CN107012137A - A kind of method that sodium alginate-chitosan fixes zytase - Google Patents
A kind of method that sodium alginate-chitosan fixes zytase Download PDFInfo
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- CN107012137A CN107012137A CN201610057210.3A CN201610057210A CN107012137A CN 107012137 A CN107012137 A CN 107012137A CN 201610057210 A CN201610057210 A CN 201610057210A CN 107012137 A CN107012137 A CN 107012137A
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- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
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Abstract
The present invention relates to a kind of method that sodium alginate-chitosan fixes zytase.Completed by following steps：Raw material mixing, microballoon shaping and cladding, low-temperature setting, elution filtering, low temperature drying, grain forming.The present invention have studied influence of the different factors such as sodium alginate concentration, chitosan concentration, calcium chloride concentration and hardening time to the enzyme activity rate of recovery using the enzyme activity rate of recovery of zytase as standard.The immobilised enzymes stability and the enzyme activity rate of recovery that this method finally gives are higher, while have the advantages that reaction condition is gentle, enzyme activity loss is small, it is significant in the commercial Applications such as feed.
Description
Technical field
The present invention relates to a kind of method that sodium alginate-chitosan fixes zytase
Background technology
Zytase is widely used in feed industry.Contain substantial amounts of xylan in the basal feeds such as wheat, corn, xylan has stronger retentiveness, small molecule adsorption activity and surface-active, causes basal feed to have strong anti-oxidant action to simple stomach or stomachless animals.And zytase is used as feed addictive, the anti-oxidant action of xylan can be effectively eliminated.Adding zytase in feed can be with degrading plant cell wall structure, animal and bird intestines content viscosity is reduced, endogenous digestive enzyme activity is improved, harmful intestinal tract micro organism quantity is reduced, so as to improve animal digesting and assimilating and feed conversion rate to nutrient, growth performance is finally improved.
But the zytase of free state there is also poor, easy in inactivation of some problems such as stability etc., causes utilization rate relatively low, cost is higher in actual applications.Therefore, can be according to enzyme immobilizatio principle, using biotechnology to solving the above problems.Enzyme immobilizatio is to fetter enzyme with solid material or be limited to can still provide for its distinctive catalytic reaction and the technology of recyclable reuse in certain area.Compared with resolvase, immobilised enzymes can not only keep that its efficient, single-minded and gentle enzymic catalytic reaction characteristic and its bin stability be high, separation and recovery easily, the series of advantages such as can repeatedly use, operate continuous and controllable, technique easy, not only developed rapidly but also because with saving resource and the energy, the Impacts on ecology and environment for reducing or preventing and remedying pollution and the strategic requirement for meeting sustainable development in the ambit such as chemistry, biology and bioengineering, medical science and life science.
Recent domestic is increasingly turned to natural macromolecular material and organic synthesis material in superior performance on the carrier selection of Immobilized Xylanase.Wherein natural macromolecular material has nontoxic, with low cost, the features such as mass-transfer performance is good, mainly there is sodium alginate, chitosan etc..The stability of zytase how is improved at present, the activity recovery of enzyme is improved, and is found applied to the fixation support of different field and suitably with new method to being fixed of zytase, is direction and the trend of research and development from now on.And sodium alginate, chitosan is most commonly seen natural macromolecular material, therefore, this method is carrier from sodium alginate, by forming water insoluble dense film with calcium ion and chitosan action face, to improve the stability of zytase, while optimizing optimum condition, to improve the enzyme activity rate of recovery.
The content of the invention
Present invention aims at providing a kind of method that sodium alginate-chitosan fixes zytase, to solve, the stability that free zytase exists in actual applications is poor, easy in inactivation is denatured, the low problem of utilization rate.
In order to realize foregoing invention purpose, the technical solution adopted in the present invention is as follows：
The method that sodium alginate-chitosan fixes zytase, is completed by following steps：
(1) raw material blend step
Weigh a certain amount of sodium alginate and be completely dissolved in formation colloid solution in distilled water, appropriate xylan enzyme liquid is added into the colloidal solution, pH is to 6.0~7.0 for regulation, be sufficiently stirred for mixing, obtain mixed liquor 1；
A certain amount of chitosan is weighed respectively and calcium chloride is dissolved in distilled water, pH is to 3.0~6.0 for regulation, is sufficiently stirred for mixing, is obtained mixed liquor 2；
(2) microballoon shaping and encapsulation steps
Using the peristaltic pump with syringe needle by mixed liquor 1 obtained by step (1), instill into mixed liquor 2 obtained by step (1), obtain 2~3mm of diameter smooth microballoon；
(3) low temperature cure step
The smooth microballoon of gained in step (2) is placed in progress low-temperature setting in 3 DEG C of -10 DEG C of environment and obtains solidified microsphere, the wherein low-temperature setting time is 1~4h；
(4) filtration step is eluted
Solidified microsphere prepared by step (3) removes remained on surface impurity with distilled water flushing and obtains eluting microballoon；
(5) low temperature drying step
Elution microballoon is placed in progress low temperature drying in 35~45 DEG C of thermostatic drying chamber and obtains approximate spherical particles, 4~8h of drying time；
(6) grain forming
The immobilized enzyme particle for the approximate ball-type that screening step (5) is obtained.
In a preferred embodiment of the invention, in the step (1), the sodium alginate mass percent concentration is 1.0~3.0%, and the chitosan mass percent concentration is 0.5~4.5%, and calcium chloride mass percent concentration is 0.5~2.5%.
In a preferred embodiment of the invention, in the step (1), the pH value in the preparation process of mixed liquor 1 uses mass percent concentration to be adjusted for 5% acetum.
In a preferred embodiment of the invention, in the step (1), the pH value in the preparation process of mixed liquor 2 uses mass percent concentration to be adjusted for 5% acetum.
In a preferred embodiment of the invention, in the step (2), the mixed liquor 1 is instilled into mixed liquor 2 obtained by step (1) with 3 drops/sec of speed.
In a preferred embodiment of the invention, the step (3) is in triplicate.
In a preferred embodiment of the invention, the Immobilized Xylanase progress embedding rate for the approximate spherical particles that step (6) is obtained, stability and enzyme activity determination of recovery rates method are as follows：
The embedding rate of immobilised enzymes is determined：Embedding rate (%)=immobilization enzyme amount (g)/enzyme total addition level (g) × 100% of immobilised enzymes；
Immobilization enzyme activity determination of recovery rates：Total activity × 100% of total activity/resolvase of the enzyme activity rate of recovery (%)=immobilised enzymes
The Stability Determination of immobilised enzymes：A certain amount of immobilised enzymes and resolvase are taken respectively；(a) vigor of immobilised enzymes and resolvase is determined respectively at a temperature of 18 DEG C, 28 DEG C, 38 DEG C, 48 DEG C, 58 DEG C, 68 DEG C, 78 DEG C；(b) vigor of immobilised enzymes and resolvase is determined in the case where pH value is 1.0,2.0,3.0,4.0,5.0,6.0,7.0 values.
Beneficial effects of the present invention
The method that a kind of sodium alginate-chitosan of the present invention fixes zytase; according to the entrapment principle of immobilised enzymes; using natural macromolecular material sodium alginate and chitosan as carrier, have the advantages that gentle reaction condition, protection enzyme molecular structure, enzyme activity loss are small；The immobilised enzymes stability and the enzyme activity rate of recovery that this method finally gives are higher, significant in the commercial Applications such as feed.
Brief description of the drawings
The relative enzyme activity comparative graph of resolvase and immobilised enzymes under the different temperatures of Fig. 1 embodiment of the present invention 1.
The relative enzyme activity comparative graph of resolvase and immobilised enzymes under the different pH condition of Fig. 2 embodiment of the present invention 1.
Embodiment
With reference to embodiment the present invention will be further explained explanation, but the present invention is not intended to limit.
Embodiment 1
A kind of method that sodium alginate-chitosan fixes zytase, is comprised the following steps that：
(1) raw material is mixed
The sodium alginate for weighing 4.0g is completely dissolved in distilled water, appropriate xylan enzyme liquid is added into the colloidal solution, adjusting pH to 6.0~7.0, (pH value is adjusted to by 5% acetum, similarly hereinafter), it is sufficiently stirred for mixing, mixed liquor 1a is obtained, wherein sodium alginate mass percent concentration is 2.0%.
3.0g chitosans are weighed respectively and 4.0g calcium chloride is dissolved in distilled water, pH is to 5.5 for regulation, are sufficiently stirred for mixing, and obtain mixed liquor 2a, wherein chitosan mass percent concentration is 1.5%, and calcium chloride mass percent concentration is 2.0%.
(2) microballoon shaping and cladding
Using the peristaltic pump with syringe needle by mixed liquor 1a obtained by (1), instilled with 3 drops/sec of speed into mixed liquor 2a obtained by step (1), obtain 2~3mm of diameter smooth microballoon.
(3) low-temperature setting
The smooth microballoon of gained in step (2) is placed in 4 DEG C of environment and carries out low-temperature setting, hardening time is 1h.
(4) elution filtering
Microballoon after step (3) is solidified removes remained on surface impurity with distilled water flushing, is repeated 3 times.
(5) low temperature drying
In the thermostatic drying chamber that microballoon after elution is placed in 40 DEG C, drying time 6h.
(6) grain forming
The immobilized enzyme particle of dried approximate ball-type is collected in screening.
The embedding rate of the immobilized enzyme particle obtained at the process conditions is 92%, and the enzyme activity rate of recovery is 64.7%
The Stability Determination of immobilised enzymes；
(a) resolvase enzyme activity relative with immobilised enzymes is compared under the different temperatures of result as shown in Figure 1; the optimum temperature range of resolvase is 37~39 DEG C; the optimum temperature range of immobilised enzymes is 47~48 DEG C; due to the protective effect of sodium alginate-chitosan wall material, the heat-resistant stability of immobilised enzymes is significantly improved.
(b) resolvase enzyme activity relative with immobilised enzymes is compared under the different pH condition of result as shown in Figure 2, the optimal pH scope of resolvase is 4.5~5.5, the optimal pH scope of immobilised enzymes is 5.5~6.5, the optimal pH scope of immobilised enzymes increases, simultaneously because sodium alginate-chitosan wall material has good stability under the conditions of low pH (pH=2.0~4.0), it is to avoid loss of the zytase in gastric juice.
Embodiment 2
A kind of method that sodium alginate-chitosan fixes zytase, is comprised the following steps that：
(1) raw material is mixed
3.0g sodium alginates are weighed to be completely dissolved in distilled water, appropriate xylan enzyme liquid is added into the colloidal solution, adjusting pH to 6.0~7.0, (pH value is adjusted to by 5% acetum, similarly hereinafter), it is sufficiently stirred for mixing, mixed liquor 1b is obtained, wherein sodium alginate mass percent concentration is 1.5%.
2.0g chitosans are weighed respectively and 5.0g calcium chloride is dissolved in distilled water, pH is to 5.5 for regulation, are sufficiently stirred for mixing, and obtain mixed liquor 2b, wherein chitosan mass percent concentration is 1.0%, and calcium chloride mass percent concentration is 2.5%；
(2) microballoon shaping and cladding
Using the peristaltic pump with syringe needle by mixed liquor 1b obtained by (1), instilled with 3 drops/sec of speed into mixed liquor 2b obtained by (1), obtain 2~3mm of diameter smooth microballoon.
(3) low-temperature setting
The smooth microballoon of gained in step (2) is placed in 4 DEG C of environment and carries out low-temperature setting, hardening time is 2h.
(4) elution filtering
Microballoon after step (3) is solidified removes remained on surface impurity with distilled water flushing, is repeated 3 times.
(5) low temperature drying
In the thermostatic drying chamber that microballoon after elution is placed in 35 DEG C, drying time 8h.
(6) grain forming
Dried approximate spherical particles are collected in screening.
The embedding rate of the immobilized enzyme particle obtained at the process conditions is 90.8%, and the enzyme activity rate of recovery is 48.5%；
The Detection of Stability method of immobilised enzymes is with reference to embodiment 1.
Embodiment 3
A kind of method that sodium alginate-chitosan fixes zytase, is comprised the following steps that：
(1) raw material is mixed
Weigh 4.0g sodium alginates to be completely dissolved in distilled water, appropriate xylan enzyme liquid is added into the colloidal solution, (pH value is adjusted to regulation pH to 6.0~7.0 by 5% acetum, similarly hereinafter), it is sufficiently stirred for mixing, obtains mixed liquor 1c, wherein sodium alginate concentration is 2.0%.4.0g chitosans are weighed respectively and 3.0g calcium chloride is dissolved in distilled water, pH is to 5.5 for regulation, are sufficiently stirred for mixing, and obtain mixed liquor 2c, wherein chitosan concentration is 2.0%, and calcium chloride concentration is 1.5%.
(2) microballoon shaping and cladding
Using the peristaltic pump with syringe needle by mixed liquor 1c obtained by (1), instilled with 3 drops/sec of speed into mixed liquor 2c obtained by (1), obtain 2~3mm of diameter smooth microballoon.
(3) low-temperature setting
It is 1.5h that thus obtained microsphere in step (2) is placed in 4 DEG C of environment into hardening time.
(4) elution filtering
Microballoon after solidification is removed into remained on surface impurity with distilled water flushing, is repeated 3 times.
(5) low temperature drying
In the thermostatic drying chamber that microballoon after elution is placed in 35 DEG C, drying time 8h.
(6) grain forming
Dried approximate spherical particles are collected in screening.
The embedding rate of the immobilized enzyme particle obtained at the process conditions is 94.1%, and the enzyme activity rate of recovery is 51.6%, and the Detection of Stability method of immobilised enzymes is with reference to embodiment 1.
In summary, it is described above be only embodiments of the present invention citing, for those skilled in the art, without departing from the technical principles of the invention, some improvement can also be made, these improvement also should be regarded as the present invention protection.
Claims (7)
1. the method that sodium alginate-chitosan fixes zytase, it is characterised in that complete by following steps
Into：
(1) raw material blend step
Weigh a certain amount of sodium alginate and be completely dissolved in formation colloid solution in distilled water, to the colloid
Appropriate xylan enzyme liquid is added in solution, pH is to 6.0~7.0 for regulation, be sufficiently stirred for mixing, mixed
Close liquid 1；
A certain amount of chitosan is weighed respectively and calcium chloride is dissolved in distilled water, regulation pH to 3.0~
6.0, it is sufficiently stirred for mixing, obtains mixed liquor 2；
(2) microballoon shaping and encapsulation steps
Using the peristaltic pump with syringe needle by mixed liquor 1 obtained by step (1), instill to step (1) gained
In mixed liquor 2,2~3mm of diameter smooth microballoon is obtained；
(3) low temperature cure step
The smooth microballoon of gained in step (2) is placed in into progress low-temperature setting in 3 DEG C of -10 DEG C of environment to be consolidated
Change microballoon, the wherein low-temperature setting time is 1~4h；
(4) filtration step is eluted
It is micro- that solidified microsphere prepared by step (3) obtains elution with distilled water flushing removal remained on surface impurity
Ball；
(5) low temperature drying step
Elution microballoon is placed in progress low temperature drying in 35~45 DEG C of thermostatic drying chamber and obtains approximate ball-type
Grain, 4~8h of drying time；
(6) grain forming
The immobilized enzyme particle for the approximate ball-type that screening step (5) is obtained.
2. sodium alginate-chitosan as claimed in claim 1 fixes the method for zytase, its feature
It is, in the step (1), the sodium alginate mass percent concentration is 1.0~3.0%, described
Chitosan mass percent concentration is 0.5~4.5%, and calcium chloride mass percent concentration is 0.5~2.5%.
3. the method that sodium alginate-chitosan as claimed in claim 1 or 2 fixes zytase, its
It is characterised by, in the step (1), the pH value in the preparation process of mixed liquor 1 uses quality hundred
Specific concentration is divided to be adjusted for 5% acetum.
4. the method that sodium alginate-chitosan as claimed in claim 1 or 2 fixes zytase, its
It is characterised by, in the step (1), the pH value in the preparation process of mixed liquor 2 uses quality hundred
Specific concentration is divided to be adjusted for 5% acetum.
5. sodium alginate-chitosan as claimed in claim 1 fixes the method for zytase, its feature
It is, in the step (2), the mixed liquor 1 is instilled to step (1) institute with 3 drops/sec of speed
Obtain in mixed liquor 2.
6. sodium alginate-chitosan as claimed in claim 1 fixes the method for zytase, its feature
It is, the step (3) is in triplicate.
7. sodium alginate-chitosan as claimed in claim 1 fixes the method for zytase, its feature
It is, the Immobilized Xylanase for the approximate spherical particles that step (6) is obtained carries out embedding rate, stability
And enzyme activity determination of recovery rates method is as follows：
The embedding rate of immobilised enzymes is determined：Embedding rate (%)=immobilization enzyme amount (g)/enzyme of immobilised enzymes
Total addition level (g) × 100%；
Immobilization enzyme activity determination of recovery rates：Total activity/resolvase of the enzyme activity rate of recovery (%)=immobilised enzymes
Total activity × 100%
The Stability Determination of immobilised enzymes：A certain amount of immobilised enzymes and resolvase are taken respectively；(a) 18
DEG C, 28 DEG C, 38 DEG C, 48 DEG C, 58 DEG C, 68 DEG C, determine respectively at a temperature of 78 DEG C immobilised enzymes with it is free
The vigor of enzyme；(b) determine solid in the case where pH value is 1.0,2.0,3.0,4.0,5.0,6.0,7.0 values
Surely the vigor of enzyme and resolvase is changed.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109180982A (en) * | 2018-08-24 | 2019-01-11 | 西安建筑科技大学 | A kind of preparation method of embedded nano material/Quorum quenching enzymes composite pellets |
| CN110669754A (en) * | 2019-10-29 | 2020-01-10 | 华侨大学 | Method for 3D printing of xylanase catalyst and application thereof |
| CN116270779A (en) * | 2023-03-14 | 2023-06-23 | 金七药业股份有限公司 | Preparation method of pseudo-ginseng extract |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1454995A (en) * | 2002-04-29 | 2003-11-12 | 天津市肝胆疾病研究所 | Method of embedding cell or tissue using sodium alginate-chitose-sodium alginate microcapsule |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109180982A (en) * | 2018-08-24 | 2019-01-11 | 西安建筑科技大学 | A kind of preparation method of embedded nano material/Quorum quenching enzymes composite pellets |
| CN110669754A (en) * | 2019-10-29 | 2020-01-10 | 华侨大学 | Method for 3D printing of xylanase catalyst and application thereof |
| CN116270779A (en) * | 2023-03-14 | 2023-06-23 | 金七药业股份有限公司 | Preparation method of pseudo-ginseng extract |
| CN116270779B (en) * | 2023-03-14 | 2023-11-10 | 金七药业股份有限公司 | Preparation method of pseudo-ginseng extract |
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Application publication date: 20170804 |