CN1109746C - Immobilized enzyme carrier - Google Patents
Immobilized enzyme carrier Download PDFInfo
- Publication number
- CN1109746C CN1109746C CN98121661A CN98121661A CN1109746C CN 1109746 C CN1109746 C CN 1109746C CN 98121661 A CN98121661 A CN 98121661A CN 98121661 A CN98121661 A CN 98121661A CN 1109746 C CN1109746 C CN 1109746C
- Authority
- CN
- China
- Prior art keywords
- polysuccinimide
- silica gel
- aminopropyl
- enzyme
- immobilized
- 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.)
- Expired - Fee Related
Links
Images
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The present invention relates to an immobilized enzyme carrier composed of an inorganic material carrier and an organic material carrier. The general chemical formula is disclosed in the specification. The immobilized enzyme carrier comprises polysuccinimide and 3-aminopropyl silica gel or 3-aminopropyl glass microspheres, which are connected with each other by a covalent bond. The preparation method of the immobilized enzyme carrier comprises: the polysuccinimide is dissolved in N, N'-dimethylformamide, the 3-aminopropyl silica gel or the 3-aminopropyl glass microspheres are added, and materials are charged according to the ratio that the amino mole number of the 3-aminopropyl silica gel or the amino mole number of the 3-aminopropyl glass microspheres to the mole number of succinimide structural units in the polysuccinimide is 1: (5 to 8); then, a product is collected from the reaction mixture. The present invention has the advantages of high practicability, etc. The present invention is favorable for industrialized production.
Description
Technical field
The present invention is a kind of fixed enzyme vector.
Background technology
At present, fixed enzyme vector mainly contains inorganic materials carrier and organic materials carrier two big classes, and the former has silica gel, activated alumina etc., and latter's majority is the polymkeric substance of synthetic.
The inorganic materials carrier has good mechanical property, the abundant and low cost and other advantages in source, but enzyme is fixed on the carrier by adsorption, owing to this Intermolecular Forces a little less than, enzyme runs off easily, has shortened the duration of service of immobilized enzyme.So, publication such as the clear 63-252544 of Japanese Patent and European patent 0109300 document and Liu Lijian is in " ion-exchange and absorption " [11 (6): 541,1995] in " activation of porous silicon ball and the silanization " article on the periodical, disclose and a kind of 3-aminopropyl silica gel or glass microsphere have been fixed enzyme by double-functional group linking agent or condensing agent, made the technology that forms covalent linkage between enzyme and the carrier.Though this can improve the stability of immobilized enzyme, but because of in immobilized reactant, using linking agent or condensing agent, the higher structure of enzyme is changed, reduced the active of enzyme or made enzyme deactivation, cause in the immobilized reactant, remaining enzyme activity is little in the reaction solution, thereby is difficult to utilize again, and this is unfavorable for suitability for industrialized production.
The shortcoming of organic materials carrier is that its mechanical property is relatively poor, and proportion is little, and compressibility is arranged.Thereby, the resistance bigger when being used on some reactor to the mobile generation of liquid, and cost is than inorganic materials height.But because this carrier can make enzyme keep higher activity, the long service life of immobilized enzyme is so the immobilized enzyme that is used for suitability for industrialized production now mostly adopts polymkeric substance to make carrier.
Summary of the invention
Technical problem to be solved by this invention is: overcome the defective of prior art, a kind of fixed enzyme vector of the advantage that integrates inorganic materials carrier and organic materials carrier is provided, be beneficial to immobilized enzyme and be used for suitability for industrialized production.
The technical solution adopted for the present invention to solve the technical problems is: comprise polysuccinimide and 3-aminopropyl silica gel or 3-aminopropyl glass microsphere, the two links to each other with covalent linkage, constitutes compound fixed enzyme vector.
Above-mentioned fixed enzyme vector is made by following method: polysuccinimide is dissolved in N, in N '-dimethyl formamide, add 3-aminopropyl silica gel or 3-aminopropyl glass microsphere, ratio 1: 5～8 by the mole number of the succimide structural unit in its amino mole number and the polysuccinimide feeds intake, and collects resultant then from reaction mixture.
Fixed enzyme vector of the present invention (hereinafter to be referred as novel vector) has been compared following major advantage with existing carrier:
One: the immobilized enzyme with novel vector makes can keep higher vigor.
Its two: in the process with the novel vector immobilized enzyme, the enzyme loss is little.Because, in immobilized reactant, enzyme directly and the active structure unit process on the novel vector, remaining enzyme can recycle in the reaction solution, has avoided this trivial step of pre-activation of carrier simultaneously.
Its three: polysuccinimide can recycling use.Because it is at N, (hereinafter to be referred as DMF) is very stable in N '-dimethyl formamide, and the solubleness in water is very little, its DMF solution is poured in the water just can be reclaimed, and be easy and simple to handle.
Its four: novel vector is easy to prepare, the low and good mechanical property of cost.
In a word, novel vector is practical, is beneficial to suitability for industrialized production, is a kind of carrier that integrates the advantage of inorganic and organic materials carrier.
Description of drawings
Fig. 1 is the synoptic diagram of preparation novel vector of the present invention.
Fig. 2 is the synoptic diagram with Fig. 1 novel vector immobilized enzyme.
Fig. 3 is a temperature of reaction and the synoptic diagram that concerns of lipase and the relative vigor of immobilized lipase.
Fig. 4 be buffered soln pH value and lipase and immobilized lipase relative vigor concern synoptic diagram.
Fig. 5 is the synoptic diagram that concerns of immobilized lipase time of storing in phosphate buffer solution and its vigor.
Embodiment
The invention will be further described below in conjunction with embodiment and accompanying drawing.
Carrier of the present invention is a composite carrier, is made of organic and inorganic materials carrier, comprises polysuccinimide and 3-aminopropyl silica gel or 3-aminopropyl glass microsphere, and the two links to each other with covalent linkage.Its preparation method is: earlier polysuccinimide is dissolved in N, in N '-dimethyl formamide, add 3-aminopropyl silica gel or 3-aminopropyl glass microsphere again, ratio (hereinafter to be referred as the ratio of mole number) 1: 5～8 by the mole number of the succimide structural unit in mole number amino in 3-aminopropyl silica gel or the 3-aminopropyl glass microsphere and the polysuccinimide feeds intake, from reaction mixture, collect resultant then, this resultant is a fixed enzyme vector of the present invention, it is polysuccinimide-silica gel or polysuccinimide-glass microsphere (see figure 1) that the applicant names it, is called for short novel vector.Sequence number 1,2,3 is respectively 3-aminopropyl silica gel or 3-aminopropyl glass microsphere, polysuccinimide, novel vector among Fig. 1.
One, novel vector prepares embodiment:
Embodiment one
10.8 the gram polysuccinimide is dissolved among the 55mL DMF, adds 15 gram 3-aminopropyl silica gel, the ratio of mole number 1: 5.At room temperature with its stir about 8 hours, make it fully reaction after, soak through suction filtration with DMF again, remove unreacted polysuccinimide by washing procedure.Then, it is inferior to give a baby a bath on the third day after its birth with ether, removes DMF.Last drying (drying naturally), getting novel vector is polysuccinimide-silica gel solid phase prod 16.69 grams, weightening finish 1.69 grams contain polysuccinimide 0.101 gram on every gram carrier.
Embodiment two
12.4 the gram polysuccinimide is dissolved among the 65mL DMF, adds 15 gram 3-aminopropyl glass microspheres, the ratio of mole number 1: 6.All the other operations are with example one.Get polysuccinimide-glass microsphere 16.98 grams, weightening finish 1.98 grams contain polysuccinimide 0.117 gram on every gram carrier.
Embodiment three
14.0 the gram polysuccinimide is dissolved among the 65mL DMF, adds 15 gram 3-aminopropyl silica gel again, the ratio of mole number 1: 6.5.All the other operations are with example one.Get polysuccinimide-silica gel solid phase prod 17.27 grams, weightening finish 2.27 grams contain polysuccinimide 0.131 gram on every gram carrier.
Embodiment four
17.2 the gram polysuccinimide is dissolved among the 75mL DMF, adds 15 gram 3-aminopropyl silica gel again, the ratio of mole number 1: 8.All the other operations are with example one.Get polysuccinimide-silica gel 17.41 grams, weightening finish 2.41 grams contain polysuccinimide 0.138 gram on every gram carrier.
Filtrate in the foregoing description can be recycled.For example, the filtrate in embodiment three is added 2 gram polysuccinimides, adds 15 gram 3-aminopropyl silica gel again, and all the other are operated with example one.Get 17.09 gram polysuccinimide-silica gel, weightening finish 2.09 grams contain polysuccinimide 0.122 gram on every gram carrier.
Two, novel vector effect:
The novel vector effect can be used Fig. 3, Fig. 4, Fig. 5 diagram shows.Wherein symbol ■ is the immobilized lipase with novel vector preparation, symbol ▲ and for being the immobilized lipase of preparing carriers with 3-aminopropyl silica gel, symbol ● be lipase.
As shown in Figure 3, the highest with the thermostability of the immobilized lipase of novel vector preparation, this is by above-mentioned three kinds of materials are placed catalysis olive oil hydrolysis under the differing temps, measures that its vigor and its relative vigor of calculating draw.Relative vigor among the figure, its definition is: energy value under certain temperature and the percentage ratio that the ratio of the highest energy value wherein obtains are the relative vigor under this temperature.
As shown in Figure 4, in the buffered soln of pH≤7.0,, more much bigger than the relative vigor of immobilized lipase that with 3-aminopropyl silica gel is preparing carriers with the relative vigor of the immobilized lipase of novel vector preparation.Relative vigor among this figure, its definition is: energy value under certain pH value and the percentage ratio that the ratio of the highest energy value wherein obtains are the relative vigor under this pH value.The buffered soln of pH among the figure≤8.0 is by KH
2PO
4With the NaOH preparation, the buffered soln of pH 〉=9.0 is by K
2HPO
4With the NaOH preparation, concentration is 0.1mol/L.
As shown in Figure 5, with the immobilized lipase of novel vector preparation in buffered soln, the time that its vigor keeps is more much longer under same condition than the immobilized lipase that with 3-aminopropyl silica gel is preparing carriers, and the vigor in its buffered soln under 30～35 ℃ is more taller than the vigor that is kept under 4 ℃, and its vigor is still than the vigor high approximately 55% that is kept under 4 ℃ when being placed into 90 days.So-called similarity condition is meant the environment that these two kinds of materials is placed 5mL buffered soln and 30～35 ℃, and surveys vigor at set intervals one time.
The mensuration process of above-mentioned enzyme activity is: 12 gram sweet oil add 40mL 2.5% polyvinyl alcohol solution, and emulsification is 5 minutes under action of ultrasonic waves, obtains white milk sap.Lipase is dissolved in the buffered soln, is made into the solution of 1mg/mL.5mL sweet oil milk sap adds in the 4mL buffered soln, adds the 1mL lipase solution again, reacts 30 minutes down in 37 ℃.(1: 1, V/V) termination reaction added 5 phenolphthalein solutions and (when surveying the vigor of enzyme in pH 〉=9 buffered soln, after the termination reaction, adds 1mL 0.1mol/L KH earlier in mixture with 20mL ethanol-acetone
2PO
4Solution), to red, this titration value is V1 with the titration of 0.04NNaOH solution.
In said determination, the 1mL lipase solution is added after termination reaction in the mixture, all the other operations are constant, use the titration of 0.04N NaOH solution to red equally, and this titration value is blank value V2.Calculate the vigor of enzyme by the difference of V1 and V2.
When surveying the immobilized lipase enzyme activity, is that the immobilized lipase of preparing carriers add in 5mL buffered soln with the immobilized lipase of polysuccinimide-preparation of silica gel or 0.7g with 3-aminopropyl silica gel with 5mL sweet oil milk sap and 0.15g, and all the other operations are with the mensuration of lipase activity.
Prepare immobilized lipase with above-mentioned novel vector, as shown in Figure 2, sequence number 3 is novel vectors, NH
2-En is a lipase, and sequence number 4 is the immobilized lipases with the novel vector preparation.Its method is: 0.6g lipase and 6g in the polysuccinimide-silica gel adding 16mL buffered soln with embodiment three preparations, were stirred 6 hours down at 4 ℃.Suction filtration, immobilized lipase is washed four times with buffered soln, vacuum-drying then.The vigor of immobilized reactant reclaims and reaches 90%, and vigor reclaims and is defined as: the vigor sum of residual enzyme occupies the percentage ratio of the lipase total activity of immobilized reactant in immobilized enzyme and the filtrate.This immobilized reactant liquid can utilize again, and for example: 24mg lipase and 0.2g novel vector add in the buffered soln, stir 6 hours down at 4 ℃.Centrifugation, centrifugate add in another part 0.2g carrier, wash immobilized lipase with 0.4mL buffered soln, and washings also adds in another part 0.2g carrier, and this a mixture stirred 6 hours down at 4 ℃ again, so repeated four times.Immobilized lipase is all washed four times with buffered soln each time, vacuum-drying then.The vigor of surveying each time immobilized lipase is respectively 13.5,11.7,8.9,6.5,5.9 units.A unit of activity is defined as: micromole's number of every gram immobilized lipase lipid acid that the hydrolysis of catalysis sweet oil milk sap is generated in one minute.
With above-mentioned 3-aminopropyl silica gel is the preparing carriers immobilized lipase, but reference literature (Shi-Jun Ge et al.Biotechnol.Appl.Biochem.24,1,1996) preparation, that is: 10g 3-aminopropyl silica gel add 30mL 2% formaldehyde phosphate buffer solution (pH8.0,0.1mol/L) in, stirred 12 hours down at 4 ℃, suction filtration with the distillation washing, is removed excessive formaldehyde.To contain in the 30mL buffered soln of 0.33g lipase with the 3-aminopropyl silica gel adding that formaldehyde reaction is crossed again, stir down at 4 ℃ and spend the night.Suction filtration, immobilized lipase is washed four times with buffered soln, and is dry under vacuum then.The vigor of enzyme is lower in the filtrate, and vigor is recovered as 34%.Vigor reclaims and is defined as: the vigor sum of residual enzyme occupies the percentage ratio of the lipase total activity of immobilized reactant in immobilized enzyme and the filtrate.
Three, used instrument, reagent illustrates in raw material among the embodiment and the relevant test:
1, raw material: polysuccinimide and 3-aminopropyl silica gel, 3-aminopropyl glass microsphere can also can be prepared as follows by external import.
Press document [Pietro Tundo, et al.Journal of the American Chemistry Society.101 (22): 6606,1979; ] and the technology in " activation of porous silicon ball and the silanization " article on the periodical of " ion-exchange and absorption " [11 (6): 541,1995], announced of publication such as Liu Lijian, preparation 3-aminopropyl silica gel (glass microsphere):
50g silica gel adds in the methanesulfonic of 150mL 7%, and heating makes it to reflux 5 hours.Cooling, suction filtration after giving a baby a bath on the third day after its birth time with distilled water, is used distilled water immersion again, and suction filtration is washed till neutrality with distilled water again, is dried to constant weight under the vacuum.Then, the silica gel of 43.2g after acid treatment is added in the mixture of 40mL 3-aminopropyltriethoxywerene werene and 120mL toluene, stir, slowly heat up and make it to reflux 10 hours.Cooling, suction filtration is used toluene washing by soaking three times, and it is inferior to give a baby a bath on the third day after its birth with anhydrous diethyl ether again, is dried to constant weight, gets 3-aminopropyl silica gel 49.27g, and weightening finish 6.08g contains 1.48mmol amino on every gram silica gel, its ir data: υ
N-H(3228-3614cm
-1, 1100cm
-1), δ
N-H(1639cm
-1, 801cm
-1).
Replace silica gel with glass microsphere, preparation 3-aminopropyl glass microsphere, its operation is the same, and every gram 3-aminopropyl glass microsphere contains 1.42mmol amino.
Press document [Neri Paolo, et al.Journal of Medicinal Chemistry.16 (8): 893,1973] preparation polysuccinimide:
The L-aspartic acid of 50g porphyrize and the phosphoric acid of 25g 85% place beaker to mix, and then mixture are evenly divided in porcelain dish, 180 ℃ of reactions 4 hours of reducing pressure down.Cooling adds crude product among the 200mL DMF, stirs to make it dissolving.Solution is slowly poured in the 1L distilled water, simultaneously vigorous stirring.Leave standstill the back suction filtration, throw out washes with water to neutrality, and vacuum-drying gets white powder solid 32.5g, productive rate 89%, limiting viscosity: 34.1mLg
-1(DMF, 25 ℃).
Its ir data: υ
C=O(1788cm
-1, 1712cm
-1).
2, instrument and reagent
Nicolet FT-IR infrared spectrometer; Ubbelohde viscometer.
Silica gel is chemical pure, the 100-200 order; Many empty glass microspheres are the chromatographic stationary phase, the 120-160 order; 3-aminopropyltriethoxywerene werene (Wuhan University's chemical plant product) is heavily steamed with preceding decompression; The L-aspartic acid is biochemical reagents; Sweet oil is a chemical pure; Toluene, DMF, formaldehyde (40%) and anhydrous diethyl ether are analytical pure; Polyvinyl alcohol is an experiment reagent, and mean polymerisation degree is 1750 ± 50; Lipase (porcine pancreas) is Sigma company product, and protein content is 25%, and vigor is 55 unit/milligrams; Buffered soln is by analytically pure KH
2PO
4, NaOH (pH≤8.0) and K
2HPO
4, NaOH (pH 〉=9.0) preparation, except that indicating especially, the pH value of buffered soln is 7.5, concentration is 0.1mol/L.
Claims (1)
1. fixed enzyme vector, it is characterized in that described carrier comprises polysuccinimide and 3-aminopropyl silica gel or 3-aminopropyl glass microsphere, the two links to each other with covalent linkage, constitute compound fixed enzyme vector, wherein said fixed enzyme vector is made by following method: polysuccinimide is dissolved in N, in N '-dimethyl formamide, add 3-aminopropyl silica gel or 3-aminopropyl glass microsphere, ratio 1: 5～8 by the mole number of the succimide structural unit in its amino mole number and the polysuccinimide feeds intake, and collects resultant then from reaction mixture.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN98121661A CN1109746C (en) | 1998-11-10 | 1998-11-10 | Immobilized enzyme carrier |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN98121661A CN1109746C (en) | 1998-11-10 | 1998-11-10 | Immobilized enzyme carrier |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1216776A CN1216776A (en) | 1999-05-19 |
| CN1109746C true CN1109746C (en) | 2003-05-28 |
Family
ID=5227224
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN98121661A Expired - Fee Related CN1109746C (en) | 1998-11-10 | 1998-11-10 | Immobilized enzyme carrier |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN1109746C (en) |
Families Citing this family (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN100389323C (en) * | 2005-07-22 | 2008-05-21 | 中国科学院上海有机化学研究所 | Capillary electrophoresis enzyme micro reactor, its making method and purposes thereof |
| CN109136201A (en) * | 2018-07-15 | 2019-01-04 | 爱必信（上海）生物科技有限公司 | A kind of purification technique of enzyme biochemical reagents |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN86106561A (en) * | 1985-09-23 | 1987-03-18 | 迈尔斯实验室公司 | Biological catalyst fixing on granular diatomaceous earth |
| JPS62216976A (en) * | 1986-03-18 | 1987-09-24 | 日立造船株式会社 | Porous ceramics |
| CN1145410A (en) * | 1996-03-19 | 1997-03-19 | 青岛海洋大学 | Method for prepn. of shell microball carrier |
| CN1190990A (en) * | 1995-07-18 | 1998-08-19 | 吉斯特·布罗卡迪斯股份有限公司 | An improved immobilized penicillin G acylase |
-
1998
- 1998-11-10 CN CN98121661A patent/CN1109746C/en not_active Expired - Fee Related
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN86106561A (en) * | 1985-09-23 | 1987-03-18 | 迈尔斯实验室公司 | Biological catalyst fixing on granular diatomaceous earth |
| JPS62216976A (en) * | 1986-03-18 | 1987-09-24 | 日立造船株式会社 | Porous ceramics |
| CN1190990A (en) * | 1995-07-18 | 1998-08-19 | 吉斯特·布罗卡迪斯股份有限公司 | An improved immobilized penicillin G acylase |
| CN1145410A (en) * | 1996-03-19 | 1997-03-19 | 青岛海洋大学 | Method for prepn. of shell microball carrier |
Also Published As
| Publication number | Publication date |
|---|---|
| CN1216776A (en) | 1999-05-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN102943069B (en) | Co-immobilization glucose oxidase/catalase microspheres and application thereof in production of gluconic acid or gluconic salt | |
| CN101724619B (en) | Application of functionalized ionic liquid modified mesoporous molecular sieve in enzyme immobilization | |
| CN101113433B (en) | Process for preparing chitosan microsphere immobilized lipolytic enzyme | |
| US6004786A (en) | Inorganic carrier containing bound silane coupling agent having carboxylic-ester group for immobilizing lipase | |
| CN1935994B (en) | Organic group functionized mesoporous molecular sieve enzyme immobilized carrier, and its preparing method | |
| CN100564523C (en) | Contain epoxy group(ing) fixed enzyme vector and preparation method and purposes | |
| CN101182507A (en) | Carrier and preparation and applications of immobilization lipase | |
| CN1109746C (en) | Immobilized enzyme carrier | |
| CN100534596C (en) | Preparation method and application for modified acrylic fibre superfine fibre film containing reactivity group phospholipid | |
| CN101508986B (en) | Immobilized penicillin acylated enzyme with silicon gel rubber as carrier and preparation method | |
| CN114479111B (en) | Novel carrier for immobilizing horseradish peroxidase | |
| CN106400466A (en) | Immobilizing carrier material prepared from carbon fibers by modification method | |
| CN105039299B (en) | A kind of immobilized HRP carrier and its preparation, application process | |
| CN1407103A (en) | Pearl chitin carrier preparation and process for using it for enzyme solidification | |
| CN110129399B (en) | Method for promoting HAU-M1 photosynthetic bacteria to efficiently produce hydrogen by utilizing chlorella biomass | |
| CN101182508B (en) | Lactose enzyme and common immobilization method of glucose isomerase | |
| CN1088755C (en) | Fixed penicillin acidylated enzyme using agar-gelatin compounded microbeads as carrier and preparation method therefor | |
| CN101260396A (en) | Immobilization lipase using loofah sponge as carrier and preparation method thereof | |
| CN101864410A (en) | Epoxy mesoporous molecular sieve for use in bio-enzyme immobilization and preparation method thereof | |
| CN114606221A (en) | Immobilized enzyme, preparation method and application thereof | |
| CN1149284C (en) | Immobilized pencillin amidase using multi-element copolymerized porous microparticles as carrier and its preparing process | |
| CN107326021B (en) | Preparation method of magnetic cellulose microsphere immobilized lipase catalyst | |
| CN1293200C (en) | Method for producing cephalexin | |
| CN1908166B (en) | Method of preparing high activity immobilization penicillin acylase hybridization carrier | |
| KR100509738B1 (en) | Method for enzyme immobilization using silicagel or composite silicagel carrier |
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 |