CN101050457B - Method for fixing chitosan enzyme by using cross-linking adsorption of chitosan - glutaraldehyde - Google Patents
Method for fixing chitosan enzyme by using cross-linking adsorption of chitosan - glutaraldehyde Download PDFInfo
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- CN101050457B CN101050457B CN2007100382148A CN200710038214A CN101050457B CN 101050457 B CN101050457 B CN 101050457B CN 2007100382148 A CN2007100382148 A CN 2007100382148A CN 200710038214 A CN200710038214 A CN 200710038214A CN 101050457 B CN101050457 B CN 101050457B
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- chitin
- chitoanase
- glutaraldehyde
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Abstract
This invention discloses a method for preparing immobilized chitosanase by chitin-glutaraldehyde crosslinking and adsorption. The method comprises: refining chitin carrier, crosslinking with glutaraldehyde, extracting chitosanase, and immobilizing. The method overcomes such problems as low adsorption strength of the carrier, easy desorption and easy deactivation of the enzyme faced by traditional methods that utilize crosslinking or adsorption only. Besides, the also has such advantages as long half-life, and little influence of environment to the immobilized system.
Description
Technical field
The present invention relates to a kind of enzyme immobilization method, particularly a kind of chitin that utilizes carries out the fixing crosslinked adsorption method of fixed enzyme for enzyme immobilization carrier, glutaraldehyde as cross linker to free chitoanase.
Background technology
Enzyme immobilization method can be divided into 4 kinds of absorption method, e, crosslinking and entrapping methods etc.Absorption method is meant the method that reaches enzyme immobilization by the secondary key interaction between carrier surface and enzyme surface; Crosslinking is to utilize method difunctional or multi-functional group reagent crosslinked bridge formation immobilized enzyme between the enzyme molecule; The carrier adsorptive power that aforesaid method uses existence separately and produced is weak, easily desorb comes off and the shortcoming of the easy inactivation of enzyme, and it is short to have the transformation period simultaneously, the immobilization system be affected by the external environment big wait not enough.
Chitin (chitin) claim chitin, chitin, chitin again, is that (N-acetyl-D-glucosamine GlcNAc) passes through β-1, the straight-chain polysaccharide that the 4-glycosidic link is formed by connecting ([GlcNAc] in N-acetylglucosamine unit
n, n is the polymerization degree, and is nontoxic for the white plates solid, tasteless, acid and alkali-resistance is corrosion-resistant, high temperature resistant, and anti-daylight, performance is very stable, is the unique natural alkaline polysaccharide of finding up to now.It is present in the particularly arthropodan crust of lower animal in a large number, is that the occurring in nature margin is only second to cellulosic second largest natural reproducible resource.Because chitin unique texture characteristics and abundant laying in naturally, since the eighties in biochemical field with it as the carrier of immobilized cell and enzyme Application and Development in addition.After the amino that chitin had is activated by glutaraldehyde, can be used as the carrier of immobilization chitoanase.Simultaneously because glutaraldehyde has very strong penetration power to material, utilize it that chitin is handled after, the surface of chitin forms organic vesicular structure, helps the absorption of enzyme.And glutaraldehyde is active bifunctional reagent, it not only with amino, the carboxyl reaction of zymoprotein, can also activate the amino that chitin has.Sorbent material with other is compared, and adopts glutaraldehyde can make the remaining vigor of chitoanase reach maximum.The absorption of chitin-glutaraldehyde cross-linking is as a kind of process for fixation of uniqueness, Application and Development in addition aspect immobilized enzyme.
Summary of the invention
The object of the present invention is to provide a kind of method of utilizing chitin-glutaraldehyde cross-linking absorption immobilized enzyme, to obtain storage and the good immobilization chitoanase of operational stability.
The object of the present invention is achieved like this:
A kind of method of utilizing chitin-glutaraldehyde cross-linking absorption set casing glycanase, characteristics are to utilize chitin to be enzyme fixed carrier, glutaraldehyde is the fixing free chitoanase of cross-linking reagent, promptly at first refining chitin carrier, then behind glutaraldehyde cross-linking, the free chitoanase that is extracted is fixed, and its concrete steps are as follows:
Making with extra care of a, fixation support chitin
With molecular weight is 2.0-2.5 * 10
4Flaky chitin grinds to form particle, cross the 20-60 mesh sieve, spend the night with distilled water immersion under the room temperature, second day suction filtration adds 3-7mol/L HCl and soaks 18-30hr, suction filtration, add 0.1-2mol/L NaOH, boil 1-3hr, extremely neutral with distilled water flushing, add 0.005-0.1mol/LNaOH solution and soak 2-6hr, suction filtration at 30-60 ℃; Add the 0.005-0.1mol/L acetum and soak 1-4hr, suction filtration in 30-60 ℃; To neutral, 60-90 ℃ dry down, obtains the purified chitin with distilled water flushing.
The preparation of b, cross-linking reagent
Select for use to be kept at below 4 ℃, the production time is that 10-30d prepares linking agent with interior glutaraldehyde analytical reagent, and be 2-5 month the working lipe of using; The glutaraldehyde reagent that with concentration is 2-6% mixes with chitoanase appropriate pH value 5.0-7.0 acetate buffer solution, is made into cross-linking reagent.
C, chitin crosslinked
The refining chitin that takes by weighing 0.1-0.5g is a carrier, adds the above-mentioned cross-linking reagent 1-5ml that obtains, and soaks and stirring 1-5hr standing over night, the centrifugal supernatant liquor that goes under the room temperature; Carrier is washed to remove remaining glutaraldehyde repeatedly with distilled water, and suction filtration promptly gets crosslinked chitin; Carrier after crosslinked is immersed in the damping fluid of pH value 5.0-7.0, leaves standstill under 4 ℃, preserves standby.
The extraction of d, free chitoanase
(1) shake flask fermentation is cultivated and is produced enzyme
Cultured pseudomonas (Pseudomonas) CUY8 seed is inserted shake flask fermentation to be cultivated; Fermention medium is: inoculum size 5-12%, glucose 2-6%, chitosan 0.5-4%, NaCl 0.1-2%, FeCl
210-30 * 10
-5G/mL, MgCl
210-35 * 10
-5G/mL, yeast extract paste 0.1-0.7%, (NH
4)
2HPO
40.8%, pH value 6.0-8.0, temperature 30-40 ℃, shaking culture 1-4d under the 100-150r/min condition.
(2) preparation of free chitoanase
After the chitoanase behind the 10-50% acetone precipitation concentrates by Sephadex G-25 column chromatography, be further purified through Sephadex G-100 column chromatography again and collect chitoanase albumen, after zymoprotein being dissolved in the acetate buffer of 0.01-0.1mol/L of 1-4ml, ice bath vacuumizes, and preservation is standby after the concentrated vacuum-drying.
The immobilization of e, free chitoanase
The crosslinked chitin solution of step (c) is removed by filter solvent, take out the crosslinked chitin carrier of 0.1-0.5g, add the free chitoanase liquid that contains 1-10mg, under 4 ℃, leave standstill, take out every 0.5-3hr during this time and stir once, the centrifuging and taking precipitation promptly gets immobilized enzyme.
The present invention adopts the absorption cross-linking method, has overcome that independent employing absorption or carrier adsorptive power that crosslinking produced are weak, easily desorb comes off and the shortcoming of the easy inactivation of enzyme; Compare simultaneously, have long half time, the immobilization system characteristics such as little that are affected by the external environment with other enzyme immobilization method.
Description of drawings
Fig. 1 is the sem photograph (750 times) of sheet chitin without glutaraldehyde cross-linking
Fig. 2 is the refining sem photograph (750 times) of chitin behind glutaraldehyde cross-linking
Fig. 3 is the refining sem photograph (1500 times) of chitin behind glutaraldehyde cross-linking
Embodiment
Embodiment 1
1. the fixation support chitin is refining
With molecular weight is 2.0 * 10
4Flaky chitin grinds to form particle, crosses 30 mesh sieves, spends the night with distilled water immersion under the room temperature.Second day suction filtration adds 4mol/L HCl and soaks 18hr, and suction filtration adds 0.5mol/L NaOH, boils 2hr,, adds 0.005mol/L NaOH solution and soaks 6hr, suction filtration at 50 ℃ to neutral with distilled water flushing; Add the 0.005mol/L acetum and soak 4hr, suction filtration in 50 ℃; To neutral, 70 ℃ dry down, obtains the purified chitin with distilled water flushing.
2. chitin is crosslinked
The refining chitin that takes by weighing 0.2g is a carrier, adds cross-linking reagent 1ml, soaks and stirring 2hr standing over night, the centrifugal supernatant liquor that goes under the room temperature.Carrier is washed to remove remaining glutaraldehyde repeatedly with distilled water, and suction filtration promptly gets crosslinked chitin.Carrier after crosslinked is immersed in the damping fluid of pH value 5.8, leaves standstill under 4 ℃, preserves standby.
3. the immobilization of free chitoanase
Standby crosslinked chitin solution is removed by filter solvent, takes out the crosslinked chitin carrier of 0.2g, add the free chitoanase liquid that contains 3mg, under 4 ℃, leave standstill, during take out every 1hr and to stir once, the centrifuging and taking precipitation promptly gets immobilized enzyme.
Embodiment 2
1. the fixation support chitin is refining
With molecular weight is 2.5 * 10
4Flaky chitin grinds to form particle, crosses 50 mesh sieves, spends the night with distilled water immersion under the room temperature.Second day suction filtration adds 5mol/L HCl and soaks 30hr, and suction filtration adds 1mol/L NaOH, boils 2hr,, adds 0.01mol/L NaOH solution and soaks 5hr, suction filtration at 40 ℃ to neutral with distilled water flushing; Add the 0.01mol/L acetum and soak 4hr, suction filtration in 40 ℃; To neutral, 90 ℃ dry down, obtains the purified chitin with distilled water flushing.
2. chitin is crosslinked
Take by weighing the refining chitin of 0.5g, add cross-linking reagent 5ml, soak and stirring 5hr standing over night, the centrifugal supernatant liquor that goes under the room temperature.Carrier is washed to remove remaining glutaraldehyde repeatedly with distilled water, and suction filtration promptly gets crosslinked chitin.Carrier after crosslinked is immersed in the damping fluid of pH value 6.0, leaves standstill under 4 ℃, preserves standby.
3. the immobilization of free chitoanase
Standby crosslinked chitin solution is removed by filter solvent, takes out the crosslinked chitin carrier of 0.5g, add the free chitoanase liquid that contains 5mg, under 4 ℃, leave standstill, during take out every 2hr and to stir once, the centrifuging and taking precipitation promptly gets immobilized enzyme.
Through scanning electron microscopic observation, the chitin carrier that utilizes the present invention to obtain presents tangible vesicular structure behind glutaraldehyde cross-linking.
Experiment confirm, utilize the immobilization chitoanase that the present invention makes and the enzyme characteristic of free chitoanase to be compared as follows:
In addition, the enzyme reaction number of operations of immobilization chitoanase surpasses 12 times, and the relative enzyme activity of its remnants is still more than 80%.
What by above experimental result as can be known, immobilization chitoanase of the present invention can prolong enzyme utilizes time and number of times.
Claims (1)
1. method of utilizing chitin-glutaraldehyde cross-linking absorption set casing glycanase, it is characterized in that it is to utilize chitin to be enzyme fixed carrier, glutaraldehyde is the fixing free chitoanase of cross-linking reagent, promptly at first refining chitin carrier, then behind glutaraldehyde cross-linking, the free chitoanase that is extracted is fixed, and its concrete steps are as follows:
Making with extra care of a, fixation support chitin
With molecular weight is 2.0-2.5 * 10
4Flaky chitin grinds to form particle, cross the 20-60 mesh sieve, spend the night with distilled water immersion under the room temperature, second day suction filtration adds 3-7mol/L HCl and soaks 18-30hr, suction filtration, add 0.1-2mol/L NaOH, boil 1-3hr, extremely neutral with distilled water flushing, add 0.005-0.1mol/LNaOH solution and soak 2-6hr, suction filtration at 30-60 ℃; Add the 0.005-0.1mol/L acetum and soak 1-4hr, suction filtration in 30-60 ℃; To neutral, 60-90 ℃ dry down, obtains the purified chitin with distilled water flushing;
The preparation of b, cross-linking reagent
Select for use to be kept at below 4 ℃, the production time is that 10-30d prepares linking agent with interior glutaraldehyde analytical reagent, and be 2-5 month the working lipe of using; The glutaraldehyde reagent that with concentration is 2-6% mixes with chitoanase appropriate pH value 5.0-7.0 acetate buffer solution, is made into cross-linking reagent;
C, chitin crosslinked
The refining chitin that takes by weighing 0.1-0.5g is a carrier, adds the above-mentioned cross-linking reagent 1-5ml that obtains, and soaks and stirring 1-5hr standing over night, the centrifugal supernatant liquor that goes under the room temperature; Carrier is washed to remove remaining glutaraldehyde repeatedly with distilled water, and suction filtration promptly gets crosslinked chitin; Carrier after crosslinked is immersed in the damping fluid of pH value 5.0-7.0, leaves standstill under 4 ℃, preserves standby;
The extraction of d, free chitoanase
(1) shake flask fermentation is cultivated and is produced enzyme
Cultured pseudomonas (Pseudomonas) CUY8 seed is inserted shake flask fermentation to be cultivated; Fermentation culture conditions is: inoculum size 5-12%, glucose 2-6%, chitosan 0.5-4%, NaCl 0.1-2%, FeCl
210-30 * 10
-5G/mL, MgCl
210-35 * 10
-5G/mL, yeast extract paste 0.1-0.7%, (NH
4)
2HPO
40.8%, pH value 6.0-8.0, temperature 30-40 ℃, shaking culture 1-4d under the 100-150r/min condition;
(2) preparation of free chitoanase
After the chitoanase behind the 10-50% acetone precipitation concentrates by Sephadex G-25 column chromatography, be further purified through Sephadex G-100 column chromatography again and collect chitoanase albumen, after zymoprotein being dissolved in the acetate buffer of 0.01-0.1mol/L of 1-4ml, ice bath vacuumizes, and preservation is standby after the concentrated vacuum-drying;
The immobilization of e, free chitoanase
The crosslinked chitin solution of step (c) is removed by filter solvent, take out the crosslinked chitin carrier of 0.1-0.5g, add the free chitoanase liquid that contains 1-10mg, under 4 ℃, leave standstill, take out every 0.5-3hr during this time and stir once, the centrifuging and taking precipitation promptly gets immobilized enzyme.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2007100382148A CN101050457B (en) | 2007-03-20 | 2007-03-20 | Method for fixing chitosan enzyme by using cross-linking adsorption of chitosan - glutaraldehyde |
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|---|---|---|---|
| CN2007100382148A CN101050457B (en) | 2007-03-20 | 2007-03-20 | Method for fixing chitosan enzyme by using cross-linking adsorption of chitosan - glutaraldehyde |
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| CN101050457B true CN101050457B (en) | 2011-02-02 |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| US9944920B2 (en) * | 2011-11-11 | 2018-04-17 | Guild Associates, Inc. | Biodegradable immobilized enzymes and methods of making the same |
| CN104498548A (en) * | 2014-11-27 | 2015-04-08 | 苏州嘉禧萝生物科技有限公司 | Production method for enriching gamma-aminobutyric acid from germinated brown rice |
| CN105820966B (en) * | 2015-12-10 | 2019-12-27 | 领先生物农业股份有限公司 | Efficient chitosanase producing strain and fermentation method thereof |
| CN105861478A (en) * | 2016-04-19 | 2016-08-17 | 青岛农业大学 | Nanometer chitin-lysozyme composite particles and preparation method thereof |
| CN111686696B (en) * | 2020-04-22 | 2023-04-07 | 杭州嘉澍环境监测有限公司 | Aminobenzene sulfonic acid modified glutaraldehyde cross-linked chitin gel material, preparation thereof and application thereof as noble metal gold adsorption material |
| CN112075547A (en) * | 2020-06-04 | 2020-12-15 | 天津农学院 | Bait for cold-formed aquatic seedling as well as preparation method and application of bait |
| CN114672475B (en) * | 2022-03-29 | 2024-02-06 | 广东海洋大学 | Immobilized alkaline protease and preparation method thereof |
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Non-Patent Citations (5)
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| 曾嘉.青霉属菌株产壳聚糖酶的分离纯化及固定化的研究.中国优秀博硕士学位论文全文数据库（硕士） 工程科技I辑.2002, * |
| 王平平，等.假单胞菌产壳聚糖酶的纯化和特性.上海水产大学学报13 2.2004,13(2),184-188. |
| 王平平等.假单胞菌产壳聚糖酶的纯化和特性.上海水产大学学报13 2.2004,13(2),184-188. * |
| 王艳，等.产壳聚糖酶菌株选育及培养条件优化.中国海洋大学学报（自然科学版）35 2.2005,35(2),293-296. |
| 王艳等.产壳聚糖酶菌株选育及培养条件优化.中国海洋大学学报(自然科学版)35 2.2005,35(2),293-296. * |
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