CN103667243A - Method for preparing cellulase immobilized carrier from straws treated by ionic liquid and modifier - Google Patents
Method for preparing cellulase immobilized carrier from straws treated by ionic liquid and modifier Download PDFInfo
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- CN103667243A CN103667243A CN201310674394.4A CN201310674394A CN103667243A CN 103667243 A CN103667243 A CN 103667243A CN 201310674394 A CN201310674394 A CN 201310674394A CN 103667243 A CN103667243 A CN 103667243A
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Abstract
The invention discloses a method for preparing cellulase immobilized carrier from straws treated by an ionic liquid and a modifier, which comprises the following steps: pretreatment of straws, modification of straws, and drying and molding of carrier. By using the cellulose material obtained by treating the straws with an ionic liquid and a modifier to prepare the enzyme immobilized carrier, the method has the advantages of green and environment-friendly treatment process, no pollution, mild operating conditions and simple and feasible treatment technique, and the ionic liquid and modifier can be completely recycled after treatment, thereby greatly lowering the production cost of the carrier. The enzyme on the enzyme immobilized carrier prepared by the method has high adsorption rate, high stability and higher catalytic activity. The enzyme immobilized carrier prepared by the method can still have higher enzyme activity after being used many times.
Description
Technical field
The invention belongs to technical field of biochemical industry, relate to a kind of straw after processing with ionic liquid and modifier for the preparation method of cellulase immobilization carrier.
Background technology
Enzyme be a kind of by amino acid, formed there is special bioactive material, it is present in the animal and plant body of all work, is to maintain body normal function digest food, a kind of essential material 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 for accelerating the speed of all kinds of biochemical reactions, but be not biochemical reaction itself.The immobilization of enzyme be exactly after enzyme being combined with carrier by some method, become be insoluble to contain substrate mutually in, thereby make enzyme be concentrated or be limited in, in certain spatial dimension, carry out enzyme digestion reaction, be therefore referred to as immobilized enzyme.Immobilized enzyme is compared with resolvase, has following advantages: 1. very easily by immobilization enzyme-to-substrate, product separately; 2. in the long period, carry out repeatedly batch reactions and dress post successive reaction again; 3. in most of the cases, can improve the stability of enzyme; 4. 5. enzyme reaction process can strictly be controlled does not have the residual of enzyme in product solution, has simplified purifying technique; 6. compared with resolvase, be more suitable in multienzymatic reaction; 7. can increase the yield of product; 8. the service efficiency of enzyme improves, cost.The process for fixation of enzyme can roughly be divided into two large 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 the high plant cell walls such as cotton, timber, flax, careless class, can produce hundred million tons of hundreds ofs every year by photosynthesis, is inexhaustible, the nexhaustible renewable resources of nature.
Ionic liquid refers to the salt being all comprised of zwitterion.Because ionic liquid has non-volatile, good ionic conductivity and thermal conductivity, high heat capacity and high thermal stability, the features such as selective dissolution power, make ionic liquid become one of focus of current chemical field research.
The enzyme adsorbing on existing carrier easily comes off, and causes thus immobilized enzyme stability and activity not high, and enzyme immobilization carrier seldom can reuse simultaneously, therefore has the aftertreatment of solid support material and the idle problem of useless carrier.
Summary of the invention
Goal of the invention: the object of this invention is to provide a kind of straw after processing with ionic liquid and modifier for high enzyme charge capacity, enzymatic activity high, can reuse and the preparation method of the cellulase immobilization carrier of cheap environmental protection.
Technical scheme: the straw after use ionic liquid of the present invention and modifier are processed, for the method for cellulase immobilization carrier, comprises the steps:
(1) pre-treatment of stalk: remove impurity on stalk, be dried and the section of cutting into;
(2) modification of stalk: first use ion liquid dissolving stalk, then add modifier to stir, obtain cellulose carrier solution, this modification is used ultrasonic wave, and the Surface and internal structure of stalk is changed;
(3) carrier drying moulding: add solvent in above-mentioned cellulose carrier solution, stir, impurity is removed in washing, then cellulase immobilization carrier is made in dry, vacuum outgas.
In step (1), stalk is chosen stipes position, and stalk stipes is cut 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 polyoxyethylene glycol or bromo 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 adding is 1-5 times of cellulose carrier liquor capacity.
Inventive principle: contain a large amount of Mierocrystalline celluloses in stalk, the ionic liquid adding can dissolve, destroy the low Mierocrystalline cellulose of degree of crystallinity in stalk, remaining is all the rigid fiber carrier of the similar nano-cellulose that degree of crystallinity is high, intensity is large, and the surface of residual fiber element carrier and the inner structure that forms porous, for the immobilization of enzyme provides a lot of sites, for subsequent reactions provides condition; The modifier adding is combined with fibrous carrier, and is connected to the surperficial and inner of fibrous carrier, can amino groups be combined on enzyme molecule, and then realize the immobilization of enzyme.
Beneficial effect: the present invention compared with prior art: its remarkable advantage is: 1, adopt and have non-volatile, 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 prepared enzyme immobilization carrier, treating processes environmental protection, pollution-free, operational condition is gentle, treatment process simple and feasible, and ionic liquid and modifier after processing can reclaim completely, can greatly reduce the production cost of carrier.2, enzyme adsorption rate on the fixed enzyme vector preparing by the inventive method is high, stability is high, has improved 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
The scanning electron microscope (SEM) photograph (amplifying 1000 times) that Fig. 1 is the carrier prepared by the inventive method.
Embodiment
Below technical solution of the present invention is elaborated.
Embodiment 1: carry out as follows:
(1) get the stipes of maize straw, wash away the impurity such as mud dust on stalk, after being dried, cut into the segment of 0.5cm left and right (0.4-0.6cm).
(2) get in the there-necked flask that 1g stalk adds 250ml, when temperature 25 ℃ (room temperature), first add 5g ionic liquid [DMIM] [DMP], with ultrasonic washing instrument (setting power 60w), process 5min, adding final concentration is the Macrogol 4000 60ml of 0.2M again, stir, ultrasonic washing instrument (setting power 60w) is processed 10min, Surface and internal structure to stalk is modified, stalk internal structure is changed, surface-area increases, the adsorption site of enzyme increases, and obtains cellulose carrier solution.
(3) to the water that adds 3 times of volumes in above-mentioned cellulose carrier solution, uniform stirring 10min, adds carrier with in deionized water, and imposes violent stirring, wash and removes impurity 3-5 time, and 60 ℃, carrier is dried, and vacuum outgas is prepared into carrier.
The scanning electron microscope (SEM) photograph of this carrier as shown in Figure 1, remove through processing by the fiber that in bamboo chip, degree of crystallinity is low (NMMO processes and easily comes off and stripping), and the carrier obtaining 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, and difference is: 1, add 1g ionic liquid [EMIM] Cl Treating straw; 2, adding 70ml final concentration is the cetomacrogol 1000 of 0.1M; 3, use 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 ℃; 5, add with the isopyknic washing with alcohol of cellulose carrier solution and remove impurity.
Embodiment 3: preparation process is substantially the same manner as Example 1, and difference is: 1, add 3g ionic liquid [EMIM] [oAc] Treating straw; 2, adding 100ml final concentration is the bromo fourth acetimide of 0.15M; 3, use 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 ℃; 5, the water adding is 5 times of cellulose carrier liquor capacity.
Embodiment 4:
Acetic acid-sodium-acetate buffer of getting the 50mM pH5.0 of 20ml, adds 0.2mg cellulase, adds the carrier of 1g embodiment 1 preparation, in 30 ℃ of shaking baths, reacts 24h.Filter and obtain immobilized enzyme, with acetic acid-sodium-acetate buffer, wash until can't detect albumen.Adopt the adsorption rate of Xylene Brilliant Cyanine G method detection fibers element enzyme, record its adsorption rate 1500mg/g(and be former adsorption rate 96%); Adopt DNS method simultaneously, using the activity of glucose as standard substance detection fibers element enzyme, the apparent activity that records this immobilized enzyme is 40IU, catalyzed degradation cellobiose substrate (0.5mg/ml), each catalysis time 2min, reuse after 15 times, carrier immobilized enzymic activity reaches 90% left and right.
Embodiment 5: with the carrier of embodiment 2 preparation, repeat embodiment 4, the adsorption rate that records cellulase be 1600mg/g(be former adsorption rate 98%); The apparent activity of this immobilized enzyme is 43IU, catalyzed degradation cellobiose substrate (1mg/ml), and each catalysis time 5min, reuses after 15 times, remaining 85% left and right of this carrier immobilized enzymic activity.
Embodiment 6: with the carrier of embodiment 3 preparation, repeat embodiment 4, the adsorption rate that records cellulase be 1800mg/g(be former adsorption rate 99%); The apparent activity of this immobilized enzyme is 45IU, catalyzed degradation cellulosic substrate (0.5mg/ml), and each catalysis time 5h, reuses after 5 times, and this carrier immobilized enzymic activity reaches 40% left and right.
Claims (9)
1. the straw after processing with ionic liquid and modifier, for a method for cellulase immobilization carrier, is characterized in that comprising the steps:
(1) pre-treatment of stalk: remove impurity on stalk, be dried and the section of cutting into;
(2) modification of stalk: first use ion liquid dissolving stalk, then add modifier to stir to obtain cellulose carrier solution, this modification is used ultrasonic wave, and the Surface and internal structure of stalk is changed;
(3) carrier drying moulding: add solvent in above-mentioned cellulose carrier solution, stir, then remove impurity with deionized water wash, then cellulase immobilization carrier is made in dry, vacuum outgas.
2. the straw after processing with ionic liquid and modifier according to claim 1, for the method for cellulase immobilization carrier, is characterized in that: 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.
3. the straw after processing with ionic liquid and modifier according to claim 1, for the method for cellulase immobilization carrier, is characterized in that: in step (2), described ionic liquid is the ionic liquid with cellulose dissolution function.
4. the straw after processing with ionic liquid and modifier according to claim 1, for the method for cellulase immobilization carrier, is characterized in that: in step (2), described modifier is polyoxyethylene glycol or bromo fourth acetimide.
5. the straw after processing with ionic liquid and modifier according to claim 1, for the method for cellulase immobilization carrier, is characterized in that: in step (1), stalk is chosen stipes position.
6. the straw after processing with ionic liquid and modifier according to claim 1, for the method for cellulase immobilization carrier, is characterized in that: in step (2), the power of described ultrasonication is 30-60w, and the treatment time is 5-10min.
7. the straw after processing with ionic liquid and modifier according to claim 1, for the method for cellulase immobilization carrier, is characterized in that: in step (1), stalk stipes is cut into the segment of 0.4-0.6cm.
8. the straw after processing with ionic liquid and modifier according to claim 1, for the method for cellulase immobilization carrier, is characterized in that: the solvent described in step (3) is water or short chain alcohol.
9. the method for cellulase immobilization carrier according to the straw after processing with ionic liquid and modifier described in claim 1 or 7, is characterized in that: the solvent adding is 1-5 times of cellulose carrier liquor capacity.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104437633A (en) * | 2014-12-12 | 2015-03-25 | 江苏科技大学 | Method for preparing simulated cellulase |
| CN110846306A (en) * | 2019-12-09 | 2020-02-28 | 江苏省中国科学院植物研究所 | Amphiphilic enzyme immobilization carrier |
| CN111534506A (en) * | 2020-05-13 | 2020-08-14 | 福州三合元生物科技有限公司 | Preparation method of ionic liquid regenerated cellulose immobilized enzyme |
| CN117960240A (en) * | 2024-03-09 | 2024-05-03 | 承德市天丰生物工程有限公司 | Preparation method of simulated cellulase |
| CN118127099A (en) * | 2024-05-06 | 2024-06-04 | 惠州市绿色能源与新材料研究院 | Straw pretreatment ionic liquid and method, regenerated straw and preparation and enzymolysis liquid thereof |
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| CN1807515A (en) * | 2005-01-20 | 2006-07-26 | 中国科学院过程工程研究所 | Method for preparing degradable material using stalk |
| 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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| CN1807515A (en) * | 2005-01-20 | 2006-07-26 | 中国科学院过程工程研究所 | Method for preparing degradable material using stalk |
| 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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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104437633A (en) * | 2014-12-12 | 2015-03-25 | 江苏科技大学 | Method for preparing simulated cellulase |
| CN110846306A (en) * | 2019-12-09 | 2020-02-28 | 江苏省中国科学院植物研究所 | Amphiphilic enzyme immobilization carrier |
| 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 |
| CN111534506A (en) * | 2020-05-13 | 2020-08-14 | 福州三合元生物科技有限公司 | Preparation method of ionic liquid regenerated cellulose immobilized enzyme |
| 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 |
| CN117960240A (en) * | 2024-03-09 | 2024-05-03 | 承德市天丰生物工程有限公司 | Preparation method of simulated cellulase |
| CN117960240B (en) * | 2024-03-09 | 2024-08-20 | 承德市天丰生物工程有限公司 | Preparation method of simulated cellulase |
| CN118127099A (en) * | 2024-05-06 | 2024-06-04 | 惠州市绿色能源与新材料研究院 | Straw pretreatment ionic liquid and method, regenerated straw and preparation and enzymolysis liquid thereof |
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