring make mixing, stand crosslinking, prepare immobilised enzymes coagulant liquid；
3) valve A is opened, and chooses whether to be passed through compressed gas according to immobilised enzymes gel fluid viscosity size, makes immobilization
Enzyme coagulant liquid is flowed out from distributor, and instillation is placed in the container equipped with salting liquid immediately below distributor；
4) immobilized enzyme particle formed in salting liquid is collected by filtration, washes, removes water, produce immobilized enzyme particle.
Preferably, the container equipped with salting liquid is placed at 20~40cm of range distribution device.
Operation using said apparatus the being fixed enzyme enzyme digestion reaction of the present invention is as follows：
A) it is by charging aperture that immobilised enzymes, buffer solution or immobilised enzymes, buffer solution and organic solvent is resistance to by proportioning input
Press in tank body, confined reaction system, open agitating device and heater, make agitating and heating, carry out enzyme digestion reaction；
B) question response terminates, and opens charging aperture and valve B, releases reaction solution.
Preferably, the bottom in autoclave body is placed with screening plant.
Compared with prior art, the present invention has following conspicuousness progress：
1st, difficult forming, the low production efficiency caused by carrier viscosity is big in the presence of immobilised enzymes preparation are solved
Under, the technical barrier such as can not accomplish scale production, not only realize and consolidated using investment or crosslinking investment prepare with scale
Surely enzyme is changed, and ratio of briquetting and preparation efficiency are all very high, have significant practical value；
2nd, apparatus of the present invention can be additionally used in single-phase or diphasic system the enzyme digestion reaction of immobilised enzymes, improve device utilization
Rate, reduce the input cost of equipment, there are a variety of application values；
3rd, apparatus of the present invention are simple in construction, easy to operate, meet industrialization production requirements.
Brief description of the drawings
Fig. 1 is a kind of structural representation of device for immobilised enzymes preparation/reaction provided by the invention；
Fig. 2 is a kind of structural representation of distributor of the present invention；
Fig. 3 is the structural representation of the distribution grid of distributor of the present invention.
In figure：1st, pressure-resistant tank body；2nd, flange lid；3rd, charging aperture；4th, visor；5th, compressed gas entrance；6th, discharging opening；7、
Agitating device；8th, flange；9th, distributor；91st, distribution cover；92nd, distribution grid；93rd, manhole；10th, valve A；11st, discharging branch
Pipe；12 valve B；13rd, muff.
Embodiment
Technical scheme is further elaborated with reference to the accompanying drawings and examples.
As shown in Figure 1 to Figure 3：A kind of device for immobilised enzymes preparation/reaction provided by the invention, including：It is pressure-resistant
Tank body 1, flange lid 2, the charging aperture 3 for being arranged on the top of flange lid 2 and visor 4, the compression for being arranged on the pressure-resistant top of tank body 1
The discharging opening 6 of gas access 5 and bottom, agitating device 7 and heater (not shown), described pressure-resistant tank body 1 and flange
O-ring seals (not shown) is provided between lid 2；Distributor is connected with by flange 8 on the pipeline of bottom discharge mouth 6
9, the distributor 9 is made up of distribution cover 91 with the distribution grid 92 for being evenly distributed with some manholes 93；Above distributor 9
Discharge pipe be provided with valve A10, on the discharge pipe above valve A10 be provided with discharging branch pipe 11, in the discharging branch pipe
11 are provided with valve B12.
The diameter of the manhole 93 is preferably 2.0~5.0mm, to meet immobilized enzyme particle size requirements, specifically
Aperture can be selected according to the immobilized enzyme particle size of required preparation.
Muff 13 is provided with the outside of the pressure-resistant tank body 1, in favor of insulation.
Charging aperture 3 and compressed gas entrance 5 are equipped with sealable locking closure, to ensure that enzyme digestion reaction enters in confined conditions
OK, the volatilization of solvent is reduced.
Embodiment 1：Immobilised enzymes is prepared using investment using apparatus of the present invention
1) sodium alginate and water are put into pressure-resistant tank body 1 by proportioning by charging aperture 3, opens agitating device 7 and heating
Device, stir at 50~60 DEG C and be completely dissolved to sodium alginate, obtain sodium alginate aqueous solution (the final concentration of 1wt% of solution)；
2) after sodium alginate aqueous solution is cooled to room temperature, beta-glucosidase enzyme powder (the 5U/mL alginic acids of proportional quantity are added
Sodium water solution), closed-type feed mouth, stirring, beta-glucosidase coagulant liquid is made；
3) opening charging aperture makes tank interior be in atmospheric pressure state, opening valve A10, make beta-glucosidase coagulant liquid from
Flowed out in distributor 9, instill the appearance for being placed on and (1~3) wt% calcium chloride waters being housed immediately below distributor 9 at 20~40cm
In device, to form smooth gelled pill；The diameter for the manhole 93 being distributed on the distribution grid 92 of the distributor 9 is
4.0mm；
4) after room temperature vulcanizing 2 hours, the gelled pill formed in calcium chloride water is collected by filtration, uses deionized water
Washing, blots surface moisture, produces immobilised enzymes beta-glucosidase enzyme granulate.
Using apparatus of the present invention immobilised enzymes molding rate can be made to reach 100mL/ minutes, and under similarity condition, use
The molding rate that syringe prepares immobilised enzymes is only 1.0mL/ minutes, it is seen then that apparatus of the present invention can make investment prepare fixation
The molding rate for changing enzyme is 100 times of prior art, and there are conspicuousness progress and industrial applications to be worth.
Embodiment 2：Immobilised enzymes is prepared using crosslinking investment using apparatus of the present invention
1) sodium alginate and water are put into pressure-resistant tank body 1 by proportioning by charging aperture 3, opens agitating device 7 and heating
Device, stir at 50~60 DEG C and be completely dissolved to sodium alginate, obtain sodium alginate aqueous solution (the final concentration of 3wt% of solution)；
2) after sodium alginate aqueous solution is cooled to room temperature, beta-glucosidase enzyme powder (the 5U/mL alginic acids of proportional quantity are added
Sodium water solution) and glutaraldehyde (making its final concentration of 0.5vol%), closed-type feed mouth, stirring, normal temperature crosslinked reaction 2 hours, system
Obtain beta-glucosidase coagulant liquid；
3) open valve A10, while be passed through proper amount of gas from compressed gas entrance, make beta-glucosidase coagulant liquid from
Quickly flowed out in distributor 9, instill and be placed at the underface 30cm of distributor 9 equipped with (0.5~3) wt% calcium chloride waters
In container, to form smooth gelled pill；The diameter for the manhole 93 being distributed on the distribution grid 92 of the distributor 9 is
2.0mm；
4) after room temperature vulcanizing 2 hours, the gelled pill formed in calcium chloride water is collected by filtration, uses deionized water
Washing, blots surface moisture, produces immobilised enzymes beta-glucosidase enzyme granulate.
Using apparatus of the present invention immobilised enzymes molding rate can be made to reach 150mL/ minutes, and under similarity condition, use
The molding rate that syringe prepares immobilised enzymes is only 0.5mL/ minutes, it is seen then that apparatus of the present invention can prepare crosslinking investment
The molding rate of immobilised enzymes is 300 times of prior art, and there are conspicuousness progress and industrial applications to be worth.
Embodiment 3：Single_phase system immobilized enzyme hydrolysis reaction is carried out using apparatus of the present invention
A) the Acetic acid-sodium acetate buffer solution of immobilised enzymes, scutelloside, pH=4.0~6.0 is pressed by proportioning by charging aperture 3
(20: 1: 500, weight ratio) put into pressure-resistant tank body 1, confined reaction system, agitating device 7 and heater are opened, 40~60
Stirring makes carry out enzyme digestion reaction at DEG C；
B) question response terminates, and opening charging aperture makes reaction system be in atmospheric pressure state, then opens valve B12, releases anti-
Answer liquid.
Preferably, the bottom in pressure-resistant tank body 1 is placed with screening plant (such as screen cloth), to screen out fixation
Change enzyme granulate, after question response liquid is released completely, flange lid 2 can be opened, screen cloth is taken out, obtain collected immobilization
Enzyme.
Embodiment 4：Diphasic system immobilized enzyme hydrolysis reaction is carried out using apparatus of the present invention
A) by charging aperture 3 by immobilised enzymes, Gardenoside, pH=3.5~5.5 Acetic acid-sodium acetate buffer solution and acetic acid
Ethyl ester is pressed proportioning (10: 1: 100: 100, weight ratio) and put into pressure-resistant tank body 1, confined reaction system, opens agitating device 7 and adds
Hot device, being stirred at 45~65 DEG C makes carry out enzyme digestion reaction；
B) question response terminates, and opening charging aperture makes reaction system be in atmospheric pressure state, then opens valve B12, releases anti-
Answer liquid.
Preferably, the bottom in pressure-resistant tank body 1 is placed with screening plant (such as screen cloth), to screen out fixation
Change enzyme granulate, after question response liquid is released completely, flange lid 2 can be opened, screen cloth is taken out, obtain collected immobilization
Enzyme.
Embodiment 5：Diphasic system immobilized enzyme hydrolysis reaction is carried out using apparatus of the present invention
A) by charging aperture 3 by immobilised enzymes, scutelloside, pH=4.0~6.0 Acetic acid-sodium acetate buffer solution and chloroform
Put into by proportioning (20: 1: 500: 500, weight ratio) in pressure-resistant tank body 1, confined reaction system, open agitating device 7 and heating
Device, being stirred at 40~60 DEG C makes carry out enzyme digestion reaction；
B) question response terminates, and opening charging aperture makes reaction system be in atmospheric pressure state, then opens valve B12, releases anti-
Answer liquid.
Preferably, the bottom in pressure-resistant tank body 1 is placed with screening plant (such as screen cloth), to screen out fixation
Change enzyme granulate, after question response liquid is released completely, flange lid 2 can be opened, screen cloth is taken out, obtain collected immobilization
Enzyme.
Finally be necessary described herein be：
Above-described embodiment is served only for the further description made to technical scheme proposed by the present invention, is only used for helping
Understand technical scheme, it is impossible to be interpreted as limiting the scope of the invention.Those skilled in the art is according to this
The non-intrinsically safe modifications and adaptations that the above of invention is made belong to the scope of protection of present invention.
Claims (4)
1. a kind of device for the preparation and enzyme digestion reaction that can be used as immobilised enzymes, including：Pressure-resistant tank body, flange lid, it is arranged on
The charging aperture and visor of flange top of closure, the compressed gas entrance for being arranged on pressure-resistant tank body top and bottom discharging opening, stir
Device and heater are mixed, O-ring seals are provided between described pressure-resistant tank body and flange lid；It is characterized in that：Go out in bottom
Distributor is connected with by flange on the pipeline of material mouth, the distributor is covered by distribution and be evenly distributed with some manholes
Distribution grid forms；Valve A is provided with the discharge pipe above distributor, discharging is provided with the discharge pipe above valve A
Branch pipe, valve B is provided with the discharging branch pipe.
2. device as claimed in claim 1, it is characterised in that：A diameter of 2.0~5.0mm of the manhole.
3. device as claimed in claim 1, it is characterised in that：Muff is provided with the outside of the pressure-resistant tank body.
4. device as claimed in claim 1, it is characterised in that：The charging aperture and compressed gas entrance are equipped with sealable
Locking closure.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410313414.XA CN105316232B (en) | 2014-07-02 | 2014-07-02 | A kind of device and its application for immobilised enzymes preparation/reaction |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201410313414.XA CN105316232B (en) | 2014-07-02 | 2014-07-02 | A kind of device and its application for immobilised enzymes preparation/reaction |
Publications (2)
| Publication Number | Publication Date |
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| CN105316232A CN105316232A (en) | 2016-02-10 |
| CN105316232B true CN105316232B (en) | 2018-04-06 |
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| CN201410313414.XA Active CN105316232B (en) | 2014-07-02 | 2014-07-02 | A kind of device and its application for immobilised enzymes preparation/reaction |
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Family Cites Families (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH0659217B2 (en) * | 1985-08-06 | 1994-08-10 | 三井東圧化学株式会社 | Method for producing immobilized enzyme and device for use in the method |
| 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 facilities |
| 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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2014
- 2014-07-02 CN CN201410313414.XA patent/CN105316232B/en active Active
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