CN102199592A - Method for preparing mixed immobilized glucose oxidase/catalase microspheres - Google Patents
Method for preparing mixed immobilized glucose oxidase/catalase microspheres Download PDFInfo
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- CN102199592A CN102199592A CN 201110084111 CN201110084111A CN102199592A CN 102199592 A CN102199592 A CN 102199592A CN 201110084111 CN201110084111 CN 201110084111 CN 201110084111 A CN201110084111 A CN 201110084111A CN 102199592 A CN102199592 A CN 102199592A
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Abstract
The invention provides a method for preparing mixed immobilized glucose oxidase (GOD)/catalase (CAT) microspheres and belongs to the technical field of immobilized enzyme. The method comprises the following steps: on the basis of using chitosan-arginine anionic microspheres as carrier, adding a mixed solution of GOD and CAT and glutaraldehyde, performing crosslinking reaction, filtering, washing, and performing freeze drying to prepare mixed immobilized GOD/CAT microspheres. The method has the characteristics of mild reaction conditions, higher recovery rate of enzyme activity, long half-life of immobilized enzyme, simplicity in operation, low cost, cleanness, safety, and the like. The product prepared by the method can be widely used in the industries of medicines, food, water treatment, cement and the like. The product can also be used as a glucose-removing reagent, an anti-oxidation reagent, a bactericide, a glucose quantitative analysis reagent and the like.
Description
One, technical field
The invention belongs to the enzyme immobilization technology field, being specifically related to modify crosslinked chitosan microsphere with arginine is carrier, the technology of preparation co-immobilization glucose oxidase (GOD)/catalase (CAT) microballoon.
Two, background technology
Immobilized enzyme is that the enzyme with solubility is bound by the new bio catalyzer on the water insoluble carrier, with can only compare by expendable resolvase, immobilized enzyme had both had the catalysis characteristics of resolvase, advantage such as have good stability again, can recycle, also can realize serialization, the automatization of production technique, be the main developing direction of biological catalyst.
Glucose oxidase (GOD) specificity catalysis β-D-glucose generates gluconic acid and hydrogen peroxide.In food service industry, be used for flour improvement, seafood freshing, the sugared content of mensuration fermented liquid; At field of medicaments, be used to prepare glucose in urine, blood sugar test test paper and oral disease prevention and control; At biological technical field, make sensor electrode etc.Catalase (CAT), the specificity catalyzing hydrogen peroxide is decomposed into water and oxygen, is used to remove residual hydrogen peroxide, is widely used in aspects such as textile printing and dyeing, papermaking, wastewater treatment, milk preservation and clinical analysis.In the catalystic converter system of GOD, introduce CAT, be about to two kinds of enzymes and be fixed in the same system, realize co-immobilization.The hydrogen peroxide of GOD catalyzed reaction generation diffuses to the distance shortening of CAT microenvironment like this, and the catalytic efficiency of two kinds of enzymes all is improved.
Existing preparation co-immobilization glucose oxidase/catalatic technology, for example in " journal of Zhejiang university (engineering version) " 2002 the 2nd interim " research of glucose oxidase-peroxidase film co-immobilization " literary compositions, disclosed method is: glucose oxidase and peroxidase are fixed on the nylon-66 thin-film carrier jointly, are used to prepare test paper that detects blood sugar concentration etc.The shortcoming of this method is that the strong-hydrophobicity of nylon causes the loss of immobilized enzyme big, and enzyme is directly fixed on the film surface in addition, and is too near with the distance on film surface, forms a large amount of tiny narrow slits, can hinder the macromolecular transmission of blood, even causes blocking; Two enzymes are fixed on the film at random in addition, and the hydrogen peroxide that glucose oxidase produces need spread the long time and distance could arrive the Peroxidase activity center, and the time of response is long.And for example on October 15th, 2008, disclosed publication number was " method of chitosan-arginine resin anion immobilizing Quimotrase " patent of CN101285060, disclosed method is: prepare chitosan-arginine negatively charged ion microballoon earlier with half crosslinking, be carrier with this microballoon then, with the dicyclohexylcarbodiimide is linking agent, the technology of preparation immobilizing chymotrypsin.The advantage of this method is the arginine residues immobilizing chymotrypsin with " flexible arm ", the immobilized enzyme that obtains has advantages such as sphere of action is big, vigor recovery height, good stability, illustrates that chitosan-arginine resin anion is the fixed enzyme vector of excellent property.The main drawback of this method is, carrier has only been fixed an enzyme, and under identical condition, enzyme of the same race has identical charges, repels mutually, is difficult to realize amino making full use of.
Three, summary of the invention
The objective of the invention is weak point at existing co-immobilization oenoxydase and peroxidase, providing a kind of is carrier with chitosan-arginine negatively charged ion microballoon, the method for preparing co-immobilization glucose oxidase/peroxidase (GOD/CAT) microballoon, this method has the reaction conditions gentleness, enzyme activity reclaims higher, and the immobilized enzyme long half time is simple to operate, cost is lower, characteristics such as clean and safe.
Principle of the present invention: the arginine residues that grafts on the cross-linked chitosan contains two free amine groups, be separated by between two amino of arginine residues 4 C-Cs and a carbon-nitrogen bond, all can accept GOD or CAT respectively, therefore, can by the linking agent glutaraldehyde respectively with the reaction of the amino on arginine residues and enzyme surface, form immobilized enzyme; Be carrier promptly, can prepare co-immobilization GOD/CAT with this microballoon; GOD oxidizing glucose in the co-immobilization enzyme is gluconic acid and produces hydrogen peroxide that the CAT decomposition of hydrogen peroxide is oxygen and water, thereby guarantees stability and the high reactivity of GOD.
The object of the present invention is achieved like this: a kind of method for preparing co-immobilization glucose oxidase/catalase microballoon, with chitosan-arginine negatively charged ion microballoon is carrier, add GOD and CAT mixing solutions and glutaraldehyde, through crosslinking reaction, filtration, washing, lyophilize, prepare co-immobilization GOD and CAT microballoon.Its concrete method steps is as follows:
(1) preparation chitosan-arginine negatively charged ion microballoon
According to volume the 7th phase disclosed " chitosan-arginine resin immobilizing chymotrypsin and character a thereof " literary composition in " applied chemistry " July the 26th in 2009, prepare chitosan-arginine negatively charged ion microballoon.
(2) preparation GOD and CAT mixing solutions
After (1) step finished, be 1: 1～6 ratio, GOD and CAT are dissolved in 0.2mol/L Sodium phosphate dibasic-citric acid solution of pH 3～8, collect the lysate that contains GOD and CAT according to the vigor ratio of GOD: CAT.The mixing solutions that contains GOD and CAT for collecting is used to prepare co-immobilization GOD/CAT.
(3) GOD and CAT co-immobilization
(2) step finish after, quality (g) according to (1) the chitosan-arginine negatively charged ion microballoon prepared of step: the volume (mL) of the mixing solutions that contains GOD and CAT that (2) step collected is than the ratio that is 1: 3～8, earlier chitosan-arginine negatively charged ion microballoon is scattered in the mixing solutions that contains GOD and CAT, after the whip attachment 30～60 minutes, be 2.5% glutaraldehyde solution again according to the glutaraldehyde volumetric concentration: the volume ratio that contains the mixing solutions of GOD and CAT is 1: 5～10 ratio, glutaraldehyde solution is added in the dispersion liquid, under 4～10 ℃, reacted 2～3 hours, the co-immobilization that is used for GOD and CAT is collected reaction solution.Reaction solution for collecting is used to prepare co-immobilization GOD/CAT microballoon.
(4) preparation co-immobilization GOD/CAT microballoon
After (3) step finished, the reaction solution with (3) step collected filtered, and abandons filtrate, collects filter residue.For the filter residue of collecting, use 0.2mol/L Sodium phosphate dibasic-citric acid solution liquid of pH 3～8 to wash 3～5 times, be used to remove loose GOD/CAT, collect washings and washed-residue respectively.For the washings of collecting, after adjusting pH is 3～8, be used for for (2) step once more; For the washed-residue of collecting, be that-40～-50 ℃, vacuum tightness are under the condition of 20～50Pa in temperature, carried out lyophilize 24～30 hours, just prepare chitosan-arginine resin co-immobilization GOD/CAT microballoon.Wherein the vigor of co-immobilization glucose oxidase reclaims and counts 60.25%～69.37% with the GOD vigor.
The present invention mainly contains following effect:
1, the inventive method has made full use of arginine as " flexible arm ", has realized glucose oxidase and catalatic co-immobilization on " flexible arm ", has reduced the space steric effect of immobilized enzyme.
2, the present invention has made full use of the synergetic property of GOD and CAT catalyzed reaction, has eliminated H
2O
2To the active destruction of GOD, the vigor of co-immobilization enzyme reclaims high, significant prolongation the work-ing life of co-immobilization GOD/CAT, can use repeatedly 29～45 times, used 45～70 hours continuously, production cost is low, helps Sustainable development.
3, the present invention has realized that two kinds of enzymes are fixed in the same system altogether.Substrate in the CAT microenvironment is generated by the very near GOD catalyzed reaction of distance, and the stability and the catalytic efficiency of two kinds of enzymes all are improved, thereby have improved industrial production efficient.
4, the present invention have that method is simple, reaction conditions is gentle, equipment be simple and easy to, do not have characteristics such as " three wastes " discharging, be a kind of production method of environmental protection.
The product that adopts the inventive method to prepare can be widely used in medicine, food, in the industries such as water treatment, cement.Can also be as removing glucose, deoxidation, sterilant, glucose quantitation analytical reagent etc.
Four, embodiment
Below in conjunction with embodiment, further specify the present invention.
Embodiment 1
A kind of method for preparing co-immobilization glucose oxidase/catalase microballoon, the concrete grammar step is as follows:
(1) preparation chitosan-arginine negatively charged ion microballoon
According to volume the 7th phase disclosed " chitosan-arginine resin immobilizing chymotrypsin and character a thereof " literary composition in " applied chemistry " July the 26th in 2009, prepare chitosan-arginine negatively charged ion microballoon.
(2) preparation GOD and CAT mixing solutions
After (1) step finished, be 1: 1～6 ratio, GOD and CAT are dissolved in 0.2mol/L Sodium phosphate dibasic-citric acid solution of pH 3～8, collect the lysate that contains GOD and CAT according to the vigor ratio of GOD: CAT.The mixing solutions that contains GOD and CAT for collecting is used to prepare co-immobilization GOD/CAT.
(3) GOD and CAT co-immobilization
(2) step finish after, quality (g) according to (1) the chitosan-arginine negatively charged ion microballoon prepared of step: the volume (mL) of the mixing solutions that contains GOD and CAT that (2) step collected is than the ratio that is 1: 3～8, earlier chitosan-arginine negatively charged ion microballoon is scattered in the mixing solutions that contains GOD and CAT, after the whip attachment 30～60 minutes, be 2.5% glutaraldehyde solution again according to the glutaraldehyde volumetric concentration: the volume ratio that contains the mixing solutions of GOD and CAT is 1: 5～10 ratio, glutaraldehyde solution is added in the dispersion liquid, under 4～10 ℃, reacted 2～3 hours, the co-immobilization that is used for GOD and CAT is collected reaction solution.Reaction solution for collecting is used to prepare co-immobilization GOD/CAT microballoon.
(4) preparation co-immobilization GOD/CAT microballoon
After (3) step finished, the reaction solution with (3) step collected filtered, and abandons filtrate, collects filter residue.For the filter residue of collecting, use 0.2mol/L Sodium phosphate dibasic-citric acid solution liquid of pH 3～8 to wash 3～5 times, be used to remove loose GOD/CAT, collect washings and washed-residue respectively.For the washings of collecting, after adjusting pH is 3～8, be used for for (2) step once more; For the washed-residue of collecting, be that-40～-50 ℃, vacuum tightness are under the condition of 20～50Pa in temperature, carried out lyophilize 24～30 hours, just prepare chitosan-arginine resin co-immobilization GOD/CAT microballoon.Wherein the vigor of co-immobilization glucose oxidase reclaims and counts 60.25%～69.37% with the GOD vigor.
Embodiment 2
A kind of method for preparing co-immobilization glucose oxidase/catalase microballoon, with embodiment 1, wherein:
In (2) step, the vigor ratio of GOD: CAT is 1: 4, and GOD and CAT are dissolved in 0.2mol/L Sodium phosphate dibasic-citric acid solution of pH 5;
In (3) step, the quality of the chitosan-arginine negatively charged ion microballoon that (1) step prepared: the volume ratio of the mixing solutions that contains GOD and CAT that (2) step collected is 1g: 5mL, whip attachment after 45 minutes the glutaraldehyde volumetric concentration be 2.5% glutaraldehyde solution: the volume ratio that contains the mixing solutions of GOD and CAT is 1: 7, under 7 ℃, reacted 2.5 hours;
In (4) step, with the 0.2mol/L Sodium phosphate dibasic of pH 5-citric acid solution liquid washing 4 times, the temperature of freeze drier is 35Pa for-45 ℃, vacuum tightness, carries out lyophilize 27 hours.
Embodiment 3
A kind of method for preparing co-immobilization glucose oxidase/catalase microballoon, with embodiment 1, wherein:
In (2) step, the vigor ratio of GOD: CAT is 1: 6, and GOD and CAT are dissolved in 0.2mol/L Sodium phosphate dibasic-citric acid solution of pH 8;
In (3) step, the quality of the chitosan-arginine negatively charged ion microballoon that (1) step prepared: the volume ratio of the mixing solutions that contains GOD and CAT that (2) step collected is 1g: 8mL, whip attachment after 60 minutes the glutaraldehyde volumetric concentration be 2.5% glutaraldehyde solution: the volume ratio that contains the mixing solutions of GOD and CAT is 1: 10, under 10 ℃, reacted 3 hours;
In (4) step, with the 0.2mol/L Sodium phosphate dibasic of pH 8-citric acid solution liquid washing 5 times, the temperature of freeze drier is 50Pa for-50 ℃, vacuum tightness, carries out lyophilize 30 hours.
Claims (4)
1. method for preparing co-immobilization glucose oxidase/catalase microballoon is characterized in that the concrete grammar step is as follows:
(1) preparation chitosan-arginine negatively charged ion microballoon
According to volume the 7th phase disclosed " chitosan-arginine resin immobilizing chymotrypsin and character a thereof " literary composition in " applied chemistry " July the 26th in 2009, prepare chitosan-arginine negatively charged ion microballoon;
(2) preparation GOD and CAT mixing solutions
After (1) step finished, be 1: 1～6 ratio, GOD and CAT are dissolved in 0.2mol/L Sodium phosphate dibasic-citric acid solution of pH 3～8, collect the lysate that contains GOD and CAT according to the vigor ratio of GOD: CAT;
(3) GOD and CAT co-immobilization
(2) step finish after, quality according to (1) the chitosan-arginine negatively charged ion microballoon prepared of step: the volume ratio of the mixing solutions that contains GOD and CAT that (2) step collected is the ratio of 1g: 3～8mL, earlier chitosan-arginine negatively charged ion microballoon is scattered in the mixing solutions that contains GOD and CAT, after the whip attachment 30～60 minutes, be 2.5% glutaraldehyde solution again according to the glutaraldehyde volumetric concentration: the volume ratio that contains the mixing solutions of GOD and CAT is 1: 5～10 ratio, glutaraldehyde solution is added in the dispersion liquid, under 4～10 ℃, reacted 2～3 hours, the co-immobilization that is used for GOD and CAT is collected reaction solution;
(4) preparation co-immobilization GOD/CAT microballoon
(3) step finish after, reaction solution with (3) step collected filters, and abandons filtrate, collect filter residue, for the filter residue of collecting, use 0.2mol/L Sodium phosphate dibasic-citric acid solution liquid of pH 3～8 to wash 3～5 times, collect washings and washed-residue respectively, for the washed-residue of collecting, in temperature is that-40～-50 ℃, vacuum tightness are under the condition of 20～50Pa, carries out lyophilize 24～30 hours, just prepares chitosan-arginine resin co-immobilization GOD/CAT microballoon.
2. according to the described a kind of method for preparing co-immobilization glucose oxidase/catalase microballoon of claim 1, it is characterized in that:
In (2) step, the vigor ratio of GOD: CAT is 1: 1, and GOD and CAT are dissolved in 0.2mol/L Sodium phosphate dibasic-citric acid solution of pH 3;
In (3) step, the quality of the chitosan-arginine negatively charged ion microballoon that (1) step prepared: the volume ratio of the mixing solutions that contains GOD and CAT that (2) step collected is 1g: 3mL, whip attachment after 30 minutes the glutaraldehyde volumetric concentration be 2.5% glutaraldehyde solution: the volume ratio that contains the mixing solutions of GOD and CAT is 1: 5, under 4 ℃, reacted 2 hours;
In (4) step, with the 0.2mol/L Sodium phosphate dibasic of pH 3-citric acid solution liquid washing 3 times, the temperature of freeze drier is 20Pa for-40 ℃, vacuum tightness, carries out lyophilize 24 hours.
3. according to the described a kind of method for preparing co-immobilization glucose oxidase/catalase microballoon of claim 1, it is characterized in that:
In (2) step, the vigor ratio of GOD: CAT is 1: 4, and GOD and CAT are dissolved in 0.2mol/L Sodium phosphate dibasic-citric acid solution of pH 5;
In (3) step, the quality of the chitosan-arginine negatively charged ion microballoon that (1) step prepared: the volume ratio of the mixing solutions that contains GOD and CAT that (2) step collected is 1g: 5mL, whip attachment after 45 minutes the glutaraldehyde volumetric concentration be 2.5% glutaraldehyde solution: the volume ratio that contains the mixing solutions of GOD and CAT is 1: 7, under 7 ℃, reacted 2.5 hours;
In (4) step, with the 0.2mol/L Sodium phosphate dibasic of pH 5-citric acid solution liquid washing 4 times, the temperature of freeze drier is 35Pa for-45 ℃, vacuum tightness, carries out lyophilize 27 hours.
4. according to the described a kind of method for preparing co-immobilization glucose oxidase/catalase microballoon of claim 1, it is characterized in that:
In (2) step, the vigor ratio of GOD: CAT is 1: 6, and GOD and CAT are dissolved in 0.2mol/L Sodium phosphate dibasic-citric acid solution of pH 8;
In (3) step, the quality of the chitosan-arginine negatively charged ion microballoon that (1) step prepared: the volume ratio of the mixing solutions that contains GOD and CAT that (2) step collected is 1g: 8mL, whip attachment after 60 minutes the glutaraldehyde volumetric concentration be 2.5% glutaraldehyde solution: the volume ratio that contains the mixing solutions of GOD and CAT is 1: 10, under 10 ℃, reacted 3 hours;
In (4) step, with the 0.2mol/L Sodium phosphate dibasic of pH 8-citric acid solution liquid washing 5 times, the temperature of freeze drier is 50Pa for-50 ℃, vacuum tightness, carries out lyophilize 30 hours.
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| CN102943069A (en) * | 2012-11-27 | 2013-02-27 | 北京化工大学 | Co-immobilization glucose oxidase/catalase microspheres and application thereof in production of gluconic acid or gluconic salt |
| CN103524569A (en) * | 2013-10-23 | 2014-01-22 | 河北工业大学 | Technological method for removing glucose in isomaltooligosaccharide product by using multiple immobilized enzymes |
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| CN104740641A (en) * | 2015-04-08 | 2015-07-01 | 烟台大学 | Glucose sensitive controlled release microballoon composition and preparation method thereof |
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| CN102943069B (en) * | 2012-11-27 | 2014-11-05 | 北京化工大学 | Co-immobilization glucose oxidase/catalase microspheres and application thereof in production of gluconic acid or gluconic salt |
| CN102943069A (en) * | 2012-11-27 | 2013-02-27 | 北京化工大学 | Co-immobilization glucose oxidase/catalase microspheres and application thereof in production of gluconic acid or gluconic salt |
| CN105146745A (en) * | 2013-10-18 | 2015-12-16 | 贵州中烟工业有限责任公司 | Immobilized catalase additive for tobacco as well as preparation method and application of additive |
| CN103524569A (en) * | 2013-10-23 | 2014-01-22 | 河北工业大学 | Technological method for removing glucose in isomaltooligosaccharide product by using multiple immobilized enzymes |
| CN103524569B (en) * | 2013-10-23 | 2015-06-10 | 河北工业大学 | Technological method for removing glucose in isomaltooligosaccharide product by using multiple immobilized enzymes |
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| CN104740641B (en) * | 2015-04-08 | 2019-02-01 | 烟台大学 | A kind of slow control-release microsphere composition of glucose sensitive and preparation method thereof |
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| CN106222159A (en) * | 2016-08-16 | 2016-12-14 | 绵阳抱朴生物技术有限公司 | A kind of defying age co-immobilization enzyme and preparation method thereof |
| CN108866036A (en) * | 2018-07-24 | 2018-11-23 | 浙江大学 | A kind of cascade enzyme reaction microballoon and preparation method thereof with antibacterial functions |
| WO2020113717A1 (en) * | 2018-12-05 | 2020-06-11 | 清华大学 | Crosslinking immobilized bienzyme-surfactant coordination complex, and preparation method therefor |
| CN111019932B (en) * | 2019-11-28 | 2023-11-14 | 湖南大学 | Preparation method of copper phosphate-enzyme mineralized material, and product and application thereof |
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| CN114984241B (en) * | 2022-04-19 | 2023-08-29 | 上海工程技术大学 | Chitosan/manganese dioxide/glucose oxidase nanocomposite and preparation and application thereof |
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