CN1724659A - Preparation method of chitin incision enzyme - Google Patents
Preparation method of chitin incision enzyme Download PDFInfo
- Publication number
- CN1724659A CN1724659A CN 200510050077 CN200510050077A CN1724659A CN 1724659 A CN1724659 A CN 1724659A CN 200510050077 CN200510050077 CN 200510050077 CN 200510050077 A CN200510050077 A CN 200510050077A CN 1724659 A CN1724659 A CN 1724659A
- Authority
- CN
- China
- Prior art keywords
- enzyme
- chitosan
- chitin
- preparation
- chitin incision
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Landscapes
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a method to make chitosan inscribing enzyme. The high yield chitosan inscribing enzyme strain inoculates into fermenter for cultivating. Filtering the suspending fungus, the chitosan inscribing raw enzyme liquid is gained. Fermenting the raw liquid, adopting ultra-filtration and concentration, organic solution or ammonia sulfate deposition, cation exchange and gel filtering, freezing, drying in vacuum, the chitosan inscribing enzyme would be gained. The fungus has stability heritability, and common fermenting condition.
Description
Technical field
The present invention relates to a kind of preparation method of chitin incision enzyme.
Background technology
Chitosan (chitosan) is the high molecular polymer of the biologically active that connected into β-1,4 glycosidic link by glucosamine, chemistry poly-2-amino-2-deoxy-D-glucose by name.It mainly is to be raw material with the shrimp shell that goes out of use, crab shell, through decalcification, remove chemical processes such as albumen and deacetylation and obtain.Its molecular structural formula is as shown below.
Chitosan has many unique biological activity.International chitin conference in 1991 is defined as the sixth-largest vital principle except sugar, protein, fat, VITAMIN and mineral substance to chitosan, has given very high evaluation to them in the effect aspect the health care.Many biological activitys of chitosan need just can embody with the form of lower molecular weight or shell oligosaccharide, as antitumor, immuno-stimulating, as genophore etc.But because the content of interior chitoanase of human body and hydrochloric acid in gastric juice seldom, the action effect of chitosan has been subjected to very big restriction.
The preparation of oligose mainly contains chemical method, oxidation style, supersonic method and enzyme liberating method.Compare with additive method, enzymic degradation prepares chitooligosaccharide-and has the reaction conditions gentleness, advantages such as secondary pollution are controlled, do not produced to relative molecular mass simple to operate, the product chitooligosaccharide-easily.
The enzyme that is used for degradation of chitosan at present has the specificity chitoanase, non-specificity enzyme such as cellulase, lipase, N,O-Diacetylmuramidase etc. are also arranged, the purity of commercially available non-specificity enzyme and vigor are all little high, and cost is higher, is not suitable for the high purity of shell oligose product of preparation relative molecular mass narrow distribution.Therefore, preparation high purity chitin incision enzyme is a hot issue of this area research.The invention discloses a kind of method for preparing the high purity chitin incision enzyme.Separation and purification obtains a kind of chitin incision enzyme from Penicillium notatum fermentation crude enzyme liquid, is applicable to the high purity of shell oligose product of preparation relative molecular mass narrow distribution.
Summary of the invention
Purpose of the present invention provides a kind of method for preparing chitin incision enzyme.
The step of method is as follows:
1) with preserving number be: the high yield chitosan enzyme-producing bacteria (Penicillium sp) of CGMCC No.0851 is seeded in the fermentor tank of 5L, substratum liquid amount 3-4L, and inoculum size is 10%-20%, culture temperature is that 26-32 ℃, stirring velocity are 200-500rmin
-1, air flow is 1.5-5Lmin
-1, ferment after 50-80 hour, remove by filter the suspension thalline and obtain the chitosan crude enzyme liquid;
2) holding back relative molecular mass is that the ultra-filtration membrane of 2-20kDa carries out ultrafiltration to crude enzyme liquid, then, with 3-5 doubly the ice ethanol sedimentation or obtain the thick enzyme throw out of chitosan with the 20%-80% ammonium sulfate precipitation;
3) use pH4.4-5.5, the thick enzyme throw out of 10-50mmol acetate buffer dissolving chitosan, go up HiPrep 16/10 SP XL cationic exchange coloum again,, obtain chitin incision enzyme solution with containing 0.5-2mol sodium-chlor, 10-50mmol acetate buffer 0-100% linear gradient wash-out;
4) filter with Sephadex G50 gel-filtration column, the acetate buffer wash-out, concentrated, freezing, vacuum-drying obtains chitin incision enzyme.
Substratum is: chitosan 5-40g/L, urea 0.5-3g/L, potassium primary phosphate 0.2-1g/L; Ammonium sulfate 0.5-3g/L, sal epsom 0.1
Advantage of the present invention is: enzymic degradation prepares chitooligosaccharide-and oligochitosan has the reaction conditions gentleness, can control degradation speed of reaction and product relative molecular mass, do not produce advantages such as secondary pollution.Particularly use the inscribe chitoanase degrade chitosan after the separation and purification, can obtain the high purity of shell oligose product of relative molecular mass narrow distribution.
Embodiment
From high yield chitoanase bacterial strain (national inventing patent ZL 02159880.0, culture presevation number is CGMCC No.0851) the fermentation crude enzyme liquid, successively adopt the method separation and purification of ultrafiltration and concentration, organic solvent or ammonium sulfate precipitation, cationic exchange and gel-filtration to obtain a kind of chitin incision enzyme.
The invention provides the method for utilizing Penicillium notatum fermentative preparation chitosan crude enzyme liquid.In the fermentor tank of 5L, pack that to cultivate base unit weight be 3-4L into, inoculum size is 10%-20%, culture temperature is that 26-32 ℃, stirring velocity are 200-450rmin
-1, air flow is 1.5-5Lmin
-1, after incubation time 50-80 hour, remove by filter the suspension thalline and obtain the chitosan crude enzyme liquid.
The present invention also provides from chitosan crude enzyme liquid separation and purification to obtain the method for chitin incision enzyme.The method that adopts ultrafiltration and concentration, organic solvent or ammonium sulfate precipitation, cationic exchange and gel-filtration, vacuum freezedrying etc. to separate purifying respectively, separation and purification obtains chitin incision enzyme from crude enzyme liquid.
Embodiment 1:
1, the preparation of crude enzyme liquid
Get 8 of test tube slant preservation bacterial classifications, activate 5 hours after, add sterilized water 15ml respectively, wash spore.The spore suspension that obtains directly inserted in the 5L fermentation tank culture medium cultivate.28 ℃ of culture temperature, rotating speed of agitator 300rmin
-1, air flow is 4.5Lmin
-1, fermentation tank culture medium is composed as follows: initial pH value is 5.
| Chitosan | 30g/L |
| Urea | 1g/L |
| Potassium primary phosphate | 0.6g/L |
| Ammonium sulfate | 1g/L |
| Sal epsom | 0.12g/L |
After the fermentor cultivation 75 hours, in refrigerator, preserved fermented liquid 2 hours down for 4 ℃, then at 6000rmin
-1Get supernatant liquor after centrifugal as crude enzyme liquid, standby.
2, the separation and purification of chitin incision enzyme
(1) concentrating of chitosan crude enzyme liquid: get above-mentioned chitoanase crude enzyme liquid, adopt hyperfiltration process to concentrate ultra-filtration membrane area 0.1m
2, holding back relative molecular mass 2kDa, working pressure 0.4MPa holds back part and is spissated crude enzyme liquid.
(2) extraction of chitoanase: get the above-mentioned crude enzyme liquid of 100ml, according to ice ethanol and 3: 1 ratio of chitosan crude enzyme liquid, add ice ethanol, leave standstill under 4 ℃ of conditions, precipitation obtains the thick enzyme of chitosan, with acetate buffer (pH 4.4) dissolving of 50ml, 20mmol.
(3) roughing out of chitoanase: adopt cationic exchange coloum HiPrep 16/10 SP XL, level pad is 30mmol acetate buffer (pH4.4), elution buffer is the 30mmol acetate buffer that contains 1mol sodium-chlor, 0-100% linear gradient wash-out, and substep is collected.This process can with the same excision enzyme of chitin incision enzyme, impurity protein separately be collected chitin incision enzyme solution.
(4) chitin incision enzyme is refining: according to the difference of chitin incision enzyme and impurity albumen relative molecular mass, adopt gel-filtration column Sephadex G-50, damping fluid is 20mmol acetate buffer (pH4.4), the refining chitin incision enzyme solution that obtains, than vigor 478U/mg, obtain the chitin incision enzyme product after the vacuum freezedrying.
Embodiment 2:
1, the preparation of crude enzyme liquid
Get 10 of test tube slant preservation bacterial classifications, activate 5 hours after, add sterilized water 15ml respectively, wash spore.The spore suspension that obtains directly inserted in the 5L fermentation tank culture medium cultivate.30 ℃ of culture temperature, rotating speed of agitator 400rmin
-1, air flow is 3.5Lmin
-1, fermentation tank culture medium is composed as follows: initial pH value is 5.
| Chitosan | 20g/L |
| Urea | 2g/L |
| Potassium primary phosphate | 0.8g/L |
| Ammonium sulfate | 2g/L |
| Sal epsom | 0.2g/L |
After the fermentor cultivation 70 hours, in refrigerator, preserved fermented liquid 2 hours down for 4 ℃, then at 6000rmin
-1Get supernatant liquor after centrifugal as crude enzyme liquid, standby.
2, the separation and purification of chitin incision enzyme
(1) concentrating of chitosan crude enzyme liquid: get above-mentioned chitoanase crude enzyme liquid, adopt hyperfiltration process to concentrate ultra-filtration membrane area 0.1m
2, holding back relative molecular mass 10kDa, working pressure 0.4MPa holds back part and is spissated crude enzyme liquid.
(2) extraction of chitoanase: get the above-mentioned crude enzyme liquid of 100ml, under 4 ℃ of conditions, slowly add ammonium sulfate and make it reach 20% saturation ratio, 6000rmin
-1The centrifugal precipitation of removing continues to add ammonium sulfate and makes its saturation ratio reach 80%, 6000rmin
-1The centrifugal collecting precipitation part is with acetate buffer (pH4.8) dissolving of 20ml 20mM.Use desalting column to carry out desalting treatment, standby.
(3) roughing out of chitoanase: adopt cationic exchange coloum HiPrep 16/10 SP XL, level pad is 20mM acetate buffer (pH4.8), elution buffer is the 20mmol acetate buffer that contains 1mol sodium-chlor, 0-100% linear gradient wash-out, and substep is collected.This process can with the same excision enzyme of chitin incision enzyme, impurity protein separately be collected chitin incision enzyme solution.
(4) chitin incision enzyme is refining: according to the difference of chitin incision enzyme and impurity albumen relative molecular mass, adopt gel-filtration column Sephadex G50, damping fluid is 20mmol acetate buffer (pH4.8), the refining chitin incision enzyme solution that obtains, than vigor 432U/mg, obtain the chitin incision enzyme product after the vacuum freezedrying.
Embodiment 3:
1, the preparation of crude enzyme liquid
Get 10 of test tube slant preservation bacterial classifications, activate 5 hours after, add sterilized water 15ml respectively, wash spore.The spore suspension that obtains directly inserted in the 5L fermentation tank culture medium cultivate.28 ℃ of culture temperature, rotating speed of agitator 500rmin
-1, air flow is 5Lmin
-1, fermentation tank culture medium is composed as follows: initial pH value is 4.5.
| Chitosan | 25g/L |
| Urea | 0.8g/L |
| Potassium primary phosphate | 0.8g/L |
| Ammonium sulfate | 1.5g/L |
| Sal epsom | 0.3g/L |
2, the separation and purification of chitin incision enzyme
(1) concentrating of chitosan crude enzyme liquid: get above-mentioned chitoanase crude enzyme liquid, adopt hyperfiltration process to concentrate ultra-filtration membrane area 0.1m
2, holding back relative molecular mass 5kDa, working pressure 0.4MPa holds back part and is spissated crude enzyme liquid.
(2) extraction of chitoanase: get the above-mentioned crude enzyme liquid of 100ml, under 4 ℃ of conditions, slowly add ammonium sulfate and make it reach 20% saturation ratio, 6000rmin
-1The centrifugal precipitation of removing continues to add ammonium sulfate and makes its saturation ratio reach 80%, 6000rmin
-1The centrifugal collecting precipitation part is with acetate buffer (pH5.0) dissolving of 20ml 10mmol.Use the dialysis band that the acetate buffer of 10mmol was dialysed 20 hours down at 4 ℃.
(3) roughing out of chitoanase: adopt cationic exchange coloum HiPrep 16/10 SP XL, level pad is 50mmol acetate buffer (pH5.0), elution buffer is the 50mmol acetate buffer that contains 1mol sodium-chlor, 0-100% linear gradient wash-out, and substep is collected.This process can with the same excision enzyme of chitin incision enzyme, impurity protein separately be collected chitin incision enzyme solution.
(4) chitin incision enzyme is refining: according to the difference of chitin incision enzyme and impurity albumen relative molecular mass, adopt gel-filtration column Sephadex G-50, damping fluid is 50mmol acetate buffer (pH5.0), the refining chitin incision enzyme solution that obtains, than vigor 453U/mg, obtain the chitin incision enzyme product after the vacuum freezedrying.
Embodiment 4:
1, the preparation of crude enzyme liquid
Get 15 of test tube slant preservation bacterial classifications, activate 5 hours after, add sterilized water 15ml respectively, wash spore.The spore suspension that obtains directly inserted in the 5L fermentation tank culture medium cultivate.32 ℃ of culture temperature, rotating speed of agitator 250rmin
-1, air flow is 5Lmin
-1, fermentation tank culture medium is composed as follows: initial pH value is 4.5.
| Chitosan | 35g/L |
| Urea | 0.8g/L |
| Potassium primary phosphate | 0.8g/L |
| Ammonium sulfate | 1g/L |
| Sal epsom | 0.4g/L |
2, the separation and purification of chitin incision enzyme
(1) concentrating of chitosan crude enzyme liquid: get above-mentioned chitoanase crude enzyme liquid, adopt hyperfiltration process to concentrate ultra-filtration membrane area 0.1m
2, holding back relative molecular mass 20kDa, working pressure 0.4MPa holds back part and is spissated crude enzyme liquid.
(2) extraction of chitoanase: get the above-mentioned crude enzyme liquid of 100ml, under 4 ℃ of conditions, slowly add ammonium sulfate and make it reach 20% saturation ratio, 6000rmin
-1The centrifugal precipitation of removing continues to add ammonium sulfate and makes its saturation ratio reach 80%, 6000rmin
-1The centrifugal collecting precipitation part is with acetate buffer (pH5.0) dissolving of 20ml 20mM.Use desalting column to carry out desalting treatment, standby.
(3) roughing out of chitoanase: adopt cationic exchange coloum HiPrep 16/10 SP XL, level pad is 20mM acetate buffer (pH5.0), elution buffer is the 20mmol acetate buffer that contains 1mol sodium-chlor, 0-100% linear gradient wash-out, and substep is collected.This process can with the same excision enzyme of chitin incision enzyme, impurity protein separately be collected chitin incision enzyme solution.
(4) chitin incision enzyme is refining: according to the difference of chitin incision enzyme and impurity albumen relative molecular mass, adopt gel-filtration column Sephadex G50, damping fluid is 20mmol acetate buffer (pH5.0), the refining chitin incision enzyme solution that obtains, than vigor 496U/mg, obtain the chitin incision enzyme product after the vacuum freezedrying.
Claims (3)
1, a kind of preparation method of chitin incision enzyme is characterized in that the step of method is as follows:
1) with preserving number be: the high yield chitosan enzyme-producing bacteria (Penicillium sp) of CGMCC No.0851 is seeded in the fermentor tank of 5L, substratum liquid amount 3-4L, and inoculum size is 10%-20%, culture temperature is that 26-32 ℃, stirring velocity are 200-450rmin
-1, air flow is 1.5-5Lmin
-1, ferment after 50-80 hour, remove by filter the suspension thalline and obtain the chitosan crude enzyme liquid;
2) holding back relative molecular mass is that the ultra-filtration membrane of 2-20kDa carries out ultrafiltration to crude enzyme liquid, then, with 2-4 doubly the ice ethanol sedimentation or obtain the thick enzyme throw out of chitosan with the 20%-80% ammonium sulfate precipitation;
3) use pH4.4-5.5, the thick enzyme throw out of 10-50mmol acetate buffer dissolving chitosan is gone up HiPrep 16/10 SP XL cationic exchange coloum, again with containing 0.5-2mol sodium-chlor, 10-50mmol acetate buffer, 0-100% linear gradient wash-out obtains chitin incision enzyme solution;
4) filter with Sephadex G 50 gel-filtration columns, the acetate buffer wash-out, concentrated, freezing, vacuum-drying obtains chitin incision enzyme.
2, according to the preparation method of the described a kind of chitin incision enzyme of claim 1,, it is characterized in that described substratum is: chitosan 5-40g/L, urea 0.5-3g/L, potassium primary phosphate 0.2-1g/L; Ammonium sulfate 0.5-3g/L, sal epsom 0.1-0.5g/L.
3, according to the preparation method of the described a kind of chitin incision enzyme of claim 1, it is characterized in that described high yield chitosan enzyme-producing bacteria (Penicillium sp) inoculation is to use the direct top fermentation jar of bacterial classification of slant culture to cultivate, the seed liquor preparation method of inoculation adopts the 100-300ml sterilized water to wash 5-15 to prop up the fungal spore that cultivation obtains in the 25ml inclined-plane, and direct inoculation is in fermentor tank.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100500770A CN1300311C (en) | 2005-06-14 | 2005-06-14 | Preparation method of chitin incision enzyme |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100500770A CN1300311C (en) | 2005-06-14 | 2005-06-14 | Preparation method of chitin incision enzyme |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1724659A true CN1724659A (en) | 2006-01-25 |
| CN1300311C CN1300311C (en) | 2007-02-14 |
Family
ID=35924289
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2005100500770A Expired - Fee Related CN1300311C (en) | 2005-06-14 | 2005-06-14 | Preparation method of chitin incision enzyme |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1300311C (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100584955C (en) * | 2006-12-27 | 2010-01-27 | 江西师范大学 | Chitosan oligosaccharide/chito-oligomer single enzyme production process |
| CN101870965A (en) * | 2010-06-01 | 2010-10-27 | 青岛康地恩生物科技有限公司 | Production method of high enzymic activity instant enzyme preparation |
| CN101845424B (en) * | 2010-02-20 | 2013-01-16 | 中诺生物科技发展江苏有限公司 | Method for preparing beta-D-galactoside galactose hydrolase preparation |
| CN103409395A (en) * | 2013-08-08 | 2013-11-27 | 上海海洋大学 | Method for fermenting microbe to prepare endo-chitosanase |
Family Cites Families (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH03280877A (en) * | 1990-03-29 | 1991-12-11 | Asahi Seibutsu Kogaku Kenkyusho:Kk | Production of saccharification-type chitosan liase |
| CN1425760A (en) * | 2001-12-12 | 2003-06-25 | 江晓路 | Fungus and method for producing chitosanase with it |
| CN1463989A (en) * | 2002-06-20 | 2003-12-31 | 中国科学院理化技术研究所 | Process for preparing low molecular weight chitosan using A.niger cellulase |
| CN1397636A (en) * | 2002-07-11 | 2003-02-19 | 江晓路 | Fungus and process for preparing chitosanase fram it |
| CN1187438C (en) * | 2002-12-24 | 2005-02-02 | 浙江大学 | Fungus strain for high yield chitinase and use thereof |
| CN1246450C (en) * | 2003-06-21 | 2006-03-22 | 北海国发海洋生物产业股份有限公司 | Process for producing chitosan enzyme producing fungus and chitosan oligomer |
-
2005
- 2005-06-14 CN CNB2005100500770A patent/CN1300311C/en not_active Expired - Fee Related
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100584955C (en) * | 2006-12-27 | 2010-01-27 | 江西师范大学 | Chitosan oligosaccharide/chito-oligomer single enzyme production process |
| CN101845424B (en) * | 2010-02-20 | 2013-01-16 | 中诺生物科技发展江苏有限公司 | Method for preparing beta-D-galactoside galactose hydrolase preparation |
| CN101870965A (en) * | 2010-06-01 | 2010-10-27 | 青岛康地恩生物科技有限公司 | Production method of high enzymic activity instant enzyme preparation |
| CN103409395A (en) * | 2013-08-08 | 2013-11-27 | 上海海洋大学 | Method for fermenting microbe to prepare endo-chitosanase |
| CN103409395B (en) * | 2013-08-08 | 2015-05-27 | 上海海洋大学 | Method for fermenting microbe to prepare endo-chitosanase |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1300311C (en) | 2007-02-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN100567500C (en) | A kind of method of from produce the Polysialic acid escherichia coli fermented broth, extracting Polysialic acid | |
| CN109251954B (en) | Production method of sea cucumber polypeptide | |
| CN104450561B (en) | One plant of application produced chitinase bacterial strain and its chitinase is produced using crab shell fermentation | |
| JP2711095B2 (en) | Production method of growth promoter of bifidobacterium | |
| CN116200433A (en) | Method for preparing ergothioneine by biosynthesis of Cordyceps fungus | |
| CN107686854B (en) | Method for degrading and modifying schizophyllan by utilizing endoprotease produced by schizophyllum commune fermentation system | |
| CN1300311C (en) | Preparation method of chitin incision enzyme | |
| CN108251476B (en) | Method for extracting vitamin B12 from enzyme preparation wastewater | |
| CN102220244B (en) | Hypocrea lixii strain and method for preparing dextran enzyme with the Hypocrea lixii strain | |
| CN111607622B (en) | Process method for producing 3-hydroxy butanone by using wheat B starch | |
| CN109504725B (en) | Method for preparing high-purity hericium erinaceus polysaccharide by fermenting hericium erinaceus and fermentation culture medium | |
| CN1515592A (en) | Process for preparing low molecular weight fucidan by using microbial enzyme method | |
| CN104774794A (en) | Strain capable of producing D-mannose isomerase and method for producing D-mannose isomerase by using same | |
| CN109456898A (en) | A kind of the fermentation preparation and its application of chaetomium globosum dextranase | |
| CN111100826B (en) | Lexella capable of producing alginate lyase and application thereof | |
| CN112094762B (en) | Corynebacteria vinifera strain and application thereof | |
| CN108531418A (en) | A kind of preparation method of chitosan enzyme | |
| CN105087427B (en) | Produce Vibrio natriegen and its application of agarase | |
| CN112481330A (en) | Fermentation production method of algae-derived beta-1, 3-glucan | |
| CN1458278A (en) | Method for producing yeast extracellular trehalose by two step fermentation method | |
| CN1187438C (en) | Fungus strain for high yield chitinase and use thereof | |
| CN112358985A (en) | Pradazobium and application thereof in preparation of water-soluble beta-1, 3 glucan | |
| CN1230553C (en) | Anti-tumour ginseng saponin preparing method | |
| CN1259427C (en) | Method for preparing grape manna oligosaccharide | |
| CN110257452A (en) | A method of the separating-purifying brown alga oligose monomer from enzymolysis liquid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C19 | Lapse of patent right due to non-payment of the annual fee | ||
| CF01 | Termination of patent right due to non-payment of annual fee |