CN103289982B - A kind of preparation of novel fixed enzyme vector - Google Patents
A kind of preparation of novel fixed enzyme vector Download PDFInfo
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- CN103289982B CN103289982B CN201210062682.XA CN201210062682A CN103289982B CN 103289982 B CN103289982 B CN 103289982B CN 201210062682 A CN201210062682 A CN 201210062682A CN 103289982 B CN103289982 B CN 103289982B
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Abstract
The present invention relates to a kind of preparation method of novel fixed enzyme vector, carrier based on macroporous adsorbent resin, adsorb chitosan under negative pressure and form surperficial chitosan film, recycling glutaraldehyde, to its surface modification, obtains a kind of novel fixed enzyme vector.By the active group on macroporous resin surface, chitosan can be combined on resin surface securely, with chitosan coat as fixed enzyme vector, is conducive to the immobilization realizing enzymic activity conformation, effectively reduces the deactivation phenomenom of enzyme in immobilization process.The advantages such as it is high that the method has stability, simple, quick.Raw material of the present invention is easy to get, cheap, and simply, activity of the immobilized enzyme is high, good stability, mild condition, and support strength is high, is easy to realize suitability for industrialized production for carrier preparation and enzyme immobilization process operation.
Description
(1) technical field
The present invention relates to a kind of technology of preparing of immobilized enzyme carrier, be one carrier based on macroporous adsorbent resin, novel carriers prepared by surface adsorption chitosan coat, for enzyme immobilizatio.
(2) background technology
Zymin is widely used in the aspects such as food, medicine, weaving, light industry, the energy, environmental protection, and achieve significant achievement, but due to free biological enzyme in reaction system poor stability, be insoluble to organic solvent, not reproducible utilization etc., add commercial biological enzyme on the high side, therefore, the application of biological enzyme in actual production is greatly limited.Enzyme immobilization technology had both overcome above-mentioned deficiency, maintained again the distinctive catalytic activity of enzyme to a certain extent, thus became one of research field the most active in biotechnology.
Enzyme immobilization technology, be with solid material by enzyme constraint or be limited in certain area, make it still can carry out distinctive catalyzed reaction, and a recyclable and reusable class technology.Compared with resolvase, immobilized enzyme while its efficient, single-minded and gentle enzymic catalytic reaction characteristic of maintenance, also present that package stability is high, Separation and Recovery easily, can repeatedly use, operate the series of advantages such as continuous and controlled, simple process.The enzyme immobilization method that people explore and study is a lot, but the real immobilized enzyme dropping into industrial applications is few, major cause is that the reagent that uses of immobilization and carrier cost are high, is secondly that immobilization efficiency is low, poor stability, the equipment more complicated that operate continuously uses.
The solid support material that the performance of immobilized enzyme depends primarily on process for fixation and uses.Enzyme immobilizatio method is divided into four kinds substantially: absorption method, covalent coupling method, entrapping method and crosslinking, wherein absorption method has that simple to operation, loading capacity is comparatively large, immobilized enzyme stability is higher, enzyme lives the advantages such as the rate of recovery is high, process costs is low, is therefore the most frequently used method of immobilized enzyme.Conventional carrier mainly contains organic carrier and inorganic carrier.Organic carrier comprises gelatin, agar powder, gluten, soybean protein, starch and ion exchange resin etc.; Inorganic carrier comprises quartz sand, diatomite, silica gel, active calcium oxide, aluminum oxide, calcium carbonate, gac, kaolin, bentonite and sintered glass etc.Although immobilization material is a lot, but all there is certain shortcoming, some reactive forces are not strong, adsorb insecure, enzyme easily comes off from carrier, and the reusing of immobilized enzyme is not high, some carrier high expensives, some methods are complicated etc., are difficult to the fixed enzyme vector finding a kind of desirable applicable suitability for industrialized production.The process for fixation that further exploitation is easier, more applicable and the more excellent solid support material of performance, significant to the industrial applications of immobilized enzyme.
(3) summary of the invention
The object of this invention is to provide a kind of preparation method of novel fixed enzyme vector, carrier based on the macroporous resin selecting suitable aperture, low pressure adsorbent chitosan forms chitosan resin carrier, recycling glutaraldehyde is to its surface modification, realize the immobilization of enzymic activity conformation, in effective minimizing immobilization process, the deactivation phenomenom of enzyme, is easy to realize suitability for industrialized production.
For achieving the above object, the technical solution used in the present invention is:
1) macroporous resin 40-80 DEG C of hot water cleans repeatedly, then uses the salt acid soak 12-48h of 1-5%, is washed to neutrality, then soaks 12-48h with the NaOH of 1-5%, being washed to neutrality, balancing, with using deionized water rinsing more before with the phosphoric acid buffer of pH7.5.
2) chitosan being dissolved in massfraction is in the acetum of 5-20%, obtains chitosan saturated solution.Instilled and be equipped with in the container of macroporous resin, negative-pressure adsorption 1-5h, chitosan is adsorbed on the surfaces externally and internally of resin.
3) filtration under diminished pressure carrier of separating, is washed till neutrality with distilled water, then adds the glutaraldehyde process of 1-10%, is washed to neutrality, vacuum-drying, obtains macroporous resin chitosan film carrier.
4) a certain amount of biological enzyme is dissolved in suitable pH buffered soln, adds the macroporous resin chitosan film carrier of a certain amount of preparation, place 1-10h for 0-20 DEG C, dry immobilized enzyme.
Fixation support prepared by the present invention has the following advantages:
1) material is easy to get, cheap, good mechanical property, and stable chemical performance is biodegradable, and immobilized enzyme efficiency is high, and preparation method is simple, easily operates.
2) use macroporous resin as upholder, with chitosan coat as fixed enzyme vector, effectively expand the chitosan scope of application.
3) by glutaraldehyde directly by biological enzyme and chitin carrier coupling, there is very high enzyme charge capacity, also can improve the stability of enzyme.
(4) embodiment
Embodiment 1
1) D301R macroporous resin 50-60 DEG C hot water cleans repeatedly, with the salt acid soak 24-48h of 3-5%, is washed to neutrality, 24-48h is soaked again with the NaOH of 3-5%, being washed to neutrality, balancing, with using deionized water rinsing more before with the phosphoric acid buffer of pH7.5.
2) chitosan being dissolved in massfraction is in the acetum of 10-15%, obtains chitosan saturated solution.Instilled in the there-necked flask that D301R macroporous resin is housed, negative-pressure adsorption 3-5h, chitosan is adsorbed on the surfaces externally and internally of resin.
3) filtration under diminished pressure carrier of separating, is washed till neutrality with distilled water, then adds the glutaraldehyde process of 5-8%, is washed to neutrality, vacuum-drying.
4) a certain amount of lipase is dissolved in pH4.5 phosphate buffer solution, adds the macroporous resin chitosan film carrier of a certain amount of preparation, place 5-10h for 0-20 DEG C, dry immobilized lipase.
Embodiment 2
1) D301G macroporous resin 50-60 DEG C hot water cleans repeatedly, with the salt acid soak 24-48h of 3-5%, is washed to neutrality, 24-48h is soaked again with the NaOH of 3-5%, being washed to neutrality, balancing, with using deionized water rinsing more before with the phosphoric acid buffer of pH7.5.
2) chitosan being dissolved in massfraction is in the acetum of 10-15%, obtains chitosan saturated solution.Instilled in the there-necked flask that D301G macroporous resin is housed, negative-pressure adsorption 3-5h, chitosan is adsorbed on the surfaces externally and internally of resin.
3) filtration under diminished pressure carrier of separating, is washed till neutrality with distilled water, then adds the glutaraldehyde process of 5-8%, is washed to neutrality, vacuum-drying.
4) a certain amount of saccharifying enzyme is dissolved in pH4.5 phosphate buffer solution, adds the macroporous resin chitosan film carrier of a certain amount of preparation, place 5-10h for 0-20 DEG C, dry immobilized glucoamylase.
Embodiment 3
1) D371 macroporous resin 40-50 DEG C hot water cleans repeatedly, with the salt acid soak 24-48h of 3%, is washed to neutrality, then soaks 24-48h with the NaOH of 3-5%, being washed to neutrality, balancing, with using deionized water rinsing more before with the phosphoric acid buffer of pH7.5.
2) chitosan being dissolved in massfraction is in the acetum of 10-15%, obtains chitosan saturated solution.Instilled in the there-necked flask that D371 macroporous resin is housed, negative-pressure adsorption 3-5h, chitosan is adsorbed on the surfaces externally and internally of resin.
3) filtration under diminished pressure carrier of separating, is washed till neutrality with distilled water, then adds the glutaraldehyde process of 5-8%, is washed to neutrality, vacuum-drying.
4) a certain amount of lipase is dissolved in pH4.5 phosphate buffer solution, adds the macroporous resin chitosan film carrier of a certain amount of preparation, place 5-10h for 0-20 DEG C, dry immobilized lipase.
Carrier based on macroporous adsorbent resin, macroporous resin chitosan film carrier prepared by negative-pressure adsorption chitosan, carrier preparation and enzyme immobilization process operation simple, activity of the immobilized enzyme is high, good stability, mild condition, support strength is high, is easy to realize suitability for industrialized production.
Claims (1)
1. a preparation method for novel fixed enzyme vector, is characterized in that, comprising:
1) macroporous resin 40-80 DEG C of hot water cleans repeatedly, then uses the salt acid soak 12-48h of 1-5%, is washed to neutrality, then soaks 12-48h with the NaOH of 1-5%, being washed to neutrality, balancing, with using deionized water rinsing more before with the phosphoric acid buffer of pH7.5;
2) chitosan being dissolved in massfraction is in the acetum of 5-20%, and obtain chitosan saturated solution, instilled and be equipped with in the container of macroporous resin, negative-pressure adsorption 1-5h, chitosan is adsorbed on the surfaces externally and internally of resin;
3) filtration under diminished pressure carrier of separating, is washed till neutrality with distilled water, then adds the glutaraldehyde process of 1-10%, is washed to neutrality, vacuum-drying, obtains macroporous resin chitosan film carrier.
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| CN201210062682.XA CN103289982B (en) | 2012-03-01 | 2012-03-01 | A kind of preparation of novel fixed enzyme vector |
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| CN201210062682.XA CN103289982B (en) | 2012-03-01 | 2012-03-01 | A kind of preparation of novel fixed enzyme vector |
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| CN103289982B true CN103289982B (en) | 2015-09-30 |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN104046609A (en) * | 2014-06-24 | 2014-09-17 | 东北农业大学 | Preparation method for efficient immobilized lipase |
| CN105037788A (en) * | 2015-07-05 | 2015-11-11 | 烟台大学 | Preparation method of chitosan macro-porous resin composite carrier |
| CN105907741A (en) * | 2016-06-06 | 2016-08-31 | 新疆大学 | Covalent binding method for preparing activated carbon immobilized lipase |
| CN111662791A (en) * | 2020-06-08 | 2020-09-15 | 江苏欣捷衬布有限公司 | Low-temperature cleaning soaping agent for reactive dye dyed lining cloth and preparation method thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1718918A (en) * | 2005-06-24 | 2006-01-11 | 华南理工大学 | Method of removing resin in paper pulp by multienzyme synergistic action |
| CN102174505A (en) * | 2011-01-14 | 2011-09-07 | 华南理工大学 | Preparation method of granular carrier immobilized bienzyme for treating papermaking white water |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1718918A (en) * | 2005-06-24 | 2006-01-11 | 华南理工大学 | Method of removing resin in paper pulp by multienzyme synergistic action |
| CN102174505A (en) * | 2011-01-14 | 2011-09-07 | 华南理工大学 | Preparation method of granular carrier immobilized bienzyme for treating papermaking white water |
Non-Patent Citations (1)
| Title |
|---|
| lowering the cationic demand caused by PGA in papermaking by solute adsorption and immobilized pectinase on chitosan beads;kai liu et al;《carbohydrate polymers》;20100610;648-652 * |
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