CN103667243B - A kind of ionic liquid and modifier process after straw for the method for cellulase immobilization carrier - Google Patents
A kind of ionic liquid and modifier process after straw for the method for cellulase immobilization carrier Download PDFInfo
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- CN103667243B CN103667243B CN201310674394.4A CN201310674394A CN103667243B CN 103667243 B CN103667243 B CN 103667243B CN 201310674394 A CN201310674394 A CN 201310674394A CN 103667243 B CN103667243 B CN 103667243B
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Abstract
The present invention discloses the straw after a kind of ionic liquid and modifier process for the method for cellulase immobilization carrier, comprises the steps: that the pre-treatment of stalk, the modification of stalk and carrier drying are shaping. The advantage of the present invention is: the cellulose materials that 1, employing ionic liquid and modifier modification stalk obtain is to prepare enzyme immobilization carrier, treating processes environmental protection, pollution-free, operational condition is gentle, treatment process simple and feasible, and ionic liquid and the modifier after process can reclaim completely, can greatly reduce the production cost of carrier. 2, the enzyme adsorption rate height on fixed enzyme vector that prepared by the inventive method, stability height, it is to increase the catalytic activity of enzyme. 3, the enzyme immobilization carrier that prepared by the inventive method can be reused repeatedly, still can keep higher enzymic activity.
Description
Technical field
The invention belongs to technical field of biochemical industry, it relates to a kind of ionic liquid and modifier process after straw for the preparation method of cellulase immobilization carrier.
Background technology
Enzyme is a kind of material with special biological activity being made up of amino acid, and it is present in the animal and plant body of all work, is maintain body normal function, digest food, the required material of the one of the vital movements such as repair tissue. Enzyme is the biomacromolecule with biocatalytic Activity, i.e. biological catalyst, and it can accelerate the speed of biochemical reaction, but does not change direction and the product of reaction. Enzyme can only be used for accelerating the speed of all kinds of biochemical reaction, but is not biochemical reaction itself. Enzyme immobilizatio be exactly become after enzyme being combined with carrier by some method be insoluble to containing substrate mutually in, thus make enzyme be concentrated or be limited in and in certain spatial dimension, carry out enzyme digestion reaction, be therefore referred to as immobilized enzyme. Immobilized enzyme, compared with resolvase, has following advantage: 1. very easily by immobilization enzyme-to-substrate, product separately; 2. batch reactions and dress post successive reaction can be carried out repeatedly again in the long period; 3. in most of the cases, it is possible to improve the stability of enzyme; 4. enzyme reaction process can strictly be controlled not have in 5. product solution the residual of enzyme, simplifies purifying technique; 6. relatively resolvase is more suitable for multienzymatic reaction; 7. the receipts rate of product can be increased; 8. the service efficiency of enzyme improves, and cost reduces. Enzyme immobilizatio method can be roughly divided into two big classes: entrapping method and connection method. Entrapping method can be further divided into two groups: gel embedding method and micro-capsule entrapping method. Connection method can be divided into again: absorption method and covalently bound method. Mierocrystalline cellulose is the main component of cotton, timber, flax, the grass high plant cell wall such as class, can produce several hundred hundred million tons by photosynthesis every year, is inexhaustible, the nexhaustible renewable resources of nature.
Ionic liquid refers to the salt being all made up of zwitterion. Owing to ionic liquid has non-volatility, good ionic conductivity and thermal conductivity, high heat capacity and high thermal stability, the features such as selective dissolution power so that ionic liquid becomes one of focus of current chemical field research.
On existing carrier, the enzyme of absorption easily comes off, and thus causes immobilized enzyme stability and activity not high, and enzyme immobilization carrier seldom can reuse simultaneously, therefore there is the aftertreatment of solid support material and the idle problem of useless carrier.
Summary of the invention
Goal of the invention: it is an object of the invention to provide a kind of ionic liquid and modifier process after straw for high enzyme charge capacity, enzymatic activity high, can reuse and the preparation method of cellulase immobilization carrier of cheap environmental protection.
Technical scheme: ionic liquid of the present invention and modifier process after straw for the method for cellulase immobilization carrier, comprise the steps:
(1) pre-treatment of stalk: remove the impurity on stalk, drying the section of cutting into;
(2) modification of stalk: first use ion liquid dissolving stalk, then add modifier stirring evenly, obtaining cellulose carrier solution, this modification uses ultrasonic wave, and the Surface and internal structure of stalk is changed;
(3) carrier drying is shaping: add solvent in above-mentioned cellulose carrier solution, stirs evenly, and washing removes impurity, and then cellulase immobilization carrier is made in dry, vacuum outgas.
In step (1), stalk chooses stipes position, and stalk stipes cuts into the segment of 0.4-0.6cm.
In step (2), the weight ratio of described stalk and ionic liquid is 1:1-5; The final concentration of described modifier is 0.1-0.2M. Described ionic liquid is the ionic liquid with cellulose dissolution function, and great majority are acidic ion liquid. Described modifier is that polyoxyethylene glycol or bromine are for fourth acetimide. The power of described ultrasonication is 30-60w, and the treatment time is 5-10min.
Solvent described in step (3) is water or short chain alcohol, and the solvent added is 1-5 times of cellulose carrier liquor capacity.
Inventive principle: containing a large amount of Mierocrystalline celluloses in stalk, the ionic liquid added can dissolve, destroy the low Mierocrystalline cellulose of degree of crystallinity in stalk, the rigid fiber carrier of the similar nano-cellulose that remaining is all degree of crystallinity height, intensity is big, and the structure of the surface of residual fiber element carrier and inner formation porous, for enzyme immobilizatio provides a lot of sites, for subsequent reactions provides condition; The modifier added is combined with fibrous carrier, and is connected to surface and the inside of fibrous carrier, it is possible to amino groups is combined on enzyme molecule, and then realizes enzyme immobilizatio.
Useful effect: the present invention compared with prior art: its remarkable advantage is: 1, adopt there is non-volatility, good ionic conductivity and thermal conductivity, high heat capacity and high thermal stability, the cellulose materials that the treatment agent modification stalks such as the ionic liquid of the advantages such as selective dissolution power obtain is to prepare enzyme immobilization carrier, treating processes environmental protection, pollution-free, operational condition is gentle, treatment process simple and feasible, and ionic liquid and the modifier after process can reclaim completely, can greatly reduce the production cost of carrier. 2, the enzyme adsorption rate height on fixed enzyme vector that prepared by the inventive method, stability height, it is to increase the catalytic activity of enzyme. 3, the enzyme immobilization carrier that prepared by the inventive method can be reused repeatedly, still can keep higher enzymic activity.
Accompanying drawing explanation
Fig. 1 is the scanning electron microscope (SEM) photograph (amplifying 1000 times) of the carrier prepared by the inventive method.
Embodiment
Below technical solution of the present invention is described in detail.
Embodiment 1: carry out as follows:
(1) get the stipes of maize straw, wash the impurity such as the mud dust on stalk, after dry, cut into the segment of about 0.5cm (0.4-0.6cm).
(2) get in the there-necked flask that 1g stalk adds 250ml, first add 5g ionic liquid [DMIM] [DMP] when temperature 25 DEG C (room temperature), 5min is processed with ultrasonic washing instrument (setting power 60w), add the Macrogol 4000 60ml that final concentration is 0.2M again, stir evenly, ultrasonic washing instrument (setting power 60w) processes 10min, the Surface and internal structure of stalk is modified, stalk internal structure is changed, surface-area increases, the adsorption site of enzyme increases, and obtains cellulose carrier solution.
(3) adding the water of 3 times of volumes in above-mentioned cellulose carrier solution, evenly stir 10min, added by carrier with in deionized water, and impose violent stirring, wash 3-5 time and remove impurity, carrier 60 DEG C of dryings, vacuum outgas is prepared into carrier.
As shown in Figure 1, the fiber (NMMO process easily comes off and stripping) that in bamboo sheet, degree of crystallinity is low removes the scanning electron microscope (SEM) photograph of this carrier through process, and the carrier obtained by above-mentioned treatment process is vesicular structure, for enzyme provides more adsorption site.
Embodiment 2: preparation process is substantially the same manner as Example 1, difference is: 1, add 1g ionic liquid [EMIM] Cl Treating straw; 2, the cetomacrogol 1000 that 70ml final concentration is 0.1M is added; 3, using ultrasonic washing instrument (setting power 30w) twice, the treatment time is 10min; 4, the whole process insulation of step (2) Treating straw, temperature is 90 DEG C; 5, add isopyknic washing with alcohol with cellulose carrier solution and remove impurity.
Embodiment 3: preparation process is substantially the same manner as Example 1, difference is: 1, add 3g ionic liquid [EMIM] [oAc] Treating straw; 2, bromine that 100ml final concentration is 0.15M is added for fourth acetimide; 3, using ultrasonic washing instrument (setting power 50w) twice, the treatment time is 8min; 4, the whole process insulation of step (2) Treating straw, temperature is 50 DEG C; 5, the water added is 5 times of cellulose carrier liquor capacity.
Embodiment 4:
Get the acetic acid-sodium-acetate buffer of the 50mMpH5.0 of 20ml, add 0.2mg cellulase, add carrier prepared by 1g embodiment 1, in 30 DEG C of water-bath shaking tables, react 24h. Filter and obtain immobilized enzyme, wash till can't detect albumen with acetic acid-sodium-acetate buffer. Adopt the adsorption rate of Coomassie Brilliant Blue detection fibers element enzyme, record that its adsorption rate 1500mg/g(is former adsorption rate 96%); Simultaneously adopt DNS method, using glucose as standard substance detection fibers element enzyme activity, the apparent activity recording this immobilized enzyme is 40IU, catalyzed degradation cellobiose substrate (0.5mg/ml), each catalysis time 2min, after reusing 15 times, carrier immobilized enzymic activity reaches about 90%.
Embodiment 5: the carrier prepared by embodiment 2 repeats embodiment 4, the adsorption rate recording cellulase is 1600mg/g(is the 98% of former adsorption rate); The apparent activity of this immobilized enzyme is 43IU, catalyzed degradation cellobiose substrate (1mg/ml), each catalysis time 5min, after reusing 15 times, and these carrier immobilized enzymic activity remnants about 85%.
Embodiment 6: the carrier prepared by embodiment 3 repeats embodiment 4, the adsorption rate recording cellulase is 1800mg/g(is the 99% of former adsorption rate); The apparent activity of this immobilized enzyme is 45IU, catalyzed degradation cellulosic substrate (0.5mg/ml), each catalysis time 5h, and after reusing 5 times, this carrier immobilized enzymic activity reaches about 40%.
Claims (5)
1. one kind process with ionic liquid and modifier after straw for the method for cellulase immobilization carrier, it is characterised in that comprise the steps:
(1) pre-treatment of stalk: remove the impurity on stalk, drying the section of cutting into;
(2) modification of stalk: first use ion liquid dissolving stalk, then add modifier stir evenly obtain cellulose carrier solution, this modification use ultrasonic wave, the Surface and internal structure of stalk is changed;
(3) carrier drying is shaping: add water or short chain alcohol in above-mentioned cellulose carrier solution, stirs evenly, then removes impurity with deionized water wash, and then cellulase immobilization carrier is made in dry, vacuum outgas;
Wherein, in step (2), the weight ratio of described stalk and ionic liquid is 1:1-5; The final concentration of described modifier is 0.1-0.2M;
Described ionic liquid is [DMIM] [DMP], [EMIM] Cl or [EMIM] OAC;
Described modifier is that polyoxyethylene glycol or bromine are for fourth acetimide.
2. the straw after processing with ionic liquid and modifier according to claim 1 is for the method for cellulase immobilization carrier, it is characterised in that: in step (1), stalk chooses stipes position.
3. the straw after processing with ionic liquid and modifier according to claim 1 is for the method for cellulase immobilization carrier, it is characterised in that: in step (2), the power of described ultrasonication is 30-60w, and the treatment time is 5-10min.
4. the straw after processing with ionic liquid and modifier according to claim 2 is for the method for cellulase immobilization carrier, it is characterised in that: in step (1), stalk stipes is cut into the segment of 0.4-0.6cm.
5. the straw after processing with ionic liquid and modifier according to claim 1 or 4 is for the method for cellulase immobilization carrier, it is characterised in that: the water added or short chain alcohol are 1-5 times of cellulose carrier liquor capacity.
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| CN104437633B (en) * | 2014-12-12 | 2016-08-24 | 江苏科技大学 | A kind of preparation method of mimic cellulose enzyme |
| CN110846306B (en) * | 2019-12-09 | 2021-08-17 | 江苏省中国科学院植物研究所 | Amphiphilic enzyme immobilization carrier |
| CN111534506B (en) * | 2020-05-13 | 2023-08-25 | 福州三合元生物科技有限公司 | Preparation method of ionic liquid regenerated cellulose immobilized enzyme |
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| CN102504285A (en) * | 2011-11-03 | 2012-06-20 | 中国科技开发院广西分院 | Regenerated cellulose microspheres and preparation method thereof |
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