CN102337258B - Pectinase immobilizing carrier, preparation thereof, and method for immobilizing pectinase - Google Patents
Pectinase immobilizing carrier, preparation thereof, and method for immobilizing pectinase Download PDFInfo
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- CN102337258B CN102337258B CN 201110303735 CN201110303735A CN102337258B CN 102337258 B CN102337258 B CN 102337258B CN 201110303735 CN201110303735 CN 201110303735 CN 201110303735 A CN201110303735 A CN 201110303735A CN 102337258 B CN102337258 B CN 102337258B
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- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
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- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
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Abstract
The invention discloses a pectinase immobilizing carrier, a preparation method thereof, and a method for immobilizing pectinase. The pectinase immobilizing carrier has the capacity of performing uniform enzymolysis, heterogeneous phase recovery and reutilization on pectinase, and is nano-scale magnetic starch microspheres which have the iron content of 50-70 percent and of which the particle size is controlled by ultrasonic waves, has a linear positive correlation with time and ranges from 10 to 80nm. The pectinase immobilizing carrier is applied to the pectinase, particularly immobilization of goose-source pectinase produced by penicillium oxalicum from goose. The preparation method comprises the following steps of: preparing starch milk, uniformly mixing FeCl3, FeCl2 and the starch milk, regulating a pH value, performing ultrasonic processing, stirring, neutralizing by using acid, washing by using 95 percent ethanol, performing magnetic separation, and performing vacuum drying. The method for immobilizing the pectinase comprises the following steps of: preparing a pectinase solution; activating the carrier; and immobilizing the pectinase and separating and drying the immobilized pectinase. The carrier greatly improves the service efficiency of enzyme, reduces the consumption of raw materials and energy, prevents environmental pollution, contributes to industrial production and can be widely applied to the fields such as fruit juice and vegetable processing and the like.
Description
Technical field
The present invention relates to a kind of method of fixation support and preparation method and immobilization polygalacturonase of polygalacturonase, belong to the enzyme engineering field.
Background technology
Magnetic macromolecular microsphere is the functional high molecule material of one type of excellent property, is to be carrier with synthetic macromolecule or biopolymer, through absorption and embedding Fe
2O
3, Fe
3O
4Or other magnetic particle, form functional high molecule material with magnetic.Magnetic macromolecular microsphere is the tiny capsules of core-shell type structure, and the magnetic nuclear of preparation magnetic microsphere mainly is Fe
3O
4Deng MOX, shell mainly is made up of two types of materials: one type is synthetic macromolecule, mainly contains Vilaterm, PS, Vestolen PP 7052 phthalein amine, Z 150PH, nitrocotton and the liquor-saturated butyral of Vilaterm etc., not biodegradable; Another kind of is biopolymer, mainly contains starch, gelatin, BSA, POLYACTIC ACID, Protanal TXF 200 etc., biodegradable.Combining of the shell of magnetic macromolecular microsphere and magnetic nuclear mainly is the effect through Van der Waals force, hydrogen bond, co-ordination bond; Polymer is by means of these reactive forces; Be bound by the metal oxide crystal surface firmly; Form solid ball-like structure, as the paramagnetism polymer microsphere of fixed enzyme vector.
Polygalacturonase (Pectinase, PE) because reaction conditions is gentle, and specificity is strong, catalytic efficiency (is high, and reaction is control easily, is widely used in food, wine brewing, environmental protection, medicine and textile industry field, has become one of the world's four big zymins.Free polygalacturonase in use is prone to the variation sex change inactivation with environment, and is difficult for from reaction system, separating repeated use.Enzyme immobilization technology is to realize that enzyme repeats to use continuously and improves its stable effective means.
The fixation support of polygalacturonase and preparation method always are important subject in the enzyme engineering field.Wherein fixation support mainly contain one, the hydrolysis method (being coprecipitation method) of chemical process, with a certain proportion of FeCl
3And FeCl
2With in synthetic macromolecule solution, add basic soln, make to generate Fe
3O
4Magnetic particle, the size distribution broad of the magnetic macromolecular microsphere particle that this method makes, inhomogeneous; Two, comprise high energy ball mill method, suspension polymerization and anti-phase suspension method of reproduction three kinds in the physical method, its magnetic particle that obtains in magnetic polymer material interacts, and very easily reunites, and is difficult to realize the superparamagnetism ability.
Research about polygalacturonase has CN102010858A (immobilized carrier of a kind of polygalacturonase and process for fixation), mainly is to be carrier with the sodium-alginate magnetic microsphere, and this carrier has Fe
3O
4Magnetic, sodium-alginate, LUTARALDEHYDE three parts are formed, because the special Fe that adds
3O
4Magnetic, it is synthetic to belong to physical method, therefore very easily reunites, and particle diameter is bigger, is difficult to realize the superparamagnetism ability, and the external morphology slabbing is unfavorable for the combination of polygalacturonase, and functional group's characteristic is not obvious, is unfavorable for the orthoselection linking agent.So seek practical more and effective polygalacturonase fixation support, further optimize enzyme immobilization technology and be very important.
Again; Because the goose source penicillium oxalicum polygalacturonase is the polygalacturonase that produces from a kind of penicillium oxalicum fermentation that the goose enteron aisle extracts; Be to belong to the animal source polygalacturonase; Different with the commercially available polygalacturonase source that is produced by fermentation of Aspergillus niger, prior art does not have the carrier that is more suitable for so far, for the scientific utilization of goose source penicillium oxalicum polygalacturonase provides foundation.
Summary of the invention
The object of the invention provides fixation support of a kind of polygalacturonase and preparation method thereof, to remedy the deficiency of prior art.
Another object of the present invention is the immobilization that this carrier is applied to polygalacturonase, particularly is applied to the immobilization of goose source penicillium oxalicum polygalacturonase, for the scientific and efficient utilization of animal source polygalacturonase provides novel method, to be used for remedying the deficiency of conventional art.
The present invention is the improvement to coprecipitation method, promptly utilizes the mixing coprecipitation method to prepare nano-scale magnetic spherex carrier, is being dissolved with Fe
2+, Fe
3+Mixing solutions in add the precipitation agent alkaline solution, and with the milk of starch hybrid reaction, generate component and precipitate uniformly, precipitation heat is decomposed and is obtained the nano-scale magnetic spherex.To reach one of which directly obtains the component homogeneous through the various chemical reactions in the solution nano-powder; It two is nano-powders that easy prepared sizes are little and be evenly distributed; To increase specific surface area; Be beneficial to combining closely of carrier functional group and the nonactive group of polygalacturonase, be beneficial to and the combining closely of polygalacturonase.
Utilize the UW effect among the present invention again; Not only remedy the uneven deficiency of particle diameter in the coprecipitation method; And can be through changing the size of ultrasonic treatment time, frequency, the next effectively control of power condition particle diameter; Thereby obtain the nano-scale magnetic spherex of the target grain size of needs,, realize that the carrier of autotelic selection different-grain diameter carries out not fixing of enzyme of the same race with the target property of further raising particulate as carrier.Processing is difficult to immobilized polygalacturonase, like goose source penicillium oxalicum polygalacturonase etc.
Carrier of the present invention can also carry out immobilization to the goose source penicillium oxalicum polygalacturonase; The goose source penicillium oxalicum polygalacturonase is that high-quality aquatic bird institute of Qingdao Agricultural University screens 1 fungal strain F67 (2006) from the goose caecum; Be accredited as penicillium oxalicum through Institute of Microorganism, Academia Sinica; With this bacterium is the polygalacturonase of fermented bacterium preparation, because system enzyme bacterial classification is different, physico chemical factors such as optimum temperuture, ph optimum are different.Repeat usage and the righttest action condition after its immobilization are studied in the immobilization of research goose source penicillium oxalicum polygalacturonase, are of great importance for economical and efficient, the scientific and reasonable goose source penicillium oxalicum polygalacturonase that utilizes.
The object of the invention is realized through following technical scheme:
A kind of polygalacturonase fixation support; Be to have polygalacturonase is realized that even enzymolysis, out-phase reclaim, the carrier of recycling ability; It is characterized in that this carrier is that iron-holder 50%～70%, particle diameter can receive UW control and is linear positive correlation with the time, particle size range is the nano-scale magnetic spherex of 10nm～80nm.
The preparation method of above-mentioned polygalacturonase fixation support (being nano-scale magnetic spherex carrier) is following:
(1) at first using deionized water to become concentration with the starch dispersed with stirring is 30%～50% milk of starch;
(2) take by weighing FeCl in 1: 1～1: 2 ratio
3, FeCl
2, be settled to 10～15 times of milk of starch with deionized water dissolving, pour milk of starch into, put in 65 ℃ of water-baths and heat up, dripping the 0.5M alkaline solution, to transfer to the pH value be 9～13;
(3) with the above-mentioned mixing liquid that mixes up pH with supersound process 20min～50min; Liquid behind the mixing is placed 65 ℃ of stirred in water bath, leave standstill and be cooled to room temperature, be neutralized to neutrality with acid solution;
(4) with the solidliquid mixture after the above-mentioned neutralization with 95% washing with alcohol 3 times; Utilize residual magnetization to make magnetic spherex and liquid separation then for the magnet of 12.3T; The supernatant that inclines, vacuum lyophilization promptly obtains nano-scale magnetic spherex carrier under-20 ℃～-45 ℃, 20Pa～100Pa.
Fixation support of the present invention is applied to the immobilization of polygalacturonase, particularly is applied to the immobilization of animal source polygalacturonase---the immobilization of goose source penicillium oxalicum polygalacturonase.
Starch in the said step (1) is cereal starch, potato starch or other kind of starch.Its milk of starch pH value is nature pH.
Soluble salt in the said step (2) is Fe
2(SO
4)
3, FeSO
4Perhaps FePO
4, Fe
3(PO
4)
2
Alkaline solution in the said step (2) is NaOH, KOH and other alkaline solutions, also can be alkaline salt solution.
Supersound process condition in the said step (3) is 10W～950W, 20KHz～25KHz, 20～50min, and the supersound process time is long more, and power is big more, and the particle diameter of nano-scale magnetic spherex carrier is more little.
Acid solution in the said step (3) is acetic acid, dilute sulphuric acid or Hydrogen chloride.
Vacuum lyophilization condition in the said step (4) is-20 ℃～-45 ℃, 20Pa～100Pa.
Compared with prior art, the present invention has following advantage:
(1) carrier of the present invention adopts biodegradable, environmentally friendly, as to contain great amount of hydroxy group natural renewable resource starch as shell, has good bio-compatibility, can keep the active and stable of enzyme well, has improved the service efficiency of enzyme.
(2) carrier of the present invention directly utilizes FeCl
3With FeCl
2Reaction generates Fe under the alkalescence effect
3O
4Magnetism molecule combines in reaction process with starch, and need not under anaerobic environment, to carry out, and makes magnetism molecule and starch binding tightr, more helps suitability for industrialized production, reduces cost.
(3) carrier of the present invention utilizes vacuum lyophilization, the loss of activity of hydroxyl, carboxyl functional group in the carrier in the minimizing air blast heat drying, and effectively functional group's recovery is higher in the carrier of preparation.
(4) carrier of the present invention can make polygalacturonase reuse; Can reduce starting material and energy consumption, reduce the industrial residue discharging of waste liquid, prevent the pollution of the environment; Can be widely used in fruit juice, vegetables juice processing and other fields, be one and have the enzyme immobilization carrier that the great exploitation of wide spectrum is worth.
Polygalacturonase process for fixation of the present invention comprises the steps and processing condition:
(1) polygalacturonase formulations prepared from solutions: take by weighing 1～3 part of polygalacturonase, deionized water dissolving is settled to 100 times of volumes.
(2) magnetic spherex carrier activation: it is microsphere supported to take by weighing 1 part of magnetic W-Gum; 2.5%～7.5% the LUTARALDEHYDE that adds 20 times of volumes; Magnetic microsphere fully is immersed in the glutaraldehyde solution, and 30 ℃, 200r/min shake crosslinked 6h～8h in shaking table, and product is used the deionized water thorough washing; Utilize residual magnetization to make magnetic spherex and liquid separation for the magnet of 12.3T; The supernatant that inclines, vacuum lyophilization under-20 ℃～-45 ℃, 20Pa～100Pa promptly obtains activatory magnetic spherex.
(3) immobilization of polygalacturonase: take by weighing 1 part of activatory magnetic spherex, 1～5 part of 1% goose source penicillium oxalicum polygalacturonase solution, using pH is that 3～5 damping fluid is settled to 25 times of volumes, under 30 ℃, rotating speed 200r/min in shaking table immobilization 2h～6h.
(4) the immobilization polygalacturonase separates dry: use residual magnetization to make magnetic spherex and liquid separation as the magnet of 12.3T step (3) products therefrom; Supernatant inclines; In supernatant, detect less than till the protein with the damping fluid repetitive scrubbing; Vacuum lyophilization under-20 ℃～-45 ℃, 20Pa～100Pa promptly gets magnetic immobilization polygalacturonase.
The volume of constant volume is lived by the enzyme of solid pectin enzyme and is determined in the middle polygalacturonase formulations prepared from solutions of said step (1), is diluted to the polygalacturonase solution enzyme 1000u/ml of being alive.
Buffered soln in the affiliated step (2) can be pH3～5 acetic acid-sodium acetate buffer solution, phosphoric acid-sodium dihydrogen phosphate buffer etc.
Compared with prior art, the present invention has following advantage:
(1) carrier of the present invention is the nano-scale magnetic spherex, size be evenly distributed, the immobilization polygalacturonase is evenly reacted.
(2) the present invention utilizes vacuum lyophilization, makes enzyme storage rate alive higher.
(3) the present invention utilizes the magnetic spherex immobilization top condition that the response surface shaker test is confirmed, the result who draws can better be used for the actual production prediction.
(4) the goose source penicillium oxalicum polygalacturonase of utilization of the present invention is the animal source polygalacturonase, the scientific and efficient of animal source polygalacturonase is utilized significant, has high economic benefit and social benefit.
Description of drawings
The grain-size graph of the nano-scale magnetic spherex carrier of Fig. 1 supersound process 50min of the present invention.(through microspherulite diameter 30～60nm of supersound process 50min large percentage, narrow distribution range)
The grain-size graph of Fig. 2 immobilization goose source penicillium oxalicum of the present invention polygalacturonase.(particle diameter 70～170nm large percentage of immobilized enzyme, distribution range is wider)
The nano-scale magnetic spherex carrier form figure of supersound process under Fig. 3 ESEM of the present invention.(particle diameter is even, sphere, and it is few to reunite, the well-crystallized, degree of scatter is good.)
Immobilization goose source penicillium oxalicum polygalacturonase microballoon aspect graph under Fig. 4 ESEM of the present invention.(it is big that the immobilized enzyme particle diameter becomes, and takes place crosslinked with LUTARALDEHYDE and polygalacturonase.)
Fig. 5 nano-scale magnetic spherex of the present invention carrier FT-IR collection of illustrative plates.(the magnetic spherex contains 1628cm
-1Hydroxyl, 3421cm
-1Carboxyl, 580cm
-1Fe
3O
4Charateristic avsorption band)
The FT-IR collection of illustrative plates of Fig. 6 immobilization goose source penicillium oxalicum of the present invention polygalacturonase.(except that containing the carrier absorption peak, also contain 1690～1500cm
-1, 1475～1000cm
-1Two key stretching vibration district X-H in-plane bending vibrations district)
Embodiment
Using deionized water to become concentration with the Zulkovsky starch dispersed with stirring is 30% milk of starch.Ratio in 1: 1.5 takes by weighing FeCl
3, FeCl
2, be settled to 10 times of milk of starch with deionized water dissolving, pour milk of starch into, put in 65 ℃ of water-baths and heat up.Reach that to drip 0.5M NaOH solution adjust pH after the temperature be 10, ultrasonic wave is handled 30min under 25KHz, 900W power.Liquid after handling is placed 65 ℃ of stirred in water bath 1.5h; Leave standstill after reaction finishes and be cooled to room temperature, be neutralized to neutrality, with 95% washing with alcohol 3 times with Glacial acetic acid min. 99.5; Utilize residual magnetization to make magnetic spherex and liquid separation for the magnet of 12.3T; The supernatant that inclines, vacuum-drying under-45 ℃, 20Pa obtains magnetic spherex carrier.This carrier detects through the laser diffraction granularity appearance, and particle diameter is 10.29nm～80.45nm, has paramagnetism, detects through Fourier infrared spectrograph again, and is as shown in Figure 5, and this carrier has significantly 580cm
-1Fe
3O
4, 1628cm
-1Hydroxyl, 3421cm
-1Therefore the carboxyl charateristic avsorption band is the immobilized good carrier of polygalacturonase, stereoscan photograph as shown in Figure 3, this carrier particle diameter evenly, reunite less, the well-crystallized, be the immobilized good carrier of polygalacturonase.
Embodiment 2
Using deionized water to become concentration with the W-Gum dispersed with stirring is 40% milk of starch.Ratio in 1: 2 takes by weighing Fe
2(SO
4)
3, FeSO
4, be settled to 15 times of milk of starch with deionized water dissolving, pour milk of starch into, put in 65 ℃ of water-baths and heat up.Reach that to drip 0.5M KOH solution adjust pH after the temperature be 11, supersound process 20min under 20KHz, the 450W power.Liquid after handling is placed 65 ℃ of stirred in water bath 2h; Leave standstill after reaction finishes and be cooled to room temperature, be neutralized to neutrality, with 95% washing with alcohol 3 times with Glacial acetic acid min. 99.5; Utilize residual magnetization to make magnetic spherex and liquid separation for the magnet of 12.3T; The supernatant that inclines, vacuum-drying under-45 ℃, 20Pa obtains magnetic spherex carrier.Detect through the laser diffraction granularity appearance, this carrier particle diameter is 14.35nm～80.31nm, has paramagnetism, detects through Fourier infrared spectrograph, and is as shown in Figure 5, and this microballoon has significantly 580cm
-1Fe
3O
4, 1628cm
-1Hydroxyl, 3421cm
-1Carboxyl charateristic avsorption band, stereoscan photograph as shown in Figure 3, this carrier particle diameter evenly, reunite less, the well-crystallized, be the immobilized good carrier of polygalacturonase.
Embodiment 3
Take by weighing 1 part of activation magnetic carrier, 3 parts of 1% the goose source penicillium oxalicum polygalacturonase solution of 5% glutaraldehyde cross-linking; Using pH is that 4 damping fluid is settled to 25 times of volumes; Under 30 ℃, rotating speed 200r/min in shaking table oscillatory reaction 3h; Use residual magnetization to make magnetic spherex and liquid separation as the magnet of 12.3T, the supernatant that inclines detects in supernatant less than till the protein with the damping fluid repetitive scrubbing; Vacuum lyophilization under-20 ℃～-45 ℃, 20～100Pa promptly gets magnetic immobilization polygalacturonase.Utilizing laser diffraction granularity appearance, ESEM, Fourier infrared spectrograph, X diffractometer to record the result can know; The immobilization polygalacturonase is compared particle diameter and is become big with magnetic spherex carrier; Functional group changes to some extent, explains that polygalacturonase has been linked on the magnetic starch carrier.Measure the enzyme recovery alive with hypoiodous acid sodium method; The enzyme recovery alive is 85.6%; Applicable pH is 3～5, and resolvase enzyme after 40 ℃ is lived and significantly descended, and immobilized enzyme significantly descends in the time of 50 ℃; It still is 69.6% that 5 relative enzyme work is reused in the polygalacturonase immobilization, and reusing 8 relative enzyme work is 60.1%.
Embodiment 4
Take by weighing 2 parts of activation magnetic carriers, 4 parts of 1% the goose source penicillium oxalicum polygalacturonase solution of 7.5% glutaraldehyde cross-linking; Using pH is that 4 damping fluid is settled to 25 times of volumes; Under 30 ℃, rotating speed 200r/min in shaking table oscillatory reaction 2h; Use residual magnetization to make magnetic spherex and liquid separation as the magnet of 12.3T, the supernatant that inclines detects in supernatant less than till the protein with the damping fluid repetitive scrubbing; Vacuum lyophilization under-20 ℃～-45 ℃, 20～100Pa promptly gets magnetic immobilization polygalacturonase.Utilizing laser diffraction granularity appearance, ESEM, Fourier infrared spectrograph, X diffractometer to record the result can know; The immobilization polygalacturonase is compared particle diameter and is become big with magnetic spherex carrier; Functional group changes to some extent, explains that polygalacturonase has been linked on the magnetic starch carrier.Measure the enzyme recovery alive with hypoiodous acid sodium method; The enzyme recovery alive is 85.2%; Applicable pH is 3～5, and resolvase enzyme after 40 ℃ is lived and significantly descended, and immobilized enzyme significantly descends in the time of 50 ℃; It still is 70.6% that 5 relative enzyme work is reused in the polygalacturonase immobilization, and reusing 8 relative enzyme work is 59.7%.
Embodiment 5
Take by weighing 5 parts of activation magnetic carriers, 5 parts of 1% the goose source penicillium oxalicum polygalacturonase solution of 2.5% glutaraldehyde cross-linking; Using pH is that 4 damping fluid is settled to 25 times of volumes; Under 30 ℃, rotating speed 200r/min in shaking table oscillatory reaction 6h; Use residual magnetization to make magnetic spherex and liquid separation as the magnet of 12.3T, the supernatant that inclines detects in supernatant less than till the protein with the damping fluid repetitive scrubbing; Vacuum lyophilization under-20 ℃～-45 ℃, 20Pa～100Pa promptly gets magnetic immobilization polygalacturonase.Utilizing laser diffraction granularity appearance, ESEM, Fourier infrared spectrograph, X diffractometer to record the result can know; The immobilization polygalacturonase is compared particle diameter and is become big with magnetic spherex carrier; Functional group changes to some extent, explains that polygalacturonase has been linked on the magnetic starch carrier.Measure the enzyme recovery alive with hypoiodous acid sodium method; The enzyme recovery alive is 86%; Applicable pH is 3～5, and resolvase enzyme after 40 ℃ is lived and significantly descended, and immobilized enzyme significantly descends in the time of 50 ℃; It still is 70.2% that 5 relative enzyme work is reused in the polygalacturonase immobilization, and reusing 8 relative enzyme work is 60.4%.
The commercially available black mold polygalacturonase of embodiment 6 immobilizations
Take by weighing 1 part of activation magnetic carrier, 3 parts of 1% the commercially available black mold polygalacturonase solution of 5% glutaraldehyde cross-linking; Using pH is that 4 damping fluid is settled to 25 times of volumes; Under 30 ℃, rotating speed 200r/min in shaking table oscillatory reaction 3h; Use residual magnetization to make magnetic spherex and liquid separation as the magnet of 12.3T, the supernatant that inclines detects in supernatant less than till the protein with the damping fluid repetitive scrubbing; Vacuum lyophilization under-20 ℃～-45 ℃, 20～100Pa promptly gets magnetic immobilization polygalacturonase.Utilizing laser diffraction granularity appearance, ESEM, Fourier infrared spectrograph, X diffractometer to record the result can know; The immobilization polygalacturonase is compared particle diameter and is become big with magnetic spherex carrier; Functional group changes to some extent, explains that polygalacturonase has been linked on the magnetic starch carrier.Measure the enzyme recovery alive with hypoiodous acid sodium method; The enzyme recovery alive is 84.7%; Applicable pH is 3～5, and resolvase enzyme after 40 ℃ is lived and significantly descended, and immobilized enzyme significantly descends in the time of 45 ℃; It still is 69.4% that 5 relative enzyme work is reused in the polygalacturonase immobilization, and reusing 8 relative enzyme work is 58.9%.Show that the present invention also can be applied to the polygalacturonase in other source.
Claims (6)
1. the preparation method of a polygalacturonase fixation support is characterized in that preparation process is following:
(1) at first using deionized water to become concentration with the starch dispersed with stirring is 30%～50% milk of starch;
(2) ratio in 1:1～1:2 takes by weighing soluble salt FeC1
3, FeCl
2, be settled to 10～15 times of milk of starch with deionized water dissolving, pour milk of starch into, put in 65 ℃ of water-baths and heat up, dripping the 0.5M alkaline solution, to transfer to the pH value be 9～13, processes mixing liquid;
(3) mixing liquid with the above-mentioned pH of mixing up places 65 ℃ of stirred in water bath again with ultrasonication 20min ~ 50min, leaves standstill and is cooled to room temperature, is neutralized to neutrality with acid solution, processes the solidliquid mixture after the neutralization;
(4) with the solidliquid mixture after the above-mentioned neutralization with 95% washing with alcohol 3 times; Utilize residual magnetization to make magnetic spherex and liquid separation then for the magnet of 12.3T; The supernatant that inclines, vacuum lyophilization promptly gets fixation support under-20 ℃～-45 ℃, 20Pa～100Pa.
2. the preparation method of fixation support as claimed in claim 1 is characterized in that the supersound process condition in the above-mentioned steps (3) does, 10W ~ 950W, 20KHz ~ 25KHz, 20min ~ 50min.
3. the preparation method of fixation support as claimed in claim 1 is characterized in that the vacuum lyophilization condition in the above-mentioned steps (4) is-20 ℃～-45 ℃, 20Pa～100Pa.
4. the preparation method of fixation support as claimed in claim 1 is characterized in that the alkaline solution in the above-mentioned steps (2) is NaOH or KOH.
5. the preparation method of fixation support as claimed in claim 1 is characterized in that the acid solution in the above-mentioned steps (3) is acetic acid, dilute sulphuric acid or Hydrogen chloride.
6. the purposes of the fixation support of claim 1 is characterized in that this fixation support is applied to the immobilization of polygalacturonase.
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|---|---|---|---|
| CN 201110303735 CN102337258B (en) | 2011-10-10 | 2011-10-10 | Pectinase immobilizing carrier, preparation thereof, and method for immobilizing pectinase |
| PCT/CN2012/070180 WO2013053210A1 (en) | 2011-10-10 | 2012-01-10 | Immobilized carrier for pectinase and methods for preparing and immobilizing pectinase |
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| CN 201110303735 CN102337258B (en) | 2011-10-10 | 2011-10-10 | Pectinase immobilizing carrier, preparation thereof, and method for immobilizing pectinase |
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