CN103667219A - Preparation method of beta-glucosaccharase cross-linked aggregates - Google Patents
Preparation method of beta-glucosaccharase cross-linked aggregates Download PDFInfo
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- CN103667219A CN103667219A CN201310664188.5A CN201310664188A CN103667219A CN 103667219 A CN103667219 A CN 103667219A CN 201310664188 A CN201310664188 A CN 201310664188A CN 103667219 A CN103667219 A CN 103667219A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2445—Beta-glucosidase (3.2.1.21)
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- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01021—Beta-glucosidase (3.2.1.21)
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Abstract
The invention provides a preparation method of beta-glucosaccharase cross-linked aggregates, which can obtain a high-enzyme-activity recovery rate. The method comprises the following steps: (1) adding a settling agent into a beta-glucosaccharase solution and oscillating; (2) adding glutaraldehyde; (3) adding a reducing agent into a mixed solution, oscillating, centrifuging and collecting precipitate, wherein the reaction temperature of the entire reaction process is controlled at 4-6 DEG C, and the pH value is 5.0-5.5; the settling agent used in the step (1) is 1,4-dioxane, and the volume ratio of 1,4-dioxane to the beta-glucosaccharase solution is (2-3):1; the concentration of glutaraldehyde in the step (2) is 38-43mM, and crosslinking time is 8-12 minutes; the reducing agent is sodium borohydride (NaBH4), and the concentration of NaBH4 is 20-25mM.
Description
Technical field
The invention belongs to immobilized enzyme production field, be specifically related to a kind of preparation method of beta-glucosidase cross-linked aggregates.
Background technology
The problems such as free β-glucose glycoside enzyme existence and stability in industrial production is poor, repeating utilization factor is low, are seriously restricting the suitability for industrialized production of biosynthesizing rhodioside.And cross-linked enzyme aggregate neither needs enzyme to carry out crystallization operation, can retain to greatest extent again the enzyme rate of recovery of living, be the best a kind of in Immobilized Enzymes Without Carriers technology.Along with cross-linked aggregates technology developing rapidly in enzyme immobilization field, in recent years, the preparation method that someone has studied β-glucose glycoside enzyme cross-linked aggregates (refers to: Ou Jie, crosslinked β-glucose glycoside enzyme aggregate preparation and the research of producing soybean isoflavones active aglycone thereof, HeFei University of Technology, Master's thesis, in June, 2007), the preparation method of this β-glucose glycoside enzyme cross-linked aggregates is: first, sinking agent (ethanol or Virahol or n-propyl alcohol) is added in beta-glucoside enzyme solution to vibration; Then, add linking agent (glutaraldehyde) crosslinked; Finally, in mixed solution, add reductive agent (sodium borohydride), after vibration, obtain crosslinked beta-glucoside enzyme aggregate (being beta-glucosidase CLEAs).And respectively the preparation condition in beta-glucosidase CLEAs preparation process (as the volume ratio of the selection of sinking agent, sinking agent and enzyme liquid, PH, temperature, settling time etc.), crosslinked condition (as glutaraldehyde concentration, temperature, crosslinking time etc.) are optimized, the enzyme rate of recovery alive of the beta-glucosidase CLEAs making under optimum preparation condition and crosslinked condition is the accessible maximum of 77.4%(), still lower, cannot meet the maximized requirement of batch production economic interests.
Summary of the invention
The present invention aims to provide the preparation method of a kind of beta-glucosidase cross-linked aggregates (beta-glucosidase CLEAs), and the beta-glucosidase CLEAs body making by the method can solve immobilization Glycosylase low problem of the enzyme rate of recovery alive in Industrial processes of prior art.
For achieving the above object, the present invention adopts following technical scheme:
A preparation method for beta-glucosidase cross-linked aggregates, comprises the following steps:
(1) sinking agent is added in beta-glucoside enzyme solution to vibration;
(2) add glutaraldehyde;
(3) in mixed solution, add reductive agent, after vibration, the centrifugal 5 ~ 10min of 300 ~ 500rpm, collecting precipitation, is beta-glucosidase cross-linked aggregates;
Wherein, the temperature of reaction of whole reaction process is controlled at 4 ~ 6 ℃, and pH value is 5.0 ~ 5.5; The sinking agent of step (1) is Isosorbide-5-Nitrae-dioxane, and the volume ratio of Isosorbide-5-Nitrae-dioxane and beta-glucosidase liquid is 2 ~ 3:1, and the beta-glucoside enzyme concn of beta-glucoside enzyme solution is 1.5 ~ 2.5mg/ml; The final concentration of the whole glutaraldehyde of step (2) is 38 ~ 43mM, and crosslinking time is 8 ~ 12min; The reductive agent of step (3) is sodium borohydride, and the final concentration of sodium borohydride is 20 ~ 25 mM.
Described beta-glucosidase is almond beta-glucosidase.
The invention has the advantages that: the common sinking agent for enzyme cross-linked aggregates preparation method has: methyl alcohol, ethanol, n-propyl alcohol, Virahol, acetone, DMF (DMF) and saturated ammonium sulphate.And the solvent that Isosorbide-5-Nitrae-dioxane is generally used as stratographic analysis reagent and acetate fiber and derivative thereof is used, up to now, have no the report used as enzyme cross-linked aggregates preparation method's sinking agent.The sinking agent of Isosorbide-5-Nitrae-dioxane as beta-glucosidase CLEAs preparation method usingd in the present invention first.Again in conjunction with crosslinked condition and the reductive condition optimized, making the enzyme of the beta-glucosidase CLEAs that finally makes live the rate of recovery can be up to more than 80% (reaching as high as 81.78%), and the enzyme of the beta-glucosidase CLEAs that the preparation method who adopts prior art makes under optimum reaction condition is lived the rate of recovery only for this enzyme of 77.4%(rate of recovery of living is the maximum that prior art reaches), i.e. the enzyme of the beta-glucosidase CLEAs that the present invention makes is lived the rate of recovery far away higher than the maximum of prior art.In the immobilization of enzyme, inevitably there will be part enzyme remain in solution in or the phenomenon of zymoprotein part inactivation, therefore, want to continue to promote on prior art enzyme is lived the basis of rate of recovery maximum 77.4%, there is very large difficulty.And the enzyme of the prepared beta-glucosidase CLEAs of the present invention rate of recovery rate of recovery of enzyme can being lived of living improves 2 ~ 5 percentage points.
Embodiment
Embodiment 1
The preparation method of a kind of beta-glucosidase cross-linked aggregates (beta-glucosidase CLEAs), comprise the following steps: (1) is by 2.5ml 1,4-dioxane joins 1 mL containing in the beta-glucoside enzyme solution of beta-glucosidase 1.5mg/ml (1, the volume ratio of 4-dioxane and beta-glucosidase liquid is 2.5:1), vibration; (2) add 25% glutaraldehyde, to the final concentration of glutaraldehyde be 43mM, crosslinking time 10min; (3) add again sodium borohydride (NaBH
4), to NaBH
4final concentration be 25 mM, reduce, after vibration, the centrifugal 5min of 400rpm, collecting precipitation, is beta-glucosidase CLEAs; The temperature of reaction of whole reaction process is controlled at 5 ℃, and pH value is 5.5.
The recycling mode of beta-glucosidase CLEAs is: after reaction solution is centrifugal, the beta-glucosidase CLEAs precipitation that collection is obtained is with buffered soln washing 3 times, add respectively again 1mL buffered soln vibration to make it to become suspension liquid, each group is the hydrolysis vigor of sampling and measuring beta-glucosidase CLEAs respectively, the enzyme work of the enzyme liquid processed without sinking agent, linking agent of take is 100%, calculates the relative hydrolysis vigor (be the enzyme of beta-glucosidase CLEAs live the rate of recovery) of beta-glucosidase CLEAs.
The hydrolysis vigour-testing method of beta-glucosidase is: 2 mL p-nitrophenyl-β-D-Glucose glycosides (pNPG) solution of getting respectively 100 μ L beta-glucosidase CLEAs to be measured solution and 10 mM, the two mixes accurately insulation 10 min of rear 50 ℃ of water-baths, add immediately afterwards sodium carbonate solution 2 mL of 1 M with termination reaction, standing 5 min of room temperature after vibration evenly, suitably after dilution, with ultraviolet spectrophotometer, measure sample at the light absorption value at wavelength 410 nm places, substitution pNP typical curve can be calculated pNP growing amount.Enzyme activity unit is defined as: in per minute, catalysis pNPG generates the enzyme amount of 1 μ moL pNP.Wherein, pNP standard curve making process is the common standard curve making method in this area.
The hydrolysis vigour-testing method of beta-glucosidase is: 2 mL p-nitrophenyl-β-D-Glucose glycosides (pNPG) solution of getting respectively 100 μ L beta-glucosidase CLEAs to be measured solution and 10 mM, the two mixes accurately insulation 10 min of rear 50 ℃ of water-baths, add immediately afterwards sodium carbonate solution 2 mL of 1 M with termination reaction, standing 5 min of room temperature after vibration evenly, suitably after dilution, with ultraviolet spectrophotometer, measure sample at the light absorption value at wavelength 410 nm places, substitution pNP typical curve can be calculated pNP growing amount.Enzyme activity unit is defined as: in per minute, catalysis pNPG generates the enzyme amount of 1 μ moL pNP.Wherein, pNP standard curve making process is the common standard curve making method in this area.Wherein, generally to select pH be citric acid-Na of 5.0 to buffered soln
2hPO
4buffered soln.
In embodiment 1, the enzyme of the beta-glucosidase CLEAs rate of recovery alive is 81.65%.
Embodiment 2
Embodiment 2 is as different from Example 1: in whole reaction process, pH value is 5.0, wherein, in step (1) 1, the volume of 4-dioxane is that 2ml(is 1, the volume ratio of 4-dioxane and beta-glucosidase liquid is 2:1), beta-glucoside enzyme solution is containing beta-glucosidase 2.5mg/ml; In step (2), the final concentration of glutaraldehyde is 40 mM; NaBH in step (3)
4final concentration be 23 mM, the centrifugal 10min of 300rpm; The enzyme rate of recovery alive that records beta-glucosidase CLEAs is 81.78%.
Embodiment 3
Embodiment 3 is as different from Example 1: in whole reaction process, pH value is 5.2, wherein, in step (1) 1, the volume of 4-dioxane is that 3ml(is 1, the volume ratio of 4-dioxane and beta-glucosidase liquid is 3:1), beta-glucoside enzyme solution is containing beta-glucosidase 2.0mg/ml; In step (2), the final concentration of glutaraldehyde is 38 mM; NaBH in step (3)
4final concentration be 20mM, the centrifugal 5min of 500rpm, the enzyme that the records beta-glucosidase CLEAs rate of recovery of living is 80.11%.
Embodiment 4
Embodiment 4 is as different from Example 1: in whole reaction process, pH value is 5.4, wherein, the volume of Isosorbide-5-Nitrae-dioxane is 3ml (1 in step (1), the volume ratio of 4-dioxane and beta-glucosidase liquid is 3:1), beta-glucoside enzyme solution is containing beta-glucosidase 2mg/ml; In step (2), the final concentration of glutaraldehyde is 10 mM; NaBH in step (3)
4final concentration be 20 mM, the enzyme that the records beta-glucosidase CLEAs rate of recovery of living is 13.21%.
Embodiment 5
Embodiment 5 is as different from Example 1: in whole reaction process, pH value is 5.0, wherein, in step (1) 1, the volume of 4-dioxane is that 3ml(is 1, the volume ratio of 4-dioxane and beta-glucosidase liquid is 3:1), beta-glucoside enzyme solution is containing beta-glucosidase 1.5mg/ml; In step (2), the final concentration of glutaraldehyde is 50 mM; NaBH in step (3)
4final concentration be 25 mM, the centrifugal 5min of 400rpm, the enzyme that the records beta-glucosidase CLEAs rate of recovery of living is 72.37%.
Embodiment 6
Embodiment 6 is as different from Example 1: in whole reaction process, pH value is 5.5, wherein, in step (1) 1, the volume of 4-dioxane is that 1.5ml(is, the volume ratio of 4-dioxane and beta-glucosidase liquid is 1.5:1), beta-glucoside enzyme solution is containing beta-glucosidase 1.5mg/ml; In step (2), the final concentration of glutaraldehyde is 38 mM; NaBH in step (3)
4final concentration be 25 mM, the centrifugal 10min of 300rpm; The enzyme rate of recovery alive that records beta-glucosidase CLEAs is 69.15%.
Embodiment 7
Embodiment 7 is as different from Example 1: in whole reaction process, pH value is 5.2, wherein, in step (1) 1, the volume of 4-dioxane is that 2.5ml(is 1, the volume ratio of 4-dioxane and beta-glucosidase liquid is 2.5:1), beta-glucoside enzyme solution is containing beta-glucosidase 2.0mg/ml; In step (2), the final concentration of glutaraldehyde is 40 mM; NaBH in step (3)
4final concentration be 23mM, the centrifugal 5min of 400rpm, the enzyme that the records beta-glucosidase CLEAs rate of recovery of living is 77.37%.
Embodiment 8
Optimum preparation condition according to prior art, is specially: get 2.0 mg/mL beta-glucoside enzyme solution 10ml, under 4 ℃ of conditions, slowly add wherein 90% ammonium sulfate saturated solution 90ml, after precipitating 30min, centrifugal, the damping fluid that is 5.0 with pH dissolves; Again to the glutaraldehyde solution that adds 10mM in lysate, under 30 ℃ of conditions, crosslinked 15min; Finally, add NaBH
4reduce, the centrifugal 10min of 300rpm, the enzyme rate of recovery alive that records beta-glucosidase CLEAs is 77.51 ± 0.03.
Contrast according to above-described embodiment 1 ~ 3 with embodiment 8, known, the enzyme of the beta-glucosidase CLEAs that the present invention is resulting is lived the rate of recovery far away higher than the enzyme of prior art (embodiment 8) rate of recovery of living, reason is: after different sinking agents precipitates resolvase, the enzyme of loss is lived different, and employing Isosorbide-5-Nitrae-dioxane of the present invention to be sinking agent can make that beta-glucosidase keeps maximum enzyme to live in settling process.What the glutaraldehyde concentration in above-described embodiment 4 adopted is the linking agent optimum concentration value (concentration of glutaraldehyde is 10 mM) of prior art, but, the enzyme rate of recovery alive of corresponding beta-glucosidase CLEAs is only 13.21%, visible, take Isosorbide-5-Nitrae-dioxane as the prepared beta-glucosidase CLEAs of sinking agent larger on crosslinked action impact in glutaraldehyde cross-linking process.Therefore, the present invention has and can make the live technique effect of rate of recovery beta-glucosidase CLEAs of high enzyme and be not merely the breakthrough change that sinking agent kind is selected, and, also to there is this special sinking agent (1 of coupling, 4-dioxane) glutaraldehyde concentration, also can be found out from embodiment 4 ~ 7 meanwhile, the volume ratio of Isosorbide-5-Nitrae-dioxane of the present invention and beta-glucosidase liquid and NaBH
4concentration the enzyme of the beta-glucosidase CLEAs rate of recovery of living is also had a certain impact, therefore, the inventor need to can draw best of breed and the optimum range value of a series of mutual couplings after testing many times, and the enzyme rate of recovery alive of beta-glucosidase CLEAs can be reached more than 80%.
Beta-glucosidase of the present invention is almond beta-glucosidase, certainly also can adopt other beta-glucosidase, as prune seed beta-glucosidase of the prior art etc.
Of the present invention to pNPG, pNP, almond beta-glucosidase (6 U/mg) all purchased from Yuan Ye bio tech ltd, Shanghai; Isosorbide-5-Nitrae-dioxane of the present invention, 25% glutaraldehyde, NaBH
4deng all purchased from traditional Chinese medicines Group Co.,Ltd.
The foregoing is only preferred embodiment of the present invention, all equalizations of doing according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
Claims (2)
1. a preparation method for beta-glucosidase cross-linked aggregates, is characterized in that: comprise the following steps:
(1) sinking agent is added in beta-glucoside enzyme solution to vibration;
(2) add glutaraldehyde;
(3) in mixed solution, add reductive agent, after vibration, the centrifugal 5 ~ 10min of 300 ~ 500rpm, collecting precipitation, is beta-glucosidase cross-linked aggregates;
The temperature of reaction of whole reaction process is controlled at 4 ~ 6 ℃, and pH value is 5.0 ~ 5.5; The sinking agent of step (1) is Isosorbide-5-Nitrae-dioxane, and the volume ratio of Isosorbide-5-Nitrae-dioxane and beta-glucosidase liquid is 2 ~ 3:1, and the beta-glucoside enzyme concn of beta-glucoside enzyme solution is 1.5 ~ 2.5mg/ml; The final concentration of the whole glutaraldehyde of step (2) is 38 ~ 43mM, and crosslinking time is 8 ~ 12min; The reductive agent of step (3) is sodium borohydride, and the final concentration of sodium borohydride is 20 ~ 25 mM.
2. the preparation method of a kind of beta-glucosidase cross-linked aggregates according to claim 1, is characterized in that: described beta-glucosidase is almond beta-glucosidase.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104911237A (en) * | 2015-06-10 | 2015-09-16 | 淮阴工学院 | Method for synthesizing salidroside by attapulgite-loaded black brin seed beta-glucosaccharase cross-linked polymers |
| WO2020113717A1 (en) * | 2018-12-05 | 2020-06-11 | 清华大学 | Crosslinking immobilized bienzyme-surfactant coordination complex, and preparation method therefor |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102533717A (en) * | 2012-03-02 | 2012-07-04 | 江南大学 | Method for immobilizing beta-glucosidase and hydrolyzing straw cellulose by cooperating beta-glucosidase with cellulase |
-
2013
- 2013-12-11 CN CN201310664188.5A patent/CN103667219A/en active Pending
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102533717A (en) * | 2012-03-02 | 2012-07-04 | 江南大学 | Method for immobilizing beta-glucosidase and hydrolyzing straw cellulose by cooperating beta-glucosidase with cellulase |
Non-Patent Citations (4)
| Title |
|---|
| INGA MATIJOSYTE 等: "Preparation and use of cross-linked enzyme aggregates (CLEAs) of laccases", 《JOURNAL OF MOLECULAR CATALYSIS B: ENZYMATIC》 * |
| 武仙山 等: "交联酶聚集体——一种无载体酶固定化方法", 《生物技术》 * |
| 罗建平 等: "交联β-葡萄糖苷酶聚集体的制备及其性质", 《食品科学》 * |
| 陈磊: "西梅籽β-葡萄糖苷酶的分离纯化、性质及固定化研究", 《中国优秀硕士学位论文全文数据库（基础科学辑）》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104911237A (en) * | 2015-06-10 | 2015-09-16 | 淮阴工学院 | Method for synthesizing salidroside by attapulgite-loaded black brin seed beta-glucosaccharase cross-linked polymers |
| CN104911237B (en) * | 2015-06-10 | 2017-09-29 | 淮阴工学院 | A kind of method that recessed soil load black cloth woods seed β glucuroides cross-linked polymer synthesizes rhodioside |
| WO2020113717A1 (en) * | 2018-12-05 | 2020-06-11 | 清华大学 | Crosslinking immobilized bienzyme-surfactant coordination complex, and preparation method therefor |
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Application publication date: 20140326 |