CN102453707A - Preparation method of immobilized cellulase - Google Patents
Preparation method of immobilized cellulase Download PDFInfo
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- CN102453707A CN102453707A CN2010105236686A CN201010523668A CN102453707A CN 102453707 A CN102453707 A CN 102453707A CN 2010105236686 A CN2010105236686 A CN 2010105236686A CN 201010523668 A CN201010523668 A CN 201010523668A CN 102453707 A CN102453707 A CN 102453707A
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Abstract
The invention discloses a preparation method of immobilized cellulase, relating to an enzyme immobilization method. The method comprises the following steps of: firstly, mixing chitosan with the degree of deacetylation of 90% with an acetic acid solution, then injecting the mixed solution into a 2% sodium tripolyphosphate solution, filtering and washing to obtain neutral spherical chitosan-polyethylene glycol carriers, then activating the carriers by a glutaraldehyde solution, carrying out immobilization reaction of the carriers and cellulase, and filtering and washing to obtain immobilized cellulase. The immobilized cellulase prepared by the method has the advantages of high mechanical strength, good enzyme stability, high enzyme activity, strong binding force between the enzyme and the carriers and high rate of immobilization.
Description
Technical field
The present invention relates to a kind of process for fixation of biological catalyst, more precisely is a kind of process for fixation of cellulase, belongs to the biological reagent preparing technical field.
Background technology
Natural cellulose is a reproducible organic substance the abundantest on the earth; The plant material every year that produces through photosynthesis in the whole world is approximately up to 10,000 hundred million tons; Wherein 89% at present by the human use, sees from human use's natural resources angle, and this is a huge waste beyond doubt.Along with world population is long suddenly, unresolved food that increasingly sharpens and energy dilemma, the utilization of cellulose resource has caused the very big concern and the great attention of countries in the world.
General biology can not the DIRECT UTILIZATION OF CELLULOSE material as food, have only to become glucose just can be utilized cellulose hydrolysis.Cellulose hydrolysis becomes the general method of glucose that acid-hydrolysis method and enzyme hydrolysis method are arranged.Mierocrystalline cellulose is compared with the starch material that is widely used in fermentation industry at present, approximately low 400 times of its acid hydrolysis abilities, and the low 100-1000 of enzymic hydrolysis ability is doubly.Acid hydrolysis is big to the corrosion on Equipment effect, but answers condition violent, produces a large amount of sour waste water simultaneously, and therefore the development and the application of this method receive very big restriction.Cellulosic enzymatic hydrolysis reaction mild condition is easy to control, product quality is high and receive much concern.But utilize cellulase catalyzed reaction band ground high temperature, graceful chemical reaction save energy, reduce production costs and improve utilization ratio of raw materials, at environment purification with to open up be that aspect such as the energy has great importance.But the substrate of cellulase and effect thereof is very complicated, and influence factor is various, and hydrolysis rate is slow; The utilization ratio of enzyme is low; Enzymolysis efficiency is not high, influences the widespread use of cellulase, therefore seeks and utilizes cellulase effectively hydrolyzing cellulose materials to have very important theory and practical significance.
The immobilized enzyme catalysis reaction technology have can reclaim, advantage such as easily separated, repeated multiple times use, for the service efficiency that improves cellulase, reduce to produce possibility can originally be provided, so the immobilization research of cellulase receives much concern for a long time.But using immobilized cellulase that traditional process for fixation carries out at present has that bad mechanical strength, stability are bad, a little less than the bonding force between enzyme and the carrier, and in use the enzyme molecule is prone to from defectives such as carrier come off.Therefore, demand exploring and set up a kind of preparation method of new immobilized cellulase now urgently.
Summary of the invention
The objective of the invention is for a little less than solving traditional fixing means and having a bonding force between bad mechanical strength, bad, the enzyme of stability and the carrier, in use the enzyme molecule is prone to a kind of cellulase immobilization method of providing from defectives such as carrier come off.
The preparation method of immobilized cellulase realizes according to the following steps::: one, taking by weighing the 2.0-4.0g deacetylation is 90% chitosan, and 0.6-0.8g PEG6000 is dissolved in the acetic acid soln of 100ml 2%, in 35-45 ℃ of blend 1.5-2.5h; Two, with syringe blend solution is injected 2% sodium tripolyphosphate solution, static 2.5-3.5h; Three, filtration washing gets neutral globular chitosan-polyethylene glycol carrier; Four, take by weighing the 9-11g carrier, add 100ml 5% glutaraldehyde solution, 45-55 ℃ of activation 5.5-6.5h; Five, filter the back and be washed till neutrality with acetone and zero(ppm) water; Six, take by weighing carrier after the 1.5-2.5g activation, add 20ml Mierocrystalline cellulose beauty liquid and 17-19 ℃ and carry out immobilized reactant 15-17h; Seven, filter the post-flush immobilized enzyme for several times, promptly get immobilized cellulase.
The present invention has following advantage: (1) the present invention utilizes the method for natural polymer support modification to utilize polyoxyethylene glycol that chitosan is carried out after the modification carrier as immobilized cellulase first; Have the advantage of safe without toxic side effect, and solved it and be dissolved in the restriction of acidic aqueous solution; (2) utilize polyoxyethylene glycol that the immobilized cellulase that chitosan carries out preparing as fixation support after the modification is had the advantage of excellent mechanical intensity, enzymic activity height and good stability; (3) the present invention adopts the covalent attachment method that chitosan modified is combined with cellulase, makes that the bonding force between enzyme and the carrier is strong, has the high advantage of immobilization rate.
Embodiment
Embodiment one: the preparation method of this embodiment immobilized cellulase realizes according to the following steps:: one, taking by weighing the 2.0-4.0g deacetylation is 90% chitosan; 0.6-0.8g PEG6000 is dissolved in the acetic acid soln of 100ml2%, in 35-45 ℃ of blend 1.5-2.5h; Two, with syringe blend solution is injected 2% sodium tripolyphosphate solution, static 2.5-3.5h; Three, filtration washing gets neutral globular chitosan-polyethylene glycol carrier; Four, take by weighing the 9-11g carrier, add 100ml 5% glutaraldehyde solution, 45-55 ℃ of activation 5.5-6.5h; Five, filter the back and be washed till neutrality with acetone and zero(ppm) water; Six, take by weighing carrier after the 1.5-2.5g activation, add 20ml Mierocrystalline cellulose beauty liquid and 17-19 ℃ and carry out immobilized reactant 15-17h; Seven, filter the post-flush immobilized enzyme for several times, promptly get immobilized cellulase.
Chitosan, sodium tripolyphosphate solution, acetic acid soln, glutaraldehyde solution and acetone are commercially available conventional reagent in this embodiment.
Embodiment two: this embodiment and embodiment one are different is that accurately to take by weighing the 3.0g deacetylation in the step 1 be 90% chitosan, and 0.75gPEG6000 is dissolved in the acetic acid soln of 100ml 2%, in 40 ℃ of blend 2h.Other step and parameter are identical with embodiment one.
Embodiment three: what this embodiment and embodiment one were different is 2% sodium tripolyphosphate solution that adopts pH=8.2 in the step 2.Other step and parameter are identical with embodiment one.
Embodiment four: what this embodiment and embodiment one were different is with No. 6 syringes blended liquid to be injected 2% sodium tripolyphosphate solution in the step 2, static 3h.Other step and parameter are identical with embodiment one.
Embodiment five: this embodiment and embodiment one are different is to filter the back in the step 3 to go out unnecessary tripoly phosphate sodium STPP with distilled water wash.Other step and parameter are identical with embodiment one.
Embodiment six: what this embodiment and embodiment one were different is the glutaraldehyde solution that adopts pH=10.0 in the step 4.Other step and parameter are identical with embodiment one.
Embodiment seven: what this embodiment and embodiment one were different is to take by weighing the 10g carrier in the step 4, adds the 100ml5% glutaraldehyde solution, 50 ℃ of activation 6h.Other step and parameter are identical with embodiment one.
Embodiment eight: wooden embodiment and embodiment one are different is the carrier that takes by weighing in the step 6 after the 2.0g activation, adds the 20ml cellulase and carries out immobilized reactant 16h in 18 ℃.Other step and parameter are identical with embodiment one.
Embodiment nine: what this embodiment and embodiment one were different is to filter the back in the step 7 with acetate-sodium acetate buffer flushing immobilized enzyme several.Other step and parameter are identical with embodiment one.
Embodiment ten: the preparation method of this embodiment immobilized cellulase realizes according to the following steps: one, accurately take by weighing the 3.0g deacetylation and be 90% chitosan, 0.75gPEG6000 is dissolved in the acetic acid soln of 100ml 2%, in 40 ℃ of blend 2h; Two, with the syringe of No. 6 syringe needles blend solution is injected 2% sodium tripolyphosphate solution of pH=8.2, static 3h; Three, filter the back and go out unnecessary tripoly phosphate sodium STPP with distilled water wash, neutral globular chitosan-polyethylene glycol carrier; Four, take by weighing the 10g carrier, add 5% glutaraldehyde solution of 100mpH=10.0,50 ℃ of activation 6h; Five, filter the back and be washed till neutrality with acetone and zero(ppm) water; Six, take by weighing carrier after the 2.0g activation, add the 20ml cellulase and carry out immobilized reactant 16h in 18 ℃; Seven, filter the back for several times, promptly get immobilized cellulase with acetate-sodium acetate buffer flushing immobilized enzyme.
Claims (9)
1. the preparation method of an immobilized cellulase; It is characterized in that: the preparation method of immobilized cellulase realizes according to the following steps: one, take by weighing the 2.0-4.0g deacetylation and be 90% chitosan; 0.6-0.8gPEG6000 be dissolved in the acetic acid soln of 100ml 2%, in 35-45 ℃ of blend 1.5-2.5h; Two, with syringe blend solution is injected 2% sodium tripolyphosphate solution, static 2.5-3.5h; Three, filtration washing gets neutral globular chitosan-polyethylene glycol carrier; Four, take by weighing the 9-11g carrier, add 100ml 5% glutaraldehyde solution, 45-55 ℃ of activation 5.5-6.5h; Five, filter the back and be washed till neutrality with acetone and zero(ppm) water; Six, take by weighing carrier after the 1.5-2.5g activation, add 20ml Mierocrystalline cellulose beauty liquid and 17-19 ℃ and carry out immobilized reactant 15-17h; Seven, filter the post-flush immobilized enzyme for several times, promptly get immobilized cellulase.
2. the preparation method of a kind of immobilized cellulase according to claim 1 is characterized in that accurately taking by weighing in the step 1 3.0g deacetylation and is 90% chitosan, and 0.75gPEG6000 is dissolved in the acetic acid soln of 100ml2%, in 40 ℃ of blend 2h.
3. the preparation method of a kind of immobilized cellulase according to claim 1 is characterized in that adopting in the step 2 2% sodium tripolyphosphate solution of pH=8.2.
4. the preparation method of a kind of immobilized cellulase according to claim 1 is characterized in that with No. 6 syringes blended liquid being injected 2% sodium tripolyphosphate solution in the step 2, static 3h.
5. the preparation method of a kind of immobilized cellulase according to claim 1 is characterized in that filtering in the step 3 back and goes out unnecessary tripoly phosphate sodium STPP with distilled water wash.
6. the preparation method of a kind of immobilized cellulase according to claim 1 is characterized in that adopting in the step 4 glutaraldehyde solution of pH=10.0.
7. the preparation method of a kind of immobilized cellulase according to claim 1 is characterized in that taking by weighing in the step 4 10g carrier, adds the 100ml5% glutaraldehyde solution, 50 ℃ of activation 6h.
8. the preparation method of a kind of immobilized cellulase according to claim 1 is characterized in that taking by weighing in the step 6 carrier after the 2.0g activation, adds the 20ml cellulase and carries out immobilized reactant 16h in 18 ℃.
9. the preparation method of a kind of immobilized cellulase according to claim 1 is characterized in that filtering in the step 7 back with acetate-sodium acetate buffer flushing immobilized enzyme for several times.
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| CN2010105236686A CN102453707A (en) | 2010-10-28 | 2010-10-28 | Preparation method of immobilized cellulase |
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| CN2010105236686A CN102453707A (en) | 2010-10-28 | 2010-10-28 | Preparation method of immobilized cellulase |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102703418A (en) * | 2012-07-02 | 2012-10-03 | 西南石油大学 | Preparation method of magnetic chitosan microsphere immobilized horse radish peroxidases |
| CN106086150A (en) * | 2016-07-25 | 2016-11-09 | 陕西科技大学 | A kind of method utilizing enzyme to combine microorganism conversion production turmeric saponin |
| CN106222228A (en) * | 2016-07-25 | 2016-12-14 | 陕西科技大学 | A kind of method utilizing enzyme immobilization technology to produce turmeric saponin |
| CN108004229A (en) * | 2017-12-25 | 2018-05-08 | 北京化工大学 | A kind of preparation of immobilized cellulase and its method for enzyme hydrolysis cellulose |
| CN111034912A (en) * | 2020-01-09 | 2020-04-21 | 广东一家亲营养科技有限公司 | Immobilized enzymolysis process of nutritional rice flour |
| CN115433557A (en) * | 2022-09-20 | 2022-12-06 | 天津君宇生物科技有限公司 | Preparation method and application of biological enzyme in oilfield production increase |
-
2010
- 2010-10-28 CN CN2010105236686A patent/CN102453707A/en active Pending
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102703418A (en) * | 2012-07-02 | 2012-10-03 | 西南石油大学 | Preparation method of magnetic chitosan microsphere immobilized horse radish peroxidases |
| CN102703418B (en) * | 2012-07-02 | 2013-10-09 | 西南石油大学 | Preparation method of magnetic chitosan microsphere immobilized horse radish peroxidases |
| CN106086150A (en) * | 2016-07-25 | 2016-11-09 | 陕西科技大学 | A kind of method utilizing enzyme to combine microorganism conversion production turmeric saponin |
| CN106222228A (en) * | 2016-07-25 | 2016-12-14 | 陕西科技大学 | A kind of method utilizing enzyme immobilization technology to produce turmeric saponin |
| CN108004229A (en) * | 2017-12-25 | 2018-05-08 | 北京化工大学 | A kind of preparation of immobilized cellulase and its method for enzyme hydrolysis cellulose |
| CN111034912A (en) * | 2020-01-09 | 2020-04-21 | 广东一家亲营养科技有限公司 | Immobilized enzymolysis process of nutritional rice flour |
| CN115433557A (en) * | 2022-09-20 | 2022-12-06 | 天津君宇生物科技有限公司 | Preparation method and application of biological enzyme in oilfield production increase |
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Application publication date: 20120516 |