CN104073483A - Enzyme-inorganic crystal compound microsphere and preparation method thereof - Google Patents
Enzyme-inorganic crystal compound microsphere and preparation method thereof Download PDFInfo
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- CN104073483A CN104073483A CN201410309010.3A CN201410309010A CN104073483A CN 104073483 A CN104073483 A CN 104073483A CN 201410309010 A CN201410309010 A CN 201410309010A CN 104073483 A CN104073483 A CN 104073483A
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Abstract
The invention discloses an enzyme-inorganic crystal compound microsphere and a preparation method thereof. The preparation method of the enzyme-inorganic crystal compound microsphere provided by the invention comprises the following step: dissolving metal ions and the enzyme in a phosphate buffer to obtain the enzyme-inorganic crystal compound microsphere, wherein the metal ions are any one of calcium ions, magnesium ions, iron ions, ferrous ions, cupric ions, nickelous ions or zinc ions, and the phosphate buffer comprises phosphate dihydrogen ions or dihydrogen phosphate ions. The preparation method of the enzyme-inorganic crystal compound microsphere provided by the invention has the advantages of simplicity in operation, low cost and the like. The stability of the enzyme in the enzyme-inorganic crystal compound microsphere provided by the invention is improved and the activity of the enzyme is substantially maintained unchanged or even improved.
Description
Technical field
The present invention relates to a kind of enzyme-mineral crystal complex microsphere and preparation method thereof, belong to enzyme immobilization technology field.
Background technology
Enzyme, as a kind of biological catalyst, has the features such as highly selective, reaction conditions gentleness because of it, be widely used in the fields such as medicine, food-processing and catalyst preparation.And multienzyme constitutes jointly by two kinds or above different enzyme, can carry out continuous catalyzed reaction.Multi-enzyme system shows high-level efficiency and Harmony in catalytic process.Yet the single enzyme of Industrial Catalysis in using or multienzyme show under actual industrial operational condition, and stability is low, tolerance is poor, the defect such as recoverable not, has limited to a great extent its industrial application.Enzyme immobilization technology is for the approach that provides is provided.
At present, enzyme immobilization technology has been widely used in industrial enzyme catalytic production process, has obtained certain effect.But there is limitation in the process for fixation of existing single enzyme and multienzyme, for example preparation method's activity yield complicated, immobilized enzyme is lower and preparation cost is more high, therefore seeks a kind of method simple to operate, that immobilized enzyme yield alive is high, with low cost very important.
Summary of the invention
The object of this invention is to provide a kind of enzyme-mineral crystal complex microsphere and preparation method thereof, specifically adopt the precipitator method to prepare enzyme-mineral crystal complex microsphere, the method is simple to operate, mild condition, with low cost, enzymic activity yield is high.
The preparation method of a kind of enzyme-mineral crystal complex microsphere provided by the invention, comprises the steps:
Metal ion and enzyme are all dissolved in phosphate buffered saline buffer, obtain described enzyme-mineral crystal complex microsphere;
Described metal ion be in calcium ion, magnesium ion, iron ion, ferrous ion, cupric ion, bivalent nickel ion or zine ion any;
Described phosphate buffered saline buffer comprises hydrogen phosphate and dihydrogen phosphate.
In above-mentioned preparation method, the molecular weight of described enzyme is 5～500kD.
In above-mentioned preparation method, described enzyme is candida antarctica lipase B (Candida antarctica Lipase B), glucose oxidase (Glucose oxidase), horseradish peroxidase (Horse radish peroxidase), ethanol dehydrogenase (Ethanol dehydrogenase), sucrase (Invertase), superoxide-dismutase (Superoxide dismutase), catalase (Catalase), Selenoperoxidase (Glutathion peroxidase), laccase (Laccase), Hexose phosphate dehydrogenase (Glucose dehydrogenase), cellobiose dehydrogenase (Cellobiose dehydrogenase), uriKoxidase (Uricase), antarctic candidia lipase A (Candida antarctica Lipase A), fold lipase from candida sp (Candida rugosa Lipase), porcine pancreatic lipase (Porcine pancreas Lipase), rhizomucor miehei lipase (Rhizomucor miehei Lipase) and dredge at least one in the thermophilic hyphomycete lipase of cotton shape (Thermomyces lanuginosus Lipase).
In above-mentioned preparation method, in the mixed solution that described enzyme and described phosphate buffered saline buffer form, the concentration of described enzyme is 0.01～10mg/mL, specifically can be 0.5mg/mL;
In described phosphoric acid buffer, the concentration of described hydrogen phosphate and described dihydrogen phosphate is 1～1000mM;
Described hydrogen phosphate specifically can be from Sodium phosphate dibasic; Described dihydrogen phosphate specifically can be from potassium primary phosphate;
The concentration of described hydrogen phosphate specifically can be 10mM; The concentration of described dihydrogen phosphate specifically can be 2mM;
The pH of described phosphoric acid buffer is 5.0～9.0, specifically can be 7.4.
In above-mentioned preparation method, described metal ion is added in described phosphate buffered saline buffer with the form of its aqueous solution;
In the described aqueous solution, the concentration of metal ion is 10～1000mM, specifically can be 200mM.
In above-mentioned preparation method, the mass ratio of described metal ion and described enzyme is 0.1～5:1, specifically can be 0.256:1.
In above-mentioned preparation method, under standing condition, at the temperature of 0～50 ℃, obtain described enzyme-mineral crystal complex microsphere, specifically can be 25 ℃ or 4 ℃;
The described standing time is 1～100 hour, specifically can be 24 hours.
In above-mentioned preparation method, described hybrid mode is any during dropping mixing and microreactor mix.
Above-mentioned preparation method also comprises the steps: dry described enzyme-mineral crystal complex microsphere;
Described drying mode be in the dry and lyophilize of vacuum Air drying, spraying any.
The present invention further provides the prepared enzyme-mineral crystal complex microsphere of above-mentioned preparation method, wherein, the mass content of enzyme is 5%～20%, specifically can be 5%～10%, 5% or 10%.
A kind of single enzyme provided by the invention or the immobilized method of multienzyme, the method has the advantages such as simple to operate, with low cost.The enzyme that the present invention prepares-be greatly improved without the stability of enzyme in crystal complex microsphere, enzymic activity substantially remains unchanged and even increases.Above feature makes the fields such as this kind of enzyme-mineral crystal complex microsphere is synthetic in enzyme catalysis, bio-sensing have great application prospect.
Accompanying drawing explanation
Fig. 1 is XRD and the standard card comparison diagram of cupric phosphate crystal and enzyme-cupric phosphate crystal complex microsphere in embodiment 6.
Fig. 2 is the scanning electron microscope (SEM) photograph of the prepared enzyme-cupric phosphate crystal complex microsphere of embodiment 1.
Fig. 3 is the scanning electron microscope (SEM) photograph of the prepared enzyme-cupric phosphate crystal complex microsphere of embodiment 2.
Fig. 4 is the scanning electron microscope (SEM) photograph of the prepared enzyme-cupric phosphate crystal complex microsphere of embodiment 3.
Fig. 5 is the scanning electron microscope (SEM) photograph of the prepared enzyme-cupric phosphate crystal complex microsphere of embodiment 4.
Fig. 6 is the scanning electron microscope (SEM) photograph of the prepared enzyme-cupric phosphate crystal complex microsphere of embodiment 5.
Fig. 7 is the scanning electron microscope (SEM) photograph of the prepared enzyme-cupric phosphate crystal complex microsphere of embodiment 6.
Fig. 8 is the comparison diagram of the prepared enzyme-cupric phosphate crystal complex microsphere of embodiment 6 and the absorbancy of enzyme powder and the relation curve of glucose concn.
Fig. 9 is the prepared enzyme-cupric phosphate crystal complex microsphere of embodiment 6 and the relative enzyme of the enzyme powder curve over time of living.
Figure 10 is the relation curve of absorbancy and different glucose concn in embodiment 7, and embedded figure is that glucose concn is at the linear relationship chart of 0～20 μ M internal absorbance and concentration.
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
The formula of PBS damping fluid (pH=7.4): 10mM HPO
4 2-, 2mM H
2pO
4 -, 137mM NaCl and 2.7mM KCl.
The activity of enzyme in following embodiment: candida antarctica lipase B (being about 9U/mg), Hexose phosphate dehydrogenase (100-200U/mg), cellobiose dehydrogenase (0.25-0.40U/mg), uriKoxidase (15-30U/mg), glucose oxidase (being about 15U/mg), horseradish peroxidase (being about 150U/mg), all enzymes are purchased from Sigma company if no special instructions.
In following embodiment, the charge capacity L of enzyme is obtained by following formula:
W wherein
1and W
2represent to be respectively carried on the quality (mg) and enzyme-mineral crystal complex microsphere total mass (mg) of enzyme on microballoon, C
1and C
2represent respectively to form the enzyme concn (mg/mL) in the solution of microballoon front and back, V represents to use the cumulative volume (mL) of enzyme solution.
The concentration of enzyme solution is all used BCA determination of protein concentration test kit to measure, and mensuration wavelength is 562nm.
The preparation of embodiment 1, candida antarctica lipase B (CALB)-cupric phosphate crystal complex microsphere
1. preparation enzyme concn is 0.5mg/mL the PBS damping fluid (pH=7.4) of CALB and the cupric sulfate pentahydrate aqueous solution of 200mM.
2. get enzyme solution 5mL in step 1 in vial, add the copper-bath in 0.05mL step 1, after mixing in 25 ℃ of standing 24h of incubator.
3. collecting precipitation after the mixture of step 2 gained being filtered, uses deionized water wash 3 times, again filters collecting precipitation, and lyophilize 5h obtains cupric phosphate crystal-candida antarctica lipase B complex microsphere.
As shown in Figure 2, as shown in Figure 2, the particle diameter of complex microsphere is about 10 μ m to the scanning electron microscope (SEM) photograph of the complex microsphere that the present embodiment prepares.In complex microsphere prepared by the present embodiment, the charge capacity of enzyme is 5%.
The preparation of embodiment 2-4, Hexose phosphate dehydrogenase-cupric phosphate crystal complex microsphere, cellobiose dehydrogenase-cupric phosphate crystal complex microsphere and uriKoxidase-cupric phosphate crystal complex microsphere
Respectively the candida antarctica lipase B (CALB) in embodiment 1 preparation process 1 is replaced with to Hexose phosphate dehydrogenase, cellobiose dehydrogenase and uriKoxidase, other operation is identical.
As shown in Figure 3, as shown in Figure 3, the particle diameter of complex microsphere is about 10 μ m to the scanning electron microscope (SEM) photograph of Hexose phosphate dehydrogenase-cupric phosphate crystal complex microsphere.In Hexose phosphate dehydrogenase-cupric phosphate crystal complex microsphere, enzyme charge capacity is 5%.
As shown in Figure 4, as shown in Figure 4, the particle diameter of complex microsphere is about 10 μ m to the scanning electron microscope (SEM) photograph of cellobiose dehydrogenase-cupric phosphate crystal complex microsphere.In cellobiose dehydrogenase-cupric phosphate crystal complex microsphere, the charge capacity of enzyme is 5%.
As shown in Figure 5, as shown in Figure 5, the particle diameter of complex microsphere is for being about 10 μ m for the scanning electron microscope (SEM) photograph of uriKoxidase-cupric phosphate crystal complex microsphere.In uriKoxidase-cupric phosphate crystal complex microsphere, the charge capacity of enzyme is 5%.
Embodiment 5, glucose oxidase (GO
xthe preparation of)-horseradish peroxidase (HRP)-cupric phosphate crystal complex microsphere
1. prepare respectively PBS damping fluid (the pH=7.4) (GO of the glucose oxidase that total concn is 0.5mg/mL (GOx) and horseradish peroxidase (HRP)
xbe 0.25mg/mL with HRP concentration) with the cupric sulfate pentahydrate aqueous solution of 200mM.
2. get enzyme solution 5mL in step 1 in vial, add the copper-bath in 0.05mL step 1, after mixing with 25 ℃ of standing 24h of incubator.
3. collecting precipitation after the mixture of step 2 gained being filtered, uses deionized water wash 3 times, again filters collecting precipitation, and lyophilize 5h obtains glucose oxidase (GOx)-horseradish peroxidase (HRP)-cupric phosphate crystal complex microsphere.
As shown in Figure 6, as shown in Figure 6, the particle diameter of complex microsphere is about 15 μ m to the scanning electron microscope (SEM) photograph of complex microsphere prepared by the present embodiment.In complex microsphere prepared by the present embodiment, the charge capacity of enzyme is 10%.
The preparation of glucose oxidase under embodiment 6, low temperature (GOx)-horseradish peroxidase (HRP)-cupric phosphate crystal complex microsphere
Temperature in embodiment 5 preparation processes 2 is replaced with to 4 ℃, and other operation is identical.
The thing phase composite of complex microsphere prepared by the present embodiment characterizes by X-ray diffraction (XRD) and compares with corresponding crystal standard card, as shown in Figure 1, enzyme load microballoon with do not have the microballoon analyzed pattern of enzyme load basically identical, and and Cu
3(PO
4)
23H
2the standard card coupling of O, confirms that the mineral crystal of microballoon consists of Copper phosphate (Cu3(PO4)2) trihydrate.
As shown in Figure 7, as shown in Figure 7, the particle diameter of complex microsphere is about 10 μ m to the scanning electron microscope (SEM) photograph of complex microsphere prepared by the present embodiment.In complex microsphere prepared by the present embodiment, the charge capacity of enzyme is 10%.
The activity of enzyme and stability and the detection to glucose in embodiment 7, enzyme-mineral crystal complex microsphere
The measuring method of enzymic activity: in order to verify the activity of enzyme in complex microsphere, the activity of the complex microsphere that the present embodiment prepares embodiment 6 and two free enzyme system enzymes of enzyme is measured respectively, concrete operation step is as follows: the phosphate buffered saline buffer (10mM that the complex microsphere (charge capacity of enzyme is 10%) of 30mg embodiment 6 preparations is joined to 5mL, pH7.4) in, resuspended evenly after, getting 50 μ L suspensions joins respectively in the phosphate buffered saline buffer (10mM, pH7.4) that 1mL glucose final concentration is respectively 0,4,8,12,16,20 μ M.In the buffered soln of the glucose of different concns, adding respectively final concentration is 22-connection nitrogen-bis-(3-ethyl-benzothiazole-6-sulfonic acid) di-ammonium salts (ABTS) of 1mM, mixes fast and be incubated (25 ℃) 5 minutes, measures the absorption value of 405nm.Blank assay is for being changed to above-mentioned complex microsphere GOx and the HRP powder (GO of 3mg enzyme total mass
xbe 1.5mg with the quality of HRP), other operation is all identical.
By difference matching complex microsphere and the absorbancy of free enzyme powder and the relation curve of glucose concn, draw respectively the slope of complex microsphere and free enzyme powder relation curve, according to slope ratio, relatively enzyme is alive.Experimental result as shown in Figure 8, compare and improved 140% with free enzyme powder as shown in Figure 8 by the total enzyme activity of complex microsphere.
The measuring method of enzyme stability: in order to verify the stability of enzyme in complex microsphere, the present invention has investigated respectively enzyme enzyme alive and free enzyme powder the situation over time alive of complex microsphere, concrete operation step is as follows: the phosphate buffered saline buffer (10mM that two enzyme complex microspheres (charge capacity of enzyme is 10%) of 30mg embodiment 6 preparations is joined to 5mL, pH7.4) in, resuspended evenly after, get 50 μ L suspensions to join glucose that 1mL final concentration is 10mM and final concentration be 1mM 2, phosphate buffered saline buffer (the 10mM of 2-connection nitrogen-bis-(3-ethyl-benzothiazole-6-sulfonic acid) di-ammonium salts (ABTS), pH7.4) in, mix fast and be incubated (25 ℃) 2 minutes, measure the absorption value of 405nm.Blank assay is for being changed to above-mentioned complex microsphere GOx and the HRP powder (GO of 3mg enzyme total mass
xbe 1.5mg with the quality of HRP), other operation is all identical.The initial absorbance that definition complex microsphere or enzyme powdered sample record is that enzyme work 100%, 24 hour is to measure once for one day, totally 7 days.
Fig. 9 is the package stability detection figure of the prepared complex microsphere of embodiment 6 and enzyme powder.As shown in Figure 9, after 24 hours, in complex microsphere, enzymic activity still remains on more than 90%, and enzyme powder only retains 20% left and right, and in complex microsphere, the stability of enzyme is compared remarkable lifting with free enzyme powder.
The detection of glucose: the complex microsphere of embodiment 6 preparations is applied to the detection of glucose, concrete operations are as follows: the phosphate buffered saline buffer (10mM that 30mg microballoon is joined to 5mL, pH7.4) in, resuspended evenly after, getting 50 μ L suspensions, to join 1mL final concentration be 0 μ M, 4 μ M, 8 μ M, 12 μ M, 16 μ M, 20 μ M, 40 μ M, 60 μ M, phosphate buffered saline buffer (the 10mM of the glucose of 80 μ M and 100 μ M, pH7.4) in, in the glucose solution of above-mentioned different concns, adding respectively final concentration is 2 of 1mM, 2-connection nitrogen-bis-(3-ethyl-benzothiazole-6-sulfonic acid) di-ammonium salts (ABTS), mix fast and be incubated (25 ℃) 5 minutes, measure the absorption value of 405nm.
Figure 10 is the relation curve of absorbancy and glucose concn.As shown in Figure 10, in the scope that is 0～100mM at glucose concn, along with the increase of concentration, absorbance increases gradually.Therefore, the complex microsphere of embodiment 6 preparations is 0～100 μ M to the sensing range of glucose, and at 0～20 μ M scope internal linear better (R=0.995), detectability is about 0.3 μ M.
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 (9)
1. a preparation method for enzyme-mineral crystal complex microsphere, comprises the steps:
Metal ion and enzyme are all dissolved in phosphate buffered saline buffer, obtain described enzyme-mineral crystal complex microsphere;
Described metal ion be in calcium ion, magnesium ion, iron ion, ferrous ion, cupric ion, bivalent nickel ion or zine ion any;
Described phosphate buffered saline buffer comprises two of phosphoric acid hydrogen and dihydrogen phosphate.
2. preparation method according to claim 1, is characterized in that: the molecular weight of described enzyme is 5～500kD.
3. according to preparation method described in claim 1 or 2, it is characterized in that: described enzyme is candida antarctica lipase B, glucose oxidase, horseradish peroxidase, ethanol dehydrogenase, sucrase, superoxide-dismutase, catalase, Selenoperoxidase, laccase, Hexose phosphate dehydrogenase, cellobiose dehydrogenase, uriKoxidase, antarctic candidia lipase A, fold lipase from candida sp, porcine pancreatic lipase, rhizomucor miehei lipase and dredges at least one in the thermophilic hyphomycete lipase of cotton shape.
4. according to preparation method described in claim 1-3 any one, it is characterized in that: in the mixed solution that described enzyme and described phosphate buffered saline buffer form, the concentration of described enzyme is 0.01～10mg/mL;
In described phosphoric acid buffer, the concentration of two of described phosphoric acid hydrogen and described dihydrogen phosphate is 1～1000mM;
The pH of described phosphoric acid buffer is 5.0～9.0.
5. according to preparation method described in claim 1-4 any one, it is characterized in that: described metal ion is added in described phosphate buffered saline buffer with the form of its aqueous solution;
In the described aqueous solution, the concentration of metal ion is 10～1000mM.
6. according to preparation method described in claim 1-5 any one, it is characterized in that: the mass ratio of described metal ion and described enzyme is 0.1～5:1.
7. according to preparation method described in claim 1-6 any one, it is characterized in that: under standing condition, at the temperature of 0～50 ℃, obtain described enzyme-mineral crystal complex microsphere;
The described standing time is 1～100 hour.
8. according to preparation method described in any one in claim 1-7, it is characterized in that: described hybrid mode is any during dropping mixing and microreactor mix.
9. the prepared enzyme-mineral crystal complex microsphere of preparation method described in any one in claim 1-8.
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Cited By (9)
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| CN104357436A (en) * | 2014-11-03 | 2015-02-18 | 西北工业大学 | Preparation method for flower-shaped immobilized enzyme with high specific surface area |
| CN105442319A (en) * | 2015-12-07 | 2016-03-30 | 武汉纺织大学 | Preparation method of nanofiber membrane material with surface fixedly provided with three-dimensional flower-shaped structure protein |
| CN105754985A (en) * | 2016-04-15 | 2016-07-13 | 清华大学 | Enzyme-metal ion nano compound and preparation method thereof |
| CN107988201A (en) * | 2016-10-26 | 2018-05-04 | 上海医药工业研究院 | A kind of preparation method of alcohol dehydrogenase and glucose dehydrogenase co-crosslinking enzyme aggregate |
| CN108342433A (en) * | 2018-02-02 | 2018-07-31 | 杭州师范大学 | A kind of compound enzyme crystal of lipase-calcium phosphate, preparation method and its method for catalyzing and synthesizing clindamycin palmitate |
| CN110609070A (en) * | 2019-08-31 | 2019-12-24 | 广东工业大学 | Nanometer flower and application thereof in detecting glucose concentration |
| CN111019932A (en) * | 2019-11-28 | 2020-04-17 | 湖南大学 | Preparation method of copper phosphate-enzyme mineralized material, product and application thereof |
| CN114574476A (en) * | 2022-04-13 | 2022-06-03 | 浙江工业大学 | Alcohol dehydrogenase-inorganic hybrid nanoflower and preparation method and application thereof |
| CN114854731A (en) * | 2022-03-02 | 2022-08-05 | 赣南师范大学 | Immobilized alkaline phosphatase, and immobilization method and application thereof |
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2014
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| CN104357436A (en) * | 2014-11-03 | 2015-02-18 | 西北工业大学 | Preparation method for flower-shaped immobilized enzyme with high specific surface area |
| CN105442319A (en) * | 2015-12-07 | 2016-03-30 | 武汉纺织大学 | Preparation method of nanofiber membrane material with surface fixedly provided with three-dimensional flower-shaped structure protein |
| CN105754985A (en) * | 2016-04-15 | 2016-07-13 | 清华大学 | Enzyme-metal ion nano compound and preparation method thereof |
| CN107988201A (en) * | 2016-10-26 | 2018-05-04 | 上海医药工业研究院 | A kind of preparation method of alcohol dehydrogenase and glucose dehydrogenase co-crosslinking enzyme aggregate |
| CN107988201B (en) * | 2016-10-26 | 2021-06-01 | 上海医药工业研究院 | Preparation method of ethanol dehydrogenase and glucose dehydrogenase co-crosslinked enzyme aggregate |
| CN108342433A (en) * | 2018-02-02 | 2018-07-31 | 杭州师范大学 | A kind of compound enzyme crystal of lipase-calcium phosphate, preparation method and its method for catalyzing and synthesizing clindamycin palmitate |
| CN108342433B (en) * | 2018-02-02 | 2020-12-22 | 杭州师范大学 | Lipase-calcium phosphate complex enzyme crystal, preparation method thereof and method for catalytically synthesizing clindamycin palmitate by using lipase-calcium phosphate complex enzyme crystal |
| CN110609070A (en) * | 2019-08-31 | 2019-12-24 | 广东工业大学 | Nanometer flower and application thereof in detecting glucose concentration |
| CN111019932A (en) * | 2019-11-28 | 2020-04-17 | 湖南大学 | Preparation method of copper phosphate-enzyme mineralized material, product and application thereof |
| 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 |
| CN114854731A (en) * | 2022-03-02 | 2022-08-05 | 赣南师范大学 | Immobilized alkaline phosphatase, and immobilization method and application thereof |
| CN114574476A (en) * | 2022-04-13 | 2022-06-03 | 浙江工业大学 | Alcohol dehydrogenase-inorganic hybrid nanoflower and preparation method and application thereof |
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