CN101367018A - Process for purifying trypsin inhibitor in soya whey wastewater with chitosan resin immobilized enzyme - Google Patents
Process for purifying trypsin inhibitor in soya whey wastewater with chitosan resin immobilized enzyme Download PDFInfo
- Publication number
- CN101367018A CN101367018A CNA2007101539033A CN200710153903A CN101367018A CN 101367018 A CN101367018 A CN 101367018A CN A2007101539033 A CNA2007101539033 A CN A2007101539033A CN 200710153903 A CN200710153903 A CN 200710153903A CN 101367018 A CN101367018 A CN 101367018A
- Authority
- CN
- China
- Prior art keywords
- trypsin inhibitor
- resin
- chitosan
- immobilized enzyme
- concentration
- 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.)
- Pending
Links
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
Abstract
The invention presents a research of a technology of Chitosan resin immobilized enzyme purifying trypsin inhibitor in soybean whey wastewater, and belongs to the fields of light industry, food and environment engineering. Soybean whey is a byproduct produced during the production of soy protein by adopting the technologies of alkaline solubility and acid-precipitation, containing a great deal of functional ingredients like trypsin inhibitor. According to statistics, domestic soybean protein isolation and production enterprises discharge soybean whey waste liquid over three million tons every year. At present, a more mature research in extracting active substance from soybean whey waste liquid is the extraction of soybean oligosaccharides and the like, but the isolation and purification of trypsin inhibitor has not yet been given enough attention to. In the technology, the prepared porous, three-dimensional mesh structure of Chitosan microspheres resin, after being activated and coupled with trypsin, is used to affinitively adsorb the trypsin inhibitor in soybean whey waste liquid and is eluted to obtain electrophoresis pure trypsin inhibitor. The method can use the waste after extraction of soy protein in high value, and is simple to operate, low in cost and easy for industrialized promotion and application.
Description
Technical field the present invention utilizes the chitosan resin immobilizing trypsinase of preparation, purifying soybean trypsin inhibitor from soya whey wastewater.This technology utilization extracts the whey wastewater after the soybean protein, can get the pure trypsin inhibitor of electrophoresis, has wide application prospect in fields such as soya whey wastewater higher value application and environmental projects.
The background technology soybean is the grain resource of China's abundant, and soy-bean whey is the accessory substance of the heavy explained hereafter soybean protein isolate of the molten acid of industrial employing alkali, wherein contains functional components such as a large amount of trypsin inhibitors, compound sugar, isoflavones, vitamin.But at present China's relevant enterprise is not generally all taked corresponding improvement, recovery measure to soybean whey liquid, and with it as wastewater treatment.According to statistics, the discarded liquid of soy-bean whey of the annual discharging of domestic soybean protein isolate manufacturing enterprise is more than 3,000,000 tons.This has not only wasted a large amount of useful resources, has also caused environmental pollution.Therefore, the discarded liquid of soy-bean whey is carried out recycling treatment, recovery functional materials wherein becomes a hot issue of China's research in recent years and engineering.At present, what the research of extraction bioactivator was comparatively ripe from the discarded liquid of soy-bean whey is the extraction of soyabean oligosaccharides, isoflavones and soyasaponins, and relevant patent documentation is also arranged.And be not subjected to abundant attention as yet for the separation and purification of wherein soybean trypsin inhibitor.
Trypsin inhibitor has different physiological roles, has a wide range of applications at biological pesticide and biomedicine field.And the separation and purification of trypsin inhibitor only takes on laboratory level that water logging, acid are carried, saltoutd, comparatively numerous and diverse method purifying such as ion-exchange chromatography or gel permeation chromatography makes, and length consuming time, productive rate and purity are not high yet; With the preparation electrophoresis method, though can obtain the high-purity trypsin inhibitor, productive rate is extremely low, spends higher.Shitosan is abundant at occurring in nature content, and safety non-toxic carries out chemical modification easily; To have rigidity big for the chitosan microball resin of preparation, inner porous, is space network, and specific area is big, and the characteristics of good hydrophilic property can satisfy the needs of various absorption and mask work.Therefore, adopt nontoxic epoxychloropropane, on the chitosan microball resin, introduce epoxy radicals as activator, so coupling trypsase, be applied to the separation and purification of trypsin inhibitor in the soya whey wastewater, have very wide prospect in industrial application.
Summary of the invention the present invention is the porous of utilizing preparation, the chitosan microball resin immobilizing trypsinase of space network, provide a kind of from soya whey wastewater the technical process of purifying soybean trypsin inhibitor.
Technological process is as follows: shitosan is dissolved in the acetum, the control temperature, add atoleine, class of department, ethyl acetate, formaldehyde, glutaraldehyde respectively, regulate pH, reacted 3～4 hours, and added sodium borohydride solution again, continue reaction after 1～1.5 hour, decompress filter, washing, drying promptly get the chitosan microball resin.Chitosan resin is placed NaOH solution, is activator with the epoxychloropropane, activated resin under the uniform temperature.Activation back resin adds trypsase and carries out coupling, and decompress filter, vacuum drying promptly get the chitosan resin immobilizing trypsinase.With immobilised enzymes resin dress post, cross post with the discarded liquid of whey, wash-out is collected the active part freeze drying, obtains the pure soybean trypsin inhibitor of electrophoresis.
The specific embodiment will be dissolved in respect to the shitosan powder of acetum 3.0～7.0% in 2.0～7.0% the acetum, constantly stir the 0.5～5.5ml of class of adding department down in 20～50 ℃, add ethyl acetate 15～55ml, react 20～50min; Regulate 50～80 ℃ of temperature, add formaldehyde 15～55ml, reaction 30～50min; Regulate 85～95 ℃ of temperature, adding glutaraldehyde 10～50ml and regulating the pH value is 7.0～10.0, continues reaction 3～5 hours.Dropwise add sodium borohydride solution 10～100ml of 0.5～8.5%, reacted 1～5 hour, decompress filter, respectively with benzinum, absolute ethanol washing, vacuum drying promptly gets the chitosan microball resin.Get chitosan resin, add 1.0～8.0mol/L NaOH solution, make the epoxychloropropane concentration of adding reach 5～40%, 40～90 ℃ of constant temperature vibration activated resin 2～10 hours, add trypsase after filtering the removal epoxychloropropane, 10～40 ℃ of couplings 10～24 hours, decompression filters, and is drying to obtain the chitosan resin immobilizing trypsinase.With the immobilised enzymes chromatographic column of packing into, cross post with sample on the discarded liquid of soy-bean whey, gradient elution is collected active peak, and freeze drying promptly gets trypsin inhibitor.
Example 1: get shitosan powder 10.0g and be dissolved in 2.0% the acetum, 30 ℃ are constantly stirred the 80 solution 1.5ml of class of adding department down, add ethyl acetate 15ml, react 20min; Adjust the temperature to 70 ℃, add formaldehyde 25ml, reaction 30min; Regulating temperature is 95 ℃, and adding glutaraldehyde 10ml and regulating the pH value is 9.0, reacts 3 hours.Dropwise add 1.5% sodium borohydride solution 100ml, reacted 1.5 hours, decompress filter, respectively with benzinum, absolute ethanol washing, vacuum drying promptly gets the chitosan microball resin.Get the 10.0g chitosan resin, the NaOH solution that adds 2.0ml epoxychloropropane and 6.0mol/L 30ml, 40 ℃ of constant temperature vibration activated resin 2 hours, add trypsase after filtering the removal epoxychloropropane, 30 ℃ of couplings 24 hours, decompression filters, and is drying to obtain the chitosan resin immobilizing trypsinase.With the immobilised enzymes 16mm * 20cm chromatographic column of packing into, cross post with sample on the discarded liquid of soy-bean whey, the pH gradient elution is collected active peak, and freeze drying promptly gets trypsin inhibitor.
Example 2: get shitosan powder 8.0 and be dissolved in 1.0% the acetum, 50 ℃ are constantly stirred the 60 solution 5.0ml of class of adding department down, add ethyl acetate 55ml, react 20min; Regulate 80 ℃ of temperature, add formaldehyde 15ml, reaction 30min; Regulate 85 ℃ of temperature, adding glutaraldehyde 50ml and regulating the pH value is 8.0, continues reaction 5 hours.Dropwise add 8.0% sodium borohydride solution 80ml, reacted 5 hours, decompress filter respectively with benzinum, acetone, water washing, is drying to obtain the chitosan microball resin.Get the 8.0g chitosan resin, the NaOH solution that adds 12.0ml epoxychloropropane and 1.0mol/L20ml, 80 ℃ of constant temperature vibration activated resin 10 hours, add trypsase after filtering the removal epoxychloropropane, 40 ℃ of couplings 24 hours, decompression filters, and is drying to obtain the chitosan resin immobilizing trypsinase.With the immobilised enzymes 45mm * 20cm chromatographic column of packing into, cross post with sample on the discarded liquid of soy-bean whey, the ionic strength gradient elution is collected active peak, and freeze drying promptly gets trypsin inhibitor.
Example 3: get shitosan 15.0g and be dissolved in 7.0% the acetum, 20 ℃ are constantly stirred the 40 solution 0.5ml of class of adding department down, add ethyl acetate 45ml, react 50min; Regulate 50 ℃ of temperature, add formaldehyde 45ml, reaction 50min; Regulate 90 ℃ of temperature, adding glutaraldehyde 35ml and regulating the pH value is 7.0, continues reaction 5 hours, dropwise adds 4.0% sodium borohydride solution 80ml, reacts 2 hours, and decompress filter with benzinum, distilled water washing, is drying to obtain the chitosan microball resin respectively.Get the 15.0g chitosan resin, the NaOH solution that adds 7.0ml epoxychloropropane and 7.0mol/L 60ml, 55 ℃ of constant temperature vibration activated resin 7 hours, add trypsase after filtering the removal epoxychloropropane, 25 ℃ of couplings 18 hours, decompression filters, and is drying to obtain the chitosan resin immobilizing trypsinase.With the immobilised enzymes 550mm * 40cm chromatographic column of packing into, cross post with sample on the discarded liquid of soy-bean whey, the pH gradient elution is collected active peak, and freeze drying promptly gets trypsin inhibitor.
Claims (5)
1. the feature process flow process of trypsin inhibitor is as follows in the chitosan resin immobilized enzyme purifying soya whey wastewater: shitosan is dissolved in the acetum, the control temperature, add atoleine, class of department, ethyl acetate, formaldehyde, glutaraldehyde respectively, regulate pH, reacted 3～4 hours, add sodium borohydride solution again, continue reaction 1～1.5 hour, decompress filter, washing, vacuum drying promptly gets the chitosan microball resin.Chitosan resin is placed NaOH solution, and the adding epoxychloropropane is an activator, activated resin; Activated resin adds trypsase and carries out coupling, and decompress filter, vacuum drying promptly get the chitosan resin immobilizing trypsinase.With immobilised enzymes resin dress post, cross post with the discarded liquid of whey, wash-out is collected the active part freeze drying, obtains the pure soybean trypsin inhibitor of electrophoresis.
2. in the technological process of trypsin inhibitor, it is 0.2～2.0% that class of adding department makes its concentration in the chitosan resin immobilized enzyme purifying soya whey wastewater according to claim 1, and the concentration of ethyl acetate is 5.0～15.0%.
3. in the technological process of trypsin inhibitor, it is 5～15% that adding formalin makes its concentration in the chitosan resin immobilized enzyme purifying soya whey wastewater according to claim 1, and the concentration of glutaraldehyde is 4.0～14.0%.
4. in the chitosan resin immobilized enzyme purifying soya whey wastewater according to claim 1 in the technological process of trypsin inhibitor, add 0.5～8.5% sodium borohydride solution, and to make its concentration be 2.0～20.0%.
5. in the technological process of trypsin inhibitor, add epoxychloropropane during activated resin and make its concentration reach 5.0～40.0% in the chitosan resin immobilized enzyme purifying soya whey wastewater according to claim 1.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007101539033A CN101367018A (en) | 2007-09-13 | 2007-09-13 | Process for purifying trypsin inhibitor in soya whey wastewater with chitosan resin immobilized enzyme |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNA2007101539033A CN101367018A (en) | 2007-09-13 | 2007-09-13 | Process for purifying trypsin inhibitor in soya whey wastewater with chitosan resin immobilized enzyme |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN101367018A true CN101367018A (en) | 2009-02-18 |
Family
ID=40411103
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNA2007101539033A Pending CN101367018A (en) | 2007-09-13 | 2007-09-13 | Process for purifying trypsin inhibitor in soya whey wastewater with chitosan resin immobilized enzyme |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN101367018A (en) |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102019213A (en) * | 2010-11-30 | 2011-04-20 | 浙江工业大学 | Method for preparing strongly-acid ion exchange medium |
| CN102770147A (en) * | 2009-12-30 | 2012-11-07 | 索莱有限责任公司 | Purified kunitz trypsin inhibitor proteins isolated from a soy processing stream |
| CN103965352A (en) * | 2014-05-09 | 2014-08-06 | 江南大学 | Method for purifying Kunitz-type trypsin inhibitor from soybean whey waste water |
| CN104761672A (en) * | 2015-04-03 | 2015-07-08 | 浙江科技学院 | Resin for enriching enzyme inhibitor as well as preparation method and application of resin |
| CN107252681A (en) * | 2017-06-29 | 2017-10-17 | 吉林省爱诺德生物工程有限公司 | A kind of aflatoxins B1The preparation method of immune affinity column |
| CN107271663A (en) * | 2017-06-29 | 2017-10-20 | 吉林省爱诺德生物工程有限公司 | A kind of preparation method of Ochratoxin A immune affinity column |
| CN110586331A (en) * | 2019-09-28 | 2019-12-20 | 北京矿冶科技集团有限公司 | Modified chitosan inhibitor and flotation separation method thereof |
-
2007
- 2007-09-13 CN CNA2007101539033A patent/CN101367018A/en active Pending
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102770147A (en) * | 2009-12-30 | 2012-11-07 | 索莱有限责任公司 | Purified kunitz trypsin inhibitor proteins isolated from a soy processing stream |
| CN102019213A (en) * | 2010-11-30 | 2011-04-20 | 浙江工业大学 | Method for preparing strongly-acid ion exchange medium |
| CN102019213B (en) * | 2010-11-30 | 2013-03-27 | 浙江工业大学 | Method for preparing strongly-acid ion exchange medium |
| CN103965352A (en) * | 2014-05-09 | 2014-08-06 | 江南大学 | Method for purifying Kunitz-type trypsin inhibitor from soybean whey waste water |
| CN104761672A (en) * | 2015-04-03 | 2015-07-08 | 浙江科技学院 | Resin for enriching enzyme inhibitor as well as preparation method and application of resin |
| CN107252681A (en) * | 2017-06-29 | 2017-10-17 | 吉林省爱诺德生物工程有限公司 | A kind of aflatoxins B1The preparation method of immune affinity column |
| CN107271663A (en) * | 2017-06-29 | 2017-10-20 | 吉林省爱诺德生物工程有限公司 | A kind of preparation method of Ochratoxin A immune affinity column |
| CN107271663B (en) * | 2017-06-29 | 2019-03-15 | 吉林省爱诺德生物工程有限公司 | A kind of preparation method of Ochratoxin A immune affinity column |
| CN107252681B (en) * | 2017-06-29 | 2019-10-18 | 吉林省爱诺德生物工程有限公司 | A kind of aflatoxins B1The preparation method of immune affinity column |
| CN110586331A (en) * | 2019-09-28 | 2019-12-20 | 北京矿冶科技集团有限公司 | Modified chitosan inhibitor and flotation separation method thereof |
| CN110586331B (en) * | 2019-09-28 | 2021-05-25 | 北京矿冶科技集团有限公司 | Modified chitosan inhibitor and flotation separation method thereof |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN101367018A (en) | Process for purifying trypsin inhibitor in soya whey wastewater with chitosan resin immobilized enzyme | |
| CN101899484B (en) | Preparation method of genipin | |
| CN104087646B (en) | Method for preparing glycyrrhetinic acid | |
| CN106381319A (en) | High-efficiency extraction and separation method of grape seed proanthocyanidin oligomer | |
| CN103288932B (en) | Method for separating and extracting pediocin PA-1 from fermentation solution | |
| CN103266154A (en) | Biological transformation method for preparing high-activity theasaponin | |
| CN106732375A (en) | A kind of preparation method and applications of clay mineral modification biological charcoal | |
| Cheng et al. | Development of immobilized cellulase through functionalized gold nano-particles for glucose production by continuous hydrolysis of waste bamboo chopsticks | |
| CN103464117A (en) | Preparation method of ethanediamine based porous dextrangel adsorbent | |
| CN110882679A (en) | Composite material for treating sewage and preparation method thereof | |
| CN103709235A (en) | Method for reducing solvent use amount and extracting high-purity enramycin | |
| CN107287183A (en) | A kind of preparation method using graphene/titanium oxide composite porous microspheres as the immobilization alpha amylase of carrier | |
| CN110051705A (en) | High efficiency extraction and the method for purifying Inonotus obliquus polyphenol | |
| CN107523558B (en) | Embedding and curing method of D-pantolactone hydrolase | |
| CN102492731B (en) | Method for preparing resveratrol by utilizing immobilized enzyme to continuously hydrolyze polydatin | |
| CN111302518A (en) | Method for recycling culture wastewater containing antibiotics by combining temperature difference concentration difference dual-driven membrane distillation and high-performance adsorbent | |
| CN101024850A (en) | Chitosan oligosaccharide/chito-oligomer single enzymatic production process | |
| CN100513561C (en) | Thrombin extracting and separating process | |
| CN101544968B (en) | Industrial production method of earthworm fibrinolytic enzyme directly extracted by utilizing ion exchange resin | |
| CN103933943A (en) | Preparation and application of 2-mercaptobenzothiazole modified cotton stalk cortex adsorbent | |
| CN101824059A (en) | Low-sugar-chain high-activity new tea saponin and biotransformation method thereof | |
| CN1224703C (en) | Process for preparing magnetic microsphere immobilized cellulase | |
| CN1127507C (en) | Process for extracting natural flavonoid and/or terpene lactones from plant by adsorbent | |
| CN101259405B (en) | Preparation and application of nylon affinity membrane with reactive blue 4 as ligand | |
| CN101792476A (en) | Method for extracting and separating fusidic acid |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Open date: 20090218 |