CN104611322A - Method for preparing immobilized cellulase - Google Patents
Method for preparing immobilized cellulase Download PDFInfo
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- CN104611322A CN104611322A CN201510039925.1A CN201510039925A CN104611322A CN 104611322 A CN104611322 A CN 104611322A CN 201510039925 A CN201510039925 A CN 201510039925A CN 104611322 A CN104611322 A CN 104611322A
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Abstract
The invention relates to a method for preparing immobilized cellulase. As free enzyme is mixed and dissolved in a reaction system and is difficult to not only separate and recover but also use repeatedly or continuously, free cellulase is immobilized. Cellulose acetate is immobilized on polypropylene composite films with different pore diameters, an effect of immobilizing the cellulose acetate to the composite films is observed through a scanning electron microscope, a polypropylene composite film with an optimal pore diameter is selected out, and the selected optimal composite film is immobilized on a roll. Conditions for immobilizing the free cellulase are that the glutaraldehyde concentration is selected to be 0.45-0.55%, the immobilization temperature is 48-49 DEG C, the pH (potential of hydrogen) value for immobilization is selected to be 3.9-4.0, the crosslinking time is 3-4 hours, the adsorption time is 3.0-3.1 hours, and the cellulase concentration is 1.0-2.0%. The activity of the free cellulase is measured to be 5.103 U/cm<2>, the enzyme activity still keeps about 80% after the roll for immobilizing the cellulase continuously presses soybean embryo slices for 24 hours, and the activity of the obtained immobilized cellulase is 4.732 U/cm<2>.
Description
Technical field
The present invention relates to a kind of method prepared by immobilized cellulase.
Background technology
Cellulase is a kind of important enzyme product, and be a kind of prozyme, primarily of compositions such as circumscribed beta-glucanase, Endo-β-glucanase and beta-glucosidases, it has very high Xylanase activity.Because cellulase has huge market potential in fields such as feed, alcohol, weaving and food, had an optimistic view of by domestic and international insider, to be the fourth-largest industrial enzyme after saccharifying enzyme, amylase and proteolytic enzyme, even completely likely become first enzyme in China, therefore cellulase is a new growth point in Enzymes Industry.
Enzyme immobilizatio be with solid material by enzyme constraint or be limited in certain area, still can carry out its distinctive catalyzed reaction, and recyclable and recycling a class technology.Compared with resolvase, immobilized enzyme is while its efficiently single-minded and gentle enzymic catalytic reaction characteristic of maintenance, overcome again the deficiency of resolvase, present that package stability is high, Separation and Recovery easily, can repeatedly use, operate continuously the series of advantages such as controlled and simple process.
The traditional preparation methods of immobilized enzyme has Physical and the large class of chemical method two.Physical method comprises crystallization process, dispersion method, physisorphtion, ionic bond method and entrapping method etc.Chemical method comprises crosslinking and combined techniques.Be that enzyme is received on polymer carrier that is natural or that synthesize by chemical bond-linking, use coupling agent to be cross-linked by enzyme by the group on enzyme surface, and form the method for larger, the insoluble immobilized enzyme of relative molecular mass.
Cellulase can make plant cell wall soften, expand or collapse, impels soybean peeling, thus improves the extraction yield of cell content.At present, in enzyme immobilizatio, more to the technique study of lipase immobilization both at home and abroad, and less for the research of Mierocrystalline cellulose enzyme immobilizatio, existing existing adopt chitosan crosslinked method immobilized cellulase, also have based on polyvinyl alcohol/Fe
2o
3nano particle immobilized cellulase, and with sodium alginate cross-linking using embedding immobilization cellulase, the novel preparation method of the present invention to immobilized cellulase is studied.
Summary of the invention
The present invention be directed to resolvase and to be miscible in reaction system not only not easily Separation and Recovery, and the enzyme of unbound state is poor to heat, strong acid, highly basic, high ionic strength and organic solvent equistability, easy in inactivation, and producing of cellulase is general more difficult, be difficult to repeatedly or continuously use, the present invention is to solve in enzymatic degumming process, and the problems such as free cellulose enzyme easy in inactivation sex change, so to being fixed of cellulase.Cellulose acetate enzyme is fixed on the polypropylene composite film of different pore size, observes by electron-microscope scanning the effect that enzyme is fixed on composite membrane, select the polypropylene composite film of optimum aperture, the best complex enzyme membrane selected is fixed on roller.The method of being fixed of cellulase is realized by following steps: one, the preparation of composite membrane; Two, enzyme immobilizatio; Three, the activity of immobilized enzyme is measured.
Accompanying drawing explanation
Fig. 1 cellulose acetate/polypropylene composite film; Fig. 2 immobilized cellulase film.
Embodiment
Embodiment one: the method for being fixed of cellulase is realized by following steps: one, the preparation of composite membrane: 5g cellulose acetate is dissolved in 100mL acetone, to be dissolved become homogeneous film liquid completely after, the polypropylene screen prepared before getting experiment is dipped in and is dissolved with in the acetone soln of cellulose acetate, nature film forming, with deionized water rinsing cellulose acetate/polypropylene composite film drying for standby for several times; Two, enzyme immobilizatio: by enzyme liquid concentration be 0.5 ~ 2.5% cellulase and Sodium phosphate dibasic-potassium phosphate buffer mix under pH is 2 ~ 6 conditions, and add cellulose acetate/polypropylene composite film, after the temperature of 35 ~ 75 DEG C stir 1 ~ 5h, the glutaraldehyde adding 0.35 ~ 0.55% again carries out crosslinking reaction 1 ~ 5h, takes out enzyme membrane and is wound around obtained immobilized cellulase roller on the surface of the cylinder; Three, immobilized enzyme relative activity is measured: the vigor of cellulase is 5.103 U/cm
2, immobilized cellulase roller is rolled soybean embryo plate 24h continuously, and result enzyme activity still remains on about 80%.
Embodiment two: the difference of present embodiment and embodiment one is to select glutaraldehyde concentration to be 0.45 ~ 0.55% in step 2.Other step is identical with embodiment one.
Embodiment three: the difference of present embodiment and embodiment one is to select glutaraldehyde concentration to be 0.45 ~ 0.55% in step 2, immobilization temperature 40 ~ 50 DEG C.Other step is identical with embodiment one.
Embodiment four: the difference of present embodiment and embodiment one is to select glutaraldehyde concentration to be 0.45 ~ 0.55% in step 2, immobilization temperature 40 ~ 50 DEG C, selects immobilization pH to be 3.0 ~ 4.0.Other step is identical with embodiment one.
Embodiment five: the difference of present embodiment and embodiment one is to select glutaraldehyde concentration to be 0.45 ~ 0.55% in step 2, immobilization temperature 40 ~ 50 DEG C, select immobilization pH to be 3.0 ~ 4.0, crosslinking time is 3 ~ 5h.Other step is identical with embodiment one.
Embodiment six: the difference of present embodiment and embodiment one is to select glutaraldehyde concentration to be 0.45 ~ 0.55% in step 2, immobilization temperature 40 ~ 50 DEG C, select immobilization pH to be 3.0 ~ 4.0, crosslinking time is 3 ~ 5h, and adsorption time is 2.5 ~ 3.5h.Other step is identical with embodiment one.
Embodiment seven: the difference of present embodiment and embodiment one is to select glutaraldehyde concentration to be 0.45 ~ 0.55% in step 2, immobilization temperature 40 ~ 50 DEG C, immobilization pH is selected to be 3.0 ~ 4.0, crosslinking time is 3 ~ 5h, adsorption time is 2.5 ~ 3.5h, and cellulase concentration is 1.0 ~ 2.0%.Other step is identical with embodiment one.
Embodiment eight: the difference of present embodiment and embodiment one is, in step 2, the result determined by single factor test is optimized being fixed condition more further, the fixing condition optimized is for selecting glutaraldehyde concentration to be 0.45 ~ 0.55%, immobilization temperature 48 ~ 49 DEG C, immobilization pH is selected to be 3.9 ~ 4.0, crosslinking time is 3 ~ 4h, adsorption time is 3.0 ~ 3.1h, and cellulase concentration is 1.0 ~ 2.0%.The immobilized cellulase vigor obtained under the optimal condition is 4.732U/cm
2.Other step is identical with embodiment one.
Claims (8)
1. the method prepared of an immobilized cellulase, it is characterized in that being fixed of cellulase to be realized by following steps: one, the preparation of composite membrane: 5g cellulose acetate is dissolved in 100mL acetone, to be dissolved become homogeneous film liquid completely after, the polypropylene screen prepared before getting experiment is dipped in and is dissolved with in the acetone soln of cellulose acetate, nature film forming, with deionized water rinsing cellulose acetate/polypropylene composite film drying for standby for several times; Two, enzyme immobilizatio: by enzyme liquid concentration be 0.5 ~ 2.5% cellulase and Sodium phosphate dibasic-potassium phosphate buffer mix under pH is 2 ~ 6 conditions, and add cellulose acetate/polypropylene composite film, after the temperature of 35 ~ 75 DEG C stir 1 ~ 5h, the glutaraldehyde adding 0.35 ~ 0.55% again carries out crosslinking reaction 1 ~ 5h, takes out enzyme membrane and is wound around obtained immobilized cellulase roller on the surface of the cylinder; Three, immobilized enzyme relative activity is measured: the vigor of cellulase is 5.103 U/cm
2, immobilized cellulase roller is rolled soybean embryo plate 24h continuously, and result enzyme activity still remains on about 80%, and the immobilized cellulase enzyme activity obtained is 4.732U/cm
2.
2. the method to being fixed of cellulase according to claim 1, is characterized in that being to being fixed of cellulase under the condition of 0.45 ~ 0.55% by selection glutaraldehyde concentration in step 2.
3. the method to being fixed of cellulase according to claim 1, when it is characterized in that selecting glutaraldehyde concentration to be 0.45 ~ 0.55% in step 2, to being fixed of cellulase under the condition that immobilization temperature is 40 ~ 50 DEG C.
4. the method to being fixed of cellulase according to claim 1, when it is characterized in that selecting glutaraldehyde concentration to be 0.45 ~ 0.55% in step 2, immobilization temperature 40 ~ 50 DEG C, selects immobilization pH to be to being fixed of cellulase under the condition of 3.0 ~ 4.0.
5. the method to being fixed of cellulase according to claim 1, when it is characterized in that selecting glutaraldehyde concentration to be 0.45 ~ 0.55% in step 2, immobilization temperature 40 ~ 50 DEG C, select immobilization pH to be 3.0 ~ 4.0, crosslinking time is to being fixed of cellulase under the condition of 3 ~ 5h.
6. the method to being fixed of cellulase according to claim 1, when it is characterized in that selecting glutaraldehyde concentration to be 0.45 ~ 0.55% in step 2, immobilization temperature 40 ~ 50 DEG C, immobilization pH is selected to be 3.0 ~ 4.0, crosslinking time is 3 ~ 5h, and adsorption time is to being fixed of cellulase under the condition of 2.5 ~ 3.5h.
7. the method to being fixed of cellulase according to claim 1, when it is characterized in that selecting glutaraldehyde concentration to be 0.45 ~ 0.55% in step 2, immobilization temperature 40 ~ 50 DEG C, immobilization pH is selected to be 3.0 ~ 4.0, crosslinking time is 3 ~ 5h, adsorption time is 2.5 ~ 3.5h, and cellulase concentration is to being fixed of cellulase under the condition of 1.0 ~ 2.0%.
8. the method to being fixed of cellulase according to claim 1, it is characterized in that determining further the condition of cellulase immobilization in step 2, when the fixing condition optimized is for selecting glutaraldehyde concentration to be 0.45 ~ 0.55%, immobilization temperature 48 ~ 49 DEG C, select immobilization pH to be 3.9 ~ 4.0, crosslinking time is 3 ~ 4h, and adsorption time is 3.0 ~ 3.1h, cellulase concentration is 1.0 ~ 2.0%, and the immobilized cellulase vigor obtained under the optimal condition is 4.732U/cm
2, carry out Mierocrystalline cellulose enzyme immobilizatio by optimum result.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107582729A (en) * | 2017-09-29 | 2018-01-16 | 中南民族大学 | A kind of preparation method and applications of belladonna extract |
| CN113881561A (en) * | 2021-05-07 | 2022-01-04 | 东北农业大学 | Method for producing rice bran protein polypeptide by using adjustable enzyme membrane reactor |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102286453A (en) * | 2011-09-19 | 2011-12-21 | 东北农业大学 | Method for immobilizing phospholipase with cellulose acetate/polypropylene composite membrane |
| CN102839045A (en) * | 2012-09-10 | 2012-12-26 | 东北农业大学 | Method of treating soybean germ flakes by compound enzyme membrane |
-
2015
- 2015-01-27 CN CN201510039925.1A patent/CN104611322A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102286453A (en) * | 2011-09-19 | 2011-12-21 | 东北农业大学 | Method for immobilizing phospholipase with cellulose acetate/polypropylene composite membrane |
| CN102839045A (en) * | 2012-09-10 | 2012-12-26 | 东北农业大学 | Method of treating soybean germ flakes by compound enzyme membrane |
Non-Patent Citations (2)
| Title |
|---|
| 时敏 等: "醋酸纤维素-聚丙烯复合膜固定化转谷氨酰胺酶的研究", 《食品科学》 * |
| 梁单琼 等: "醋酸纤维素-聚四氟乙烯复合膜固定化脂肪酶的研究", 《食品科学》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107582729A (en) * | 2017-09-29 | 2018-01-16 | 中南民族大学 | A kind of preparation method and applications of belladonna extract |
| CN113881561A (en) * | 2021-05-07 | 2022-01-04 | 东北农业大学 | Method for producing rice bran protein polypeptide by using adjustable enzyme membrane reactor |
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Application publication date: 20150513 |