CN101508985A - Process for producing chitosanase carrier and fixing method for the chitosanase - Google Patents
Process for producing chitosanase carrier and fixing method for the chitosanase Download PDFInfo
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- CN101508985A CN101508985A CNA2009101151120A CN200910115112A CN101508985A CN 101508985 A CN101508985 A CN 101508985A CN A2009101151120 A CNA2009101151120 A CN A2009101151120A CN 200910115112 A CN200910115112 A CN 200910115112A CN 101508985 A CN101508985 A CN 101508985A
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- carrier
- chitoanase
- enzyme
- inscribe
- cotton
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Abstract
The invention relates to a method for preparing a carrier of endo-chitosanase and a method for fixing the endo-chitosanase. Fiber ribbons of cotton obtained by treatments by NaOH, glacial acetic acid, paratoluensulfonyl chloride and pyridine can be used as the carrier of the endo-chitosanase. Adopting the technical proposal of the invention has the advantages that immobilized enzyme is convenient for being separated from products in a liquid system after a reaction, and can be repeatedly used after the immobilized enzyme is taken out; compared with free enzyme, after the enzyme is immobilized, the enzyme can be easily separated from substrate and the products, and can carry out column packing continuous reactions in a long term; residue of the enzyme can not be remained in a product of Chitosan oligosaccharide solution so as to improve the quality of the chitosan oligosaccharide; and the enzyme has high stability and longer service life and retention period, and can be repeatedly used, and cost is greatly reduced.
Description
Technical field
The present invention relates to the fixing means of the preparation method of enzyme carrier and the fixing means of enzyme, the particularly preparation method of inscribe chitoanase carrier and inscribe chitoanase.
Background technology
After chitosan need be dissolved in the solution, the processing through chitoanase obtained micromolecular oligochitosan again, and oligochitosan is widely used in industries such as medicine and food with other advantageous properties because of molecular weight is little.Traditional prepares the process of oligochitosan from chitosan, adopts the zymotechnic of liquid state usually, though the efficient of the enzyme of liquid state can satisfy production requirement, following drawback is arranged: the less stable of enzyme.Because enzyme is the protein with certain space conformation, under the influence that is subjected to extraneous factors such as heat, pH value, mineral ion, organic solvent, easy sex change inactivation, chitoanase and oligochitosan product after reaction finishes all still exist in the liquid system, the two separation difficulty has brought certain difficulty for further separation and purification.Particularly the oligochitosan product need go out, and enzyme is alive makes solid phase prod again, and so liquid enzymes also is inactivated, thereby chitoanase has only used once.
The inscribe chitoanase, be called for short the eCSN enzyme in the industrial production that can be used for oligochitosan (Chitooligosaccharides), because oligochitosan has better water solubility, be more conducive to absorption of human body, particularly the polymerization degree has stronger anti-infective, the activity that suppresses tumour especially at 5,6 oligochitosan.But utilize chemical synthesis process to produce oligochitosan, especially have than Johnson ﹠ Johnson and manage active few pentasaccharides and six sugar, very difficult, productive rate is low, production cost is higher and easy contaminate environment, and use the inscribe chitoanase to produce advantages such as oligochitosan has efficiently, environmental protection by degraded, therefore utilizing the inscribe chitoanase to produce oligochitosan has become the focus that present research is used.The bacterial classification that collection from lake silt, mutagenesis and separation obtain can produce exocytosis type inscribe chitoanase, crude enzyme liquid after its fermentation is carried out separation and purification, can obtain a kind of greater activity, narrow spectrum inscribe chitoanase (endo-Chitosanase, eCSN) pure product.Because the chemical nature of eCSN enzyme is a protein, resolvase is less stable in use, and enzyme mixes with substrate and product in reactive system, has brought certain difficulty for the separation and purification of product, and enzyme can not be reused.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of inscribe chitoanase carrier and the fixing means of inscribe chitoanase.
Technical scheme of the present invention is:
The preparation method of inscribe chitoanase carrier, wherein: handle the converted top that obtains cotton through NaOH, glacial acetic acid, Tosyl chloride, pyridine and can be used as inscribe chitoanase carrier.
The preparation method of inscribe chitoanase carrier, wherein: it is after handling 10min in 15% the NaOH solution that the converted top of cotton immerses the 200ml mass percentage concentration, with distilled water wash for several times, in mass percentage concentration is 10% glacial acetic acid, soak 30min again, be washed till neutrality with deionized water at last; Ratio in the converted top of 15ml pyridine/g cotton adds pyridine, at 25 ℃, acts on 0.5h in the 250r/min water bath with thermostatic control vibrator; In the ratio of the converted top of 15g Tosyl chloride/g cotton, in the converted top of the cotton that pyridine was handled, add Tosyl chloride with acetone solution, every 1g Tosyl chloride adopts the 2ml acetone solution; At 25 ℃, act on 4h in the 250r/min water bath with thermostatic control vibrator, add acetone again and clean, clean with 0.05mol/L HCl again, HCl can unnecessary Tosyl chloride and the pyridine of flush away, and the converted top of the cotton of taking-up can be used as inscribe chitoanase carrier.This carrier is put in 4 ℃ of preservations among the HCl of 0.05mol/L.
The converted top of the cotton that the present invention relates to can be the converted top of the medical cotton of pharmaceutical manufacturer's production.It also can be other converted top.
The fixing means of inscribe chitoanase, wherein: deionized water wash carrier 3 times, with 250U/g carrier ratio, with 0.2mol/LPH5.5 NaH
2PO
4One Na
2HPO
4The inscribe chitoanase of damping fluid dilution adds in the carrier, fixing 12h in the water bath with thermostatic control vibrator of 250r/min, and deionized water wash is preserved in 4 ℃ of damping fluids of immobilized enzyme.
" U " that the present invention relates to is the unit of enzyme activity.
Resolvase vitality test: get 100 μ L1% chitosan-acetic acid solutions, add 5 μ L inscribe chitoanases (being dissolved in the 95 μ L water), continuous oscillation 10min in 45 ℃ of 300r/min rotary type constant temperature water bath vibrators (SHZ-88 type).After DeR finished, with reference to 3, the fixed sugared method of 5-dinitrosalicylic acid method (DNS) colorimetric was measured the reducing sugar amount, calculates the vigor of resolvase.
Immobilization enzyme activity determination: get 200 μ L1% chitosan-acetic acid solutions, add the immobilized inscribe chitoanase of the inventive method 0.01g, at 45 ℃ of 300r/min rotary type constant temperature water bath vibrators (SHZ-88 type) internal reaction 10min.After DeR finishes, get 100 μ L reducing sugars with reference to 3, the fixed sugared method of 5-dinitrosalicylic acid method (DNS) colorimetric is measured concentration of reduced sugar, calculates the immobilized enzyme vigor.
1 enzyme unit definition of living: 1 enzyme activity (U) unit definition is under 7.0 conditions in (45 ± 1) ℃, pH value, and hydrolyzing chitosan discharged the needed enzyme amount of 1 μ mol reducing sugar that is equivalent in one minute.3, the fixed sugared method of 5-dinitrosalicylic acid method (DNS) colorimetric is measured reducing sugar content.
Institute's fixed enzyme activity (U) on the converted top of the immobilization chitoanase activity recovery=cotton/fixing preceding initial enzyme activity (U) that adds
The unit immobilized enzyme is lived (U/g): institute's fixed enzyme activity on the converted top of every gram butt cotton.
The calculating of chitosan hydrolyzate rate: chitosan hydrolyzate rate (%)=reducing sugar content/initial chitosan content 100%
Immobilization inscribe chitoanase zymologic property research:
Immobilization chitoanase optimal reactive temperature:
The enzyme liquid of purifying after fixing, at PH5.5,0.2mol/L NaH
2PO
4One Na
2HPO
4In the damping fluid, respectively under 30 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃, 60 ℃, measuring the immobilized enzyme vigor, is 100% in high enzymatic activity, and this enzyme is after immobilization, and optimal reactive temperature still is 45 ℃.
Immobilization chitoanase optimal reaction PH;
The enzyme liquid of purifying is after fixing, respectively at (PH4.5-6.5) 0.2mol/L NaH
2PO
4One Na
2HPO
4In the damping fluid, measure the immobilized enzyme vigor for 45 ℃.It is 6.5 that experiment records resolvase reaction optimal pH.Enzyme is after immobilization, and optimal reaction PH oxytropism direction is offset 1 unit.Therefore, after the chitoanase immobilization, can be used to handle neutral slant acidity material.
The stability in storage of immobilization eCSN enzyme:
Resolvase and immobilization inscribe chitoanase place 0.2mol/LNaH respectively
2PO
4One Na
2HPO
4In the buffered soln (pH6.5), store down, under the suitableeest hydrolysising condition of enzyme, measure the remaining vigor that stores the back enzyme after 30 days at 4 ℃.The result shows, stores the higher enzyme activity of immobilization inscribe chitoanase maintenance after 30 days.
The operational stability of immobilization eCSN enzyme:
Operational stability is to weigh the leading indicator of immobilized enzyme practical value, characterizes its operational stability with the repetition hydrolysising experiment of immobilized enzyme.With immobilized enzyme wet method dress post, bed adds 1% chitosan solution, is an enzymic hydrolysis process with every 24hr.After one time hydrolysis reaction is finished, use the deionized water wash immobilized enzyme, remove degraded substrate and degraded product remaining on the immobilized enzyme.Again the reaction that is hydrolyzed is then measured concentration of reduced sugar with reference to the fixed sugared method of DNS colorimetric under the suitableeest hydrolysising condition of enzyme, calculate the residue vigor of immobilized enzyme.The result shows, after immobilized enzyme repeated hydrolyzing chitosan 10 times, enzyme was lived and still kept 40%.
Packed bed reactor immobilization inscribe chitoanase enzymolysis chitosan:
At PH is 5.5,45 ℃, 20ml, and the continuous circulating reaction of 1% chitosan solution and immobilized enzyme, different time be (6h, 12h, 18h, 24h) sampling at interval, detects the variation of oligochitosan in the enzymolysis solution composition by thin-layer chromatography or HPLC.
Inscribe chitoanase eCSN is effective commercial enzyme preparation that hydrolyzing chitosan prepares low-molecular weight chitoglycan and oligochitosan.In this research, the inscribe chitoanase is fixed on the Tosyl chloride activatory cotton fibre carrier, the optimum condition that draws preparation immobilization eCSN enzyme is pH6.5, ionic concn is 0.2mol/L NaH
2PO
4One Na
2HPO
4Damping fluid, enzyme concentration are the 250U/g carrier, and the immobilized reactant time is 12hr, and activity recovery can reach 60%.The optimal reactive temperature of immobilized enzyme does not change, 1 unit of optimal reaction PH oxytropism direction skew, the stability in storage and the operational stability of immobilized enzyme show that applying immobilized inscribe chitoanase can continuous degradation Preparation of Chitosan low-molecular weight chitoglycan and oligochitosan.
Adopt technical scheme of the present invention that following advantage is arranged:
Solidify enzyme and be convenient to separate, and solidify and to use repeatedly after enzyme takes out with product in the reacted liquid system.Compare with resolvase, behind the enzyme immobilization, very easily separate, can adorn the post successive reaction in a long time, do not have the residual of enzyme in the product oligochitosan solution, improved the quality of oligochitosan with substrate, product; Operation lifetime and preservation period that enzyme has advantages of higher stability, grows, and enzyme is reusable, and cost significantly reduces.The present invention is an initiator with NaOH and pyridine, Tosyl chloride is as intermediate, with the hydroxyl on the converted top surface of the cotton formation covalent linkage that reacts, the inscribe chitoanase is in fixation procedure, and the free N-terminal is covalently bound on the converted top of cotton by replacing p-toluenesulfonyl on the amino-acid residue; Studied the zymologic property of immobilized enzyme; And inquired into the operational condition of immobilized enzyme continuous degradation chitosan in the packed bed column type reactor, this production application to immobilized enzyme has than higher reference value.
Embodiment
The preparation method of embodiment 1, inscribe chitoanase carrier, wherein: it is after handling 10min in 15% the NaOH solution that the converted top of cotton immerses the 200ml mass percentage concentration, with distilled water wash for several times, in mass percentage concentration is 10% glacial acetic acid, soak 30min again, be washed till neutrality with deionized water at last; Ratio in the converted top of 15ml pyridine/g cotton adds pyridine, at 25 ℃, acts on 0.5h in the 250r/min water bath with thermostatic control vibrator; In the ratio of the converted top of 15g Tosyl chloride/g cotton, in the converted top of the cotton that pyridine was handled, add Tosyl chloride with acetone solution, every 1g Tosyl chloride adopts the 2ml acetone solution; At 25 ℃, act on 4h in the 250r/min water bath with thermostatic control vibrator, add acetone again and clean, clean with 0.05mol/L HCl again, HCl can unnecessary Tosyl chloride and the pyridine of flush away, and the converted top of the cotton of taking-up can be used as inscribe chitoanase carrier.This carrier is put in 4 ℃ of preservations among the HCl of 0.05mol/L.
The converted top of the cotton that the present invention relates to can be the converted top of the medical cotton of pharmaceutical manufacturer's production.It also can be other converted top.
The fixing means of embodiment 2, inscribe chitoanase, wherein: deionized water wash carrier 3 times, with 250U/g carrier ratio, with 0.2mol/L PH6.5NaH
2PO
4One Na
2HPO
4The inscribe chitoanase of damping fluid dilution adds in the carrier, fixing 12h in the water bath with thermostatic control vibrator of 250r/min, and deionized water wash is preserved in 4 ℃ of damping fluids of immobilized enzyme.Carrier wherein obtains by embodiment 1 method.
Claims (3)
1, the preparation method of inscribe chitoanase carrier is characterized in that: handle the converted top that obtains cotton through NaOH, glacial acetic acid, Tosyl chloride, pyridine and can be used as inscribe chitoanase carrier.
2, the preparation method of inscribe chitoanase carrier, it is characterized in that: it is after handling 10min in 15% the NaOH solution that the converted top of cotton immerses the 200ml mass percentage concentration, with distilled water wash for several times, in mass percentage concentration is 10% glacial acetic acid, soak 30min again, be washed till neutrality with deionized water at last; Ratio in the converted top of 15ml pyridine/g cotton adds pyridine, at 25 ℃, acts on 0.5h in the 250r/min water bath with thermostatic control vibrator; In the ratio of the converted top of 15g Tosyl chloride/g cotton, in the converted top of the cotton that pyridine was handled, add Tosyl chloride with acetone solution, every 1g Tosyl chloride adopts the 2ml acetone solution; At 25 ℃, act on 4h in the 250r/min water bath with thermostatic control vibrator, add acetone again and clean, clean with 0.05mol/L HCl again, the converted top of the cotton of taking-up can be used as inscribe chitoanase carrier.
3, the preparation method of the inscribe chitoanase carrier that obtains of the preparation method of inscribe chitoanase carrier as claimed in claim 2 is applied to the fixing means of inscribe chitoanase, it is characterized in that: deionized water wash carrier 3 times, with 250U/g carrier ratio, with 0.2mol/L PH6.5 NaH
2PO
4One Na
2HPO
4The inscribe chitoanase of damping fluid dilution adds in the carrier, fixing 12h in the water bath with thermostatic control vibrator of 250r/min, and deionized water wash is preserved in 4 ℃ of damping fluids of immobilized enzyme.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009101151120A CN101508985B (en) | 2009-03-27 | 2009-03-27 | Process for producing chitosanase carrier and fixing method for the chitosanase |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2009101151120A CN101508985B (en) | 2009-03-27 | 2009-03-27 | Process for producing chitosanase carrier and fixing method for the chitosanase |
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| Publication Number | Publication Date |
|---|---|
| CN101508985A true CN101508985A (en) | 2009-08-19 |
| CN101508985B CN101508985B (en) | 2012-07-25 |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104532571A (en) * | 2015-01-19 | 2015-04-22 | 重庆大学 | Preparation process of chitosan oligosaccharide modifying cotton fiber |
| CN107446910A (en) * | 2017-09-29 | 2017-12-08 | 江西师范大学 | A kind of preparation method of fibroin fiber immobilization chitosanase |
| CN113862250A (en) * | 2021-10-14 | 2021-12-31 | 黄河三角洲京博化工研究院有限公司 | Immobilization method of chitosanase |
-
2009
- 2009-03-27 CN CN2009101151120A patent/CN101508985B/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104532571A (en) * | 2015-01-19 | 2015-04-22 | 重庆大学 | Preparation process of chitosan oligosaccharide modifying cotton fiber |
| CN104532571B (en) * | 2015-01-19 | 2017-01-18 | 重庆大学 | Preparation process of chitosan oligosaccharide modifying cotton fiber |
| CN107446910A (en) * | 2017-09-29 | 2017-12-08 | 江西师范大学 | A kind of preparation method of fibroin fiber immobilization chitosanase |
| CN113862250A (en) * | 2021-10-14 | 2021-12-31 | 黄河三角洲京博化工研究院有限公司 | Immobilization method of chitosanase |
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| Publication number | Publication date |
|---|---|
| CN101508985B (en) | 2012-07-25 |
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