CN110628756A - Co-immobilized enzyme and preparation method and application thereof - Google Patents
Co-immobilized enzyme and preparation method and application thereof Download PDFInfo
- Publication number
- CN110628756A CN110628756A CN201910979731.8A CN201910979731A CN110628756A CN 110628756 A CN110628756 A CN 110628756A CN 201910979731 A CN201910979731 A CN 201910979731A CN 110628756 A CN110628756 A CN 110628756A
- Authority
- CN
- China
- Prior art keywords
- enzyme
- solution
- immobilized enzyme
- catalase
- laccase
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F3/00—Biological treatment of water, waste water, or sewage
- C02F3/34—Biological treatment of water, waste water, or sewage characterised by the microorganisms used
- C02F3/342—Biological treatment of water, waste water, or sewage characterised by the microorganisms used characterised by the enzymes used
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/04—Enzymes or microbial cells immobilised on or in an organic carrier entrapped within the carrier, e.g. gel or hollow fibres
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/10—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a carbohydrate
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/18—Multi-enzyme systems
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
- C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
- C12N9/0061—Laccase (1.10.3.2)
-
- C—CHEMISTRY; METALLURGY
- 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
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0065—Oxidoreductases (1.) acting on hydrogen peroxide as acceptor (1.11)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y110/00—Oxidoreductases acting on diphenols and related substances as donors (1.10)
- C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
- C12Y110/03002—Laccase (1.10.3.2)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y111/00—Oxidoreductases acting on a peroxide as acceptor (1.11)
- C12Y111/01—Peroxidases (1.11.1)
- C12Y111/01006—Catalase (1.11.1.6)
-
- C—CHEMISTRY; METALLURGY
- C02—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F—TREATMENT OF WATER, WASTE WATER, SEWAGE, OR SLUDGE
- C02F2101/00—Nature of the contaminant
- C02F2101/30—Organic compounds
- C02F2101/38—Organic compounds containing nitrogen
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biochemistry (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Dispersion Chemistry (AREA)
- Inorganic Chemistry (AREA)
- Biodiversity & Conservation Biology (AREA)
- Hydrology & Water Resources (AREA)
- Environmental & Geological Engineering (AREA)
- Water Supply & Treatment (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention relates to a co-immobilized enzyme and a preparation method and application thereof, wherein the co-immobilized enzyme is sodium alginate-CaCl2And (3) taking the gel and the active carbon as carriers, and immobilizing catalase and laccase. The co-immobilized enzyme adopts an embedding method and physicsThe two methods of the adsorption method are combined for immobilization, the heat resistance, acid and alkali resistance, storage stability and operation stability of the obtained co-immobilized enzyme are all obviously improved, the co-immobilized enzyme has an excellent treatment effect on the aniline wastewater, the operation stability of the aniline wastewater is good, and the cost of the co-immobilized enzyme actually applied to wastewater treatment can be greatly reduced.
Description
Technical Field
The invention relates to an enzyme immobilization technology, in particular to a co-immobilized enzyme and a preparation method and application thereof.
Background
The co-immobilized enzyme refers to an immobilization technique in which two or more enzymes are immobilized simultaneously on the same carrier to produce a co-immobilized system. The co-immobilized enzyme can fully exert respective catalytic characteristics of different enzymes, combines respective enzyme catalytic characteristics, and synergistically and fully utilizes the two enzymes to increase the catalytic reaction efficiency of the co-immobilized enzyme on complex substrates. Meanwhile, the reaction steps and time can be reduced, and the efficiency of the product can be better improved.
In the prior art, a scholars co-immobilizes cellulase and lysozyme on the surface of amino-functionalized magnetic nanoparticles for cell wall degradation reaction. The competition between cellulase and lysozyme during the immobilization process is due to the limited active sites of MNPs. 78.9% cellulase and 69.6% lysozyme in the maximum active site were synergistic effects of the two-enzyme co-immobilization. The heat stability of the half-life period of the co-immobilized enzyme is 3h, and the co-immobilized enzyme has higher catalytic efficiency on cell wall hydrolysis. In addition, co-immobilized enzymes have higher thermostability and broader pH tolerance than free enzymes. After 6 recycles, the co-immobilized enzyme retained 60% of the residual activity.
Patent application CN107012138A discloses a silicon/carbon-based composite immobilized enzyme environment-friendly material and a preparation method thereof. The environment-friendly material is prepared by immobilizing compound biological enzyme on modified silicon-carbon-based carriers, wherein the carriers contain nano-scale microcrystal structural copolymer to form the modified silicon-carbon-based immobilized enzyme biological material. The material must be modified first, and the operation is complex.
Disclosure of Invention
The invention aims to overcome the problems of insufficient enzyme immobilization technology and poor material stability in the prior art, and provides a co-immobilized enzyme, a preparation method and application thereof.
The key point of the invention is to prepare the co-immobilized enzyme with good enzyme and carrier combination effect, and the inventor finds that the influencing factors are arranged from primary to secondary: adding amount of active carbon>Sodium alginate concentration>CaCl2And (4) concentration. As the influence of the activated carbon is the greatest, the more the activated carbon is added, the better the adsorption is, and the sodium alginate solution and CaCl are treated by the activated carbon2In the solution, the higher the concentration of the two components is, the more compact the components in the rubber ball are, thus being not beneficial to the mass transfer of the substrate and generating steric effect. Therefore, the optimal scheme is as follows: 200mL of complex enzyme solution, 0.15g of active carbon, 2 percent of sodium alginate and CaCl2The concentration was 2%. Under the condition, the adsorption performance and the mass transfer effect of the hollow carbon rubber ball are optimal, if the immobilized enzyme is prepared by attaching the free enzyme into the rubber ball, the substrate can enter and exit the rubber ball more easily, namely the mass transfer effect is better, and the internal enzyme and the substrate can obtain better contact effect, so that the catalytic reaction rate is higher, and better enzyme activity can be expressed, therefore, the condition is adopted in the research to prepare the immobilized enzyme.
The immobilization carrier used in the invention is a sodium alginate-active carbon composite carrier, laccase and catalase are co-immobilized by adopting an adsorption and embedding method, and the method is obviously different from the conventional immobilization carrier and immobilization method. Therefore, the immobilization method of the invention has simpler operability and simpler immobilization material; and the composite carrier of the invention does not need to be modified.
The specific scheme is as follows:
the co-immobilized enzyme is sodium alginate-CaCl2And (3) taking the gel and the active carbon as carriers, and immobilizing catalase and laccase.
Further, the optimal temperature of the co-immobilized enzyme is 45 ℃;
optionally, the co-immobilized enzyme has high operational stability, and catalase and laccase still retain more than 61% and more than 74% of activity after 5 times of recycling at 25 ℃.
Further, the co-immobilized enzyme has good storage stability, and the immobilized catalase and laccase still retain more than 65% and more than 61% of activity after being stored for 30 days at 4 ℃.
The preparation method of the co-immobilized enzyme comprises the following steps:
step 1: preparing a mixed buffer solution of catalase and laccase;
step 2: mixing the complex enzyme solution with active carbon, adding sodium alginate after complete adsorption, stirring, cooling, granulating with injector, and adding CaCl2In the aqueous solution of (A), smooth beads are generated, and the immobilized gel beads are in CaCl2Is hardened, and then washed with water and dried to obtain a co-immobilized enzyme.
Further, the step 1 comprises the steps of 1a dissolving catalase freeze-dried powder by using a phosphate buffer solution to obtain a catalase solution, 1b dissolving laccase freeze-dried powder by using an acetic acid-sodium acetate buffer solution to obtain a laccase solution, and 1c mixing the catalase solution and the laccase solution.
Further, the phosphate buffer has a pH of 6 to 7 and a concentration of 0.05 to 0.1M;
optionally, the concentration of catalase in the catalase solution is 0.5-1.25 mg/mL;
optionally, the acetic acid-sodium acetate buffer has a pH of 4-5 and a concentration of 0.2-0.3M;
optionally, the concentration of laccase in the laccase solution is 0.5-1.25 mg/mL;
optionally, when the catalase solution and the laccase solution are mixed, the mass ratio of the catalase to the laccase is 1:2-2: 1.
Further, in the step 2, the ratio of the addition amount of the activated carbon to the w: v ratio of the complex enzyme solution is 1:100, and the volume ratio of the addition amount of the sodium alginate solution to the complex enzyme solution is 1: 5;
optionally, the sodium alginate is 1-3% aqueous solution.
Further, in the step 2, CaCl2The mass concentration of the aqueous solution is 1-3%.
Further, in the step 2, the temperature of the ice water bath is reduced to 0-4 ℃, and the immobilization time is 30-60 min.
The invention also protects the application of the co-immobilized enzyme, and the co-immobilized enzyme is used for treating aniline wastewater.
Has the advantages that:
the invention combines the embedding method and the physical adsorption method to carry out enzyme immobilization, the heat resistance, the acid and alkali resistance, the storage stability and the operation stability of the obtained co-immobilized enzyme are obviously improved, the co-immobilized enzyme has excellent treatment effect on the aniline wastewater, the operation stability of the aniline wastewater is better, and the cost of the co-immobilized enzyme actually applied to wastewater treatment can be greatly reduced.
Drawings
In order to illustrate the technical solution of the present invention more clearly, the drawings will be briefly described below, and it is apparent that the drawings in the following description relate only to some embodiments of the present invention and are not intended to limit the present invention.
FIG. 1 is a graph showing the effect of free enzyme concentration on the activity of an immobilized enzyme, as provided in example 2 of the present invention;
FIG. 2 is a graph showing the effect of immobilization time on the activity of an immobilized enzyme as provided in example 3 of the present invention;
FIG. 3 is a graph showing the effect of the ratio of catalase and laccase on the co-immobilized enzyme activity, as provided in example 4 of the present invention;
FIG. 4 is a graph showing the effect of reaction temperature on co-immobilized enzyme activity, as provided in example 5 of the present invention;
FIG. 5 is a graph showing the effect of reaction pH on co-immobilized enzyme activity as provided in example 6 of the present invention;
FIG. 6 is a graph showing the effect of storage time on co-immobilized enzyme activity as provided in example 7 of the present invention;
FIG. 7 is a graph of the effect of the number of reuses on the activity of co-immobilized enzymes provided in one embodiment 8 of the present invention;
FIG. 8 is a graph of the reciprocal of the free catalase and the immobilized catalase provided in example 9 of the present invention;
FIG. 9 is a reciprocal plot of the free laccase and the immobilized laccase provided in example 9 of the present invention.
Detailed Description
Preferred embodiments of the present invention will be described in more detail below. While the following describes preferred embodiments of the present invention, it should be understood that the present invention may be embodied in various forms and should not be limited by the embodiments set forth herein. The examples do not specify particular techniques or conditions, and are performed according to the techniques or conditions described in the literature in the art or according to the product specifications. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available. In the following examples, "%" means weight percent, unless otherwise specified.
The test methods used below included:
firstly, enzyme activity determination: because the enzymes used in the embodiment of the invention are all commercial industrial products, the pure products are difficult to produce, and the enzyme freeze-dried powder contains more or less certain impurities, the enzyme content in the prepared enzyme solution can be expressed by the catalytic reaction capability, namely the enzyme activity. Enzyme activity is also the rate of catalytic reaction. The rate of an enzyme-catalyzed reaction refers to the amount of product produced or the amount of substrate reduced per unit time. The enzyme activity is not equal to the absolute content of the enzyme, but is merely a representation of the relative content.
First, a standard curve is drawn as follows:
(1) drawing a hydrogen peroxide standard curve:
accurately preparing H with different concentrations2O2Solution in H2O2And measuring the absorbance value A of the hydrogen peroxide at the characteristic wavelength of 240nm, taking the solution concentration (mmol/L) as an abscissa and the corresponding absorbance value A as an ordinate, and performing linear fitting analysis to obtain a hydrogen peroxide standard curve.
(2) ABTS (Chinese name: 2, 2-diaza-bis (3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt) free radical standard curve drawing:
accurately preparing ABTS solutions with different concentrations, decomposing the ABTS with laccase catalysis at different concentrations, obtaining ABTS free radical solutions with different concentrations after reacting for a long enough time, measuring the absorbance value A of the ABTS free radical under the characteristic wavelength of 420nm, taking the solution concentration (mmol/L) as a horizontal coordinate, and taking the corresponding absorbance value A as a vertical coordinate to obtain an ABTS free radical standard curve.
Determination of free catalase enzyme activity:
0.05mL of catalase at a certain concentration was added dropwise to 10mL of H2O2Oscillating the substrate solution (10mM) at constant temperature of 25 ℃ and 140rpm for 10min, taking out the substrate solution immediately after the reaction is finished, using phosphate buffer solution as reference solution, and measuring H in the solution by using an ultraviolet spectrophotometer2O2The change in absorbance at 240nm due to the catalysis of catalase. The enzyme activity was calculated as the linear range of the reaction, 1 enzyme activity unit (U) being the amount of enzyme required to degrade 1. mu. mol of hydrogen peroxide per minute.
Determination of immobilized catalase enzyme activity:
0.05mL of catalase enzyme solution in the above step was replaced with 0.5g of immobilized enzyme, and finally the immobilized enzyme was filtered from the substrate solution to terminate the reaction. The remaining steps are the same as the measurement of the enzyme activity of the free enzyme. The enzyme activity was calculated as the linear range of the reaction, degrading 1. mu. mol H per minute2O2The amount of enzyme required is defined as one activity unit, expressed in U.
And (3) measuring the enzyme activity of the free laccase:
0.05mL of laccase solution with a certain concentration is dripped into 10mL of ABTS substrate solution, the mixture is vibrated for 10min at a constant temperature of 25 ℃ and at a rotating speed of 140rpm, the mixture is immediately taken out after the reaction is finished, acetic acid-sodium acetate buffer solution is used as reference solution, and an ultraviolet spectrophotometer is used for measuring the change value of the absorbance value of ABTS free radicals in the solution at 420nm, which is generated by laccase catalytic oxidation. The enzyme activity was calculated as the linear range of the reaction, and 1 enzyme activity unit (U) means the amount of enzyme required to increase the absorbance of 1. mu. moL of substrate by 0.001 per minute.
And (3) measuring enzyme activity of the immobilized laccase:
and replacing 0.05mL of laccase enzyme solution in the step with 0.5g of immobilized enzyme, finally filtering the immobilized enzyme from the substrate, and stopping the reaction. The remaining steps are the same as the procedure for measuring the activity of the free enzyme. The enzyme activity was calculated as the linear range of the reaction, and 1 enzyme activity unit (U) means the amount of enzyme required to increase the absorbance of 1. mu. moL of substrate by 0.001 per minute.
The relative enzyme activity calculation method of the immobilized enzyme comprises the following steps:
the relative enzyme activity of the immobilized enzyme means the ratio of the highest activity of 100 in the same set of experiments to the activity of other immobilized enzymes or free enzymes, expressed as a percentage (%).
Determination of the two, Mil constant
(1) Michael constant of Catalase
Hydrogen peroxide solutions were prepared in which the substrate concentrations were 2mM, 4mM, 6mM, 8mM, 10mM, and 12mM, respectively, and the changes in absorbance at 240nm were measured for each of the free catalase and the co-immobilized enzyme, respectively, to calculate the initial rates of the enzyme reaction kinetics. According to the Lineweaer-Burk method, a reciprocal plot can be obtained by plotting the reciprocal of the initial rate of the enzyme reaction as the ordinate and the reciprocal of the substrate concentration as the abscissa. The absorbance value A after the reaction (3min) was measured, and the reciprocal 1/A of the absorbance value was taken as the ordinate, which represents the reciprocal 1/V of the reaction rate. Using the intercept of the straight line in the X axis and the Y axis and using the Mie equation to obtain the Mie constant KmAnd maximum reaction velocity Vmax。
(2) Michael's constant on a scale of
ABTS solution reaction systems with substrate concentrations of 0.2mM, 0.4mM, 0.6mM, 0.8mM, 1.0mM and 1.2mM are prepared respectively, the absorbance change of the free laccase and the co-immobilized enzyme at 420nm is measured respectively, and the initial speed of the enzyme reaction kinetics is calculated. According to the Lineweaer-Burk method, plots were made with the reciprocal of the initial velocity of the enzyme reaction as the ordinate and the reciprocal of the substrate concentration as the abscissa, and as described above, a double reciprocal plot was obtained and the Michael constant was obtained from the Michael equationKmAnd maximum reaction velocity Vmax。
Calculation of K for free and Co-immobilized enzymesmThe formula (1) can be seen.
Wherein v is the initial reaction rate, and μmol/(L.min); v is the maximum reaction speed, mu mol/(L.min); [ S ]]Substrate concentration, mol/L; kmIs the Michaelis constant, mol/L.
Example 1
Preparing co-immobilized enzyme according to the following steps:
step 1: preparing a mixed buffer solution of catalase and laccase.
Preparation of phosphate buffer solution:
weighing Na2HPO4 3.4092g，NaH2PO41.5944g, dissolved in distilled water, adjusted to pH7.0, and made to volume of 500mL to give 0.05M phosphate buffer, which was stored at 4 deg.C (boiled before use).
Preparation of catalase solution:
accurately weighing catalase freeze-dried powder on an electronic balance, dissolving the catalase freeze-dried powder in 0.05M phosphate buffer solution with the pH of 7 to obtain an enzyme solution with a certain concentration, placing the enzyme solution in a refrigerator at 4 ℃ after preparation, and using the enzyme solution as soon as possible.
Preparing an acetic acid-sodium acetate buffer solution:
solution 1: 0.2mol/L sodium acetate solution, 8.2g sodium acetate, deionized water are dissolved and the volume is determined in a 500mL volumetric flask.
Solution 2: 0.2mol/L acetic acid solution, 6g glacial acetic acid is taken and added with deionized water to be dissolved in a 500mL volumetric flask.
The solution 1 and the solution 2 are mixed according to a specific volume ratio, the pH value of the prepared buffer solution is measured by a pH meter, and the volume of the mixture ratio is shown in the following table. The volume ratio of pH 4.5 was selected, 500mL of buffer was prepared, and the buffer was stored at 4 ℃.
Preparing a laccase solution:
accurately weighing laccase freeze-dried powder by a certain mass on an electronic balance, dissolving the laccase freeze-dried powder by using 0.2M acetic acid-sodium acetate buffer solution with the pH value of 4.5 to obtain an enzyme solution with a certain concentration, placing the enzyme solution in a refrigerator at 4 ℃ after preparation, and using the enzyme solution as soon as possible.
And mixing the catalase solution and the laccase solution to obtain the compound enzyme solution.
Step 2: mixing the complex enzyme solution with active carbon, adding sodium alginate after complete adsorption, stirring, cooling, granulating with injector, and adding CaCl2In the aqueous solution of (A), smooth beads are generated, and the immobilized gel beads are in CaCl2Is hardened, and then washed with water and dried to obtain a co-immobilized enzyme. The specific adding amount is as follows: 200mL of complex enzyme solution, 0.15g of active carbon, 2% of sodium alginate solution and 2% of CaCl2Solution, enzyme solution concentration of 0.75mg/mL, immobilization time of 40min, and catalase-laccase volume ratio of 1:1.8 (i.e. enzyme activity ratio 233: 1).
Example 2
On the basis of example 1, the concentration of the enzyme solution is changed, and the result is shown in figure 1, and as can be seen from figure 1, the relative enzyme activities of the immobilized laccase and the catalase continuously increase along with the increase of the concentration of the enzyme solution, and when the concentration of the immobilized laccase and the catalase reaches 0.75mg/mL, the relative enzyme activities of the two enzymes after the immobilization are maximum; however, as the concentration of the enzyme solution is increased, the relative enzyme activities of the immobilized enzymes are reduced, which may be caused by the fact that the internal space of the carrier particle is constant and the number of active groups is limited under the condition of constant particle size and carrier mass. Before the binding sites of the immobilized carriers are not saturated, the activities of the two immobilized enzymes are continuously increased; however, when the active sites in the internal space of the carrier are saturated, the enzyme carrying amount will reach the limit, and the activity will not be increased any more when the enzyme amount is increased. Firstly, certain enzyme molecules enter the deep part of micropores in carrier activated carbon during immobilization, so that the diffusion resistance of a substrate is increased, and the catalytic capability of the enzyme is reduced; secondly, when the number of the enzyme molecules is too large, the enzyme proteins can also generate a blocking effect, the plasticity of the space structure of the enzyme molecules is also reduced, the enzyme molecules cannot fully play a catalytic role, and the activity of the immobilized enzyme is reduced. Therefore, in the process of producing an immobilized enzyme, the concentration of the enzyme solution to be added is preferably selected to be 0.75 mg/mL.
Example 3
The results are shown in FIG. 2, which shows that CaCl is present in CaCl, on the basis of example 1, with varying immobilization times2When the immobilization time in the solution is 40min, the relative enzyme activity of the immobilized laccase and the immobilized catalase is the highest, and the relative activity of the immobilized laccase and the immobilized catalase begins to decrease after the immobilization time is increased. The reason is that after the reaction of Ca ions and sodium alginate, the carrier forms a gridding model, the active carbon absorbed with free enzyme is stably fixed in the carrier, and after a certain hardening time, enzyme molecules are in CaCl2The stability of the solution is not too high, Ca ions in the solution have certain inhibition effect on enzyme molecules, and the relative activity of the immobilized enzyme is reduced. In addition, the longer the hardening time is, the tighter the combination of the components in the immobilized enzyme is, the more crowded the enzyme molecules and other components are, the plasticity of the spatial structure of the enzyme molecules is reduced, the steric effect is generated, and the mass transfer of the substrate is not facilitated, so that the whole enzyme activity is reduced. Therefore, in the preparation process of the immobilized enzyme, the immobilization time of 40min is selected as the best time.
Example 4
Based on example 1, different enzyme ratios of catalase and laccase are respectively adopted, the result is shown in FIG. 3, and as can be seen from FIG. 3, in the co-immobilized enzyme, the enzyme activity is correspondingly increased along with the increase of the ratio of one enzyme, and the activities of the two enzymes are increased along with the increase of the ratio of the respective enzyme solutions in the mixed enzyme solution. Both catalase and laccase had better activity when the mixing ratio of the two enzymes was 1:1.8 (w/w). Therefore, in the preparation process of the co-immobilized enzyme, the ratio of catalase to laccase is optimally selected to be 1:1.8 (w/w).
Furthermore, even when the mixing ratio of catalase and laccase is the same (1:1), the relative activities of catalase and laccase in the co-immobilized enzyme are not consistent, because of the competitive effect of catalase and laccase in the co-immobilization process. Of catalase and laccaseThe active sites interact with each other, so that free electrons are mutually transmitted and competitively absorbed, and mutually compete for free oxygen atoms to carry out coordination and combination. Catalase catalysis H2O2The reaction generates a large amount of OH free radicals, and the catalytic reduction state substrate reaction of the laccase leads the substrate to be oxidized to generate the free radicals. Meanwhile, various non-enzymatic secondary reactions (hydroxylation, disproportionation, polymerization and the like) of the laccase absorb electrons to be transferred to laccase active centers, and superoxide transition bodies are formed through double-electron reactions and then are reduced into water. The overall process of the reaction requires continuous one-electron oxidation to satisfy the full reduction of the laccase. Catalase can rapidly scavenge some of the H generated by the reaction of the substrate in solution2O2And the peroxide is used for reducing other influences on the main catalytic reaction of the laccase. After the two enzymes are combined into a double-enzyme system, the existence of the competitive effect also enables the transfer of free electrons and oxygen atoms between each active site to be more efficient, and reduces some secondary reactions, thereby improving the efficiency of catalytic oxidation. In conclusion, the addition of a certain amount of catalase makes the overall catalytic performance of the co-immobilized enzyme more efficient.
Example 5
The co-immobilized enzyme prepared in example 1 was taken, the reaction temperature of the enzyme was changed in the range of 15-65 ℃ and kept at the temperature in the water bath, and the effect of the temperature on the activities of catalase and laccase after dissociation and immobilization was investigated (the highest of the respective enzyme activities was taken as 100%), and the results are shown in FIG. 4. from FIG. 4, it can be seen that in the lower temperature region, similar to a general catalyst, the activity of the enzyme increased with the increase of the reaction temperature, and when the reaction temperature of the free enzyme reached 35 ℃, both free enzymes began to be inactivated, and the activity began to decrease; the optimal temperature of the immobilized catalase and laccase is 45 ℃, so that the immobilized enzyme can adapt to temperature change and has higher stability. This is mainly due to the fact that high temperatures can destroy higher order structures in the enzyme protein, resulting in thermal denaturation of the enzyme molecules and eventual inactivation. Because the immobilized enzyme is stably immobilized in the carrier bead through the physicochemical action, the physicochemical property of the co-immobilized enzyme is changed, and the change of the enzymological property is caused, therefore, the immobilized enzyme can still keep the tertiary structure of the protein unchanged at higher temperature and continuously carry out catalytic reaction, the enzyme activity keeps higher level, and the immobilized enzyme has stronger adaptability to high temperature, thereby creating the possibility for the co-immobilized enzyme in practical application.
Example 6
The co-immobilized enzyme prepared in example 1 was reacted in a constant temperature water bath at 25 ℃ while changing the pH of the reaction in the range of pH 2 to pH 9, and the effect of pH on the activities of catalase and laccase after the release and immobilization was examined (the highest of the respective enzyme activities was 100%), and the results are shown in FIG. 5, in which the optimum pH of the catalase after the release was 7 and the optimum pH of the catalase after the immobilization was 8; the pH optimum of the free laccase is 5, and the pH optimum of the immobilized free laccase is 4. It can be seen that the immobilized enzyme can adapt to the change of pH value, is more resistant to acid and alkali and has higher stability. The process of enzyme immobilization generally has certain influence on the ionization state, dissociation state and enzyme molecular conformation, which is related to the pH value, stability and catalytic activity of the immobilized enzyme. This indicates that the immobilization process provides a stable structure for the enzyme, preventing irreversible unfolding of the enzyme protein molecules, thereby making the pH of the immobilized enzyme more stable relative to the pH of the free enzyme. Changes in the inhibition of enzyme activity at relatively low or high pH ranges may be due to two reasons, one being that the conformation of the enzyme molecule may change, giving rise to unfavorable charge assignments to the amino acid residues of the enzyme; secondly, the change of the pH environment can affect hydrogen bonds in enzyme molecules, so that the conformation of the enzyme is inaccurate, and the enzyme activity is reduced.
Both enzymes have their own optimum pH reaction conditions, i.e., the pH at which they exhibit the highest catalytic reaction rate. Changing the pH value in the reaction system changes the charged property of the carrier and causes the group at the enzyme active center to be ionized, resulting in the decrease of the enzyme activity. When the change of pH value in the reaction system is large, some chemical bonds for maintaining the tertiary structure of the enzyme molecule may be affected, so that the enzyme protein molecule itself causes irreversible denaturation. In a word, the change of pH can change the enzyme activity of free enzyme to a great extent, and the immobilized carrier changes the microenvironment [41] around enzyme molecules, so that the immobilized enzyme has higher stability to the change of pH value, and simultaneously creates the possibility for the co-immobilized enzyme in practical application.
Example 7
The co-immobilized enzymes prepared in example 1 were stored at 4 ℃ for 30 days, and then the effects of storage time on the activities of catalase and laccase after dissociation and immobilization were examined (the respective enzyme activities immediately after storage were taken as 100%), and the difference in the activity of the co-immobilized enzymes was analyzed by comparison after 30 days of storage. The results are shown in FIG. 6, and it can be seen from FIG. 6 that the storage stability of the immobilized enzyme at 4 ℃ is better than that of the free enzyme. At day 30, 39.5% of free catalase enzyme activity and 45.76% of free laccase enzyme activity remain, while the enzyme activity remaining after immobilized catalase is 65.51% and the enzyme activity remaining after immobilized laccase is 61.35%. During storage, the free enzyme gradually lost its activity, and it was found that the enzyme solution became somewhat turbid, and some floc was present due to the protein substances precipitated by enzyme denaturation. After the enzyme is fixed in the carrier, a relatively stable microenvironment is created around the enzyme molecules and the structure of the enzyme molecules can be maintained due to the existence of physical embedding and chemical bonds, so that the denaturation speed of the enzyme protein is reduced. Therefore, the storage stability of the co-immobilized enzyme is much higher than that of the free enzyme, which is also one of the evaluation factors of the possibility of practical application of the co-immobilized enzyme.
Example 8
The co-immobilized enzyme prepared in example 1 was recycled at 25 ℃ for 5 times, and then the effects of the recycling times on the activities of the catalase and laccase after dissociation and immobilization (the respective enzyme activities for the first use were taken as 100%) were examined, and the difference in the activity of the co-immobilized enzyme before and after each recycling was comparatively analyzed. The results are shown in FIG. 7, and it can be seen from FIG. 7 that the enzymatic activities of catalase and laccase in the co-immobilized enzyme gradually decreased with the increase of the number of repeated use, but the operation stabilities of both enzymes were good, and after 5 cycles of recycling, 61.44% of the catalase remainedAnd the laccase still has 74.24 percent of activity. Meanwhile, as can be seen from the figure, the curve does not drop immediately, and the activity of the co-immobilized enzyme is not reduced immediately, which indicates that the carrier and the internal structure of the co-immobilized enzyme are relatively stable and the phenomenon of enzyme leakage is less, and also indicates that the immobilized carrier gel ball can continuously absorb water and swell during recycling, and continuously expose new catalytic active sites to participate in the catalytic action of the enzyme to catalyze the reaction of the substrate. However, the prepared co-immobilized enzyme can be recycled only 5 times because the catalase in the co-immobilized enzyme causes strong H2O2The catalytic reaction generates a large amount of oxygen, which is easy to generate the rapid extrusion effect of the internal space of the immobilized carrier gel ball, so that the gel ball is expanded and dissolved, and the enzyme is leaked out and can not be reused. In conclusion, the most important advantage of the co-immobilized enzyme compared with the free enzyme is that the co-immobilized enzyme can be recycled for many times, which can greatly reduce the production cost in practical application, and is also an important evaluation factor of the co-immobilized enzyme in practical application.
Example 9
The Km is a basic kinetic constant in an enzyme reaction and indicates the magnitude of affinity between a substrate and an enzyme, and the smaller the Km of the enzyme, the greater the affinity between the substrate and the enzyme. The speed of any enzymatic reaction is critically affected by the concentration of the particular enzyme substrate, and this relationship is consistent with Michaelis-Menten kinetics. Km of free enzyme and co-immobilized enzyme were calculated by drawing a Lineweaver-Burk plot, and subjected to reaction kinetic analysis, and their double reciprocal plots are shown in FIGS. 8 and 9.
As calculated from fig. 8, the Km of the catalase was 8.58mmol/L in free form and 8.65mmol/L in immobilized catalase. This result indicates that free catalase was present on substrate H compared to immobilized catalase2O2With greater affinity therebetween. This is due to diffusion limitations and steric hindrance effects inside the immobilized support.
The Km of the free laccase is 0.19mmol/L and the Km of the immobilized laccase is 0.52mmol/L as calculated from FIG. 9. This result indicates that the free laccase has a greater affinity for the substrate ABTS than the immobilized laccase. This is due to diffusion limitations and steric hindrance effects inside the immobilized support.
In the enzyme catalytic reaction, free enzyme is easier to synthesize an activated intermediate compound with a substrate than immobilized enzyme, enzyme molecules are surrounded by a carrier after immobilization, the reaction space is relatively narrow, and the mass transfer of the substrate is limited to a certain extent. The substrate concentration in the microenvironment near the carrier will be relatively lower than that in the substrate solution, making the probability of enzyme binding to the substrate lower, thereby reducing the affinity between the two. The problem of diffusion limitation can be divided into inner diffusion limitation and outer diffusion limitation: the internal diffusion limitation occurs in a large number of pores inside the carrier, and the substrate is transported from the particle surface of the co-immobilized enzyme to the active center of the enzyme; out-diffusion limitation occurs in the microenvironment near the co-immobilized enzyme particles, and the substrate is transferred from the macroscopic system to the particle surface. The reaction system is composed of co-immobilized enzymes, and the substrate has the problem of diffusion limitation in a macroscopic environment, and the effect is obvious when the diffusion speed is low and the enzyme catalytic activity is strong.
Example 10
The co-immobilized enzyme prepared in example 1, 5g of co-immobilized enzyme, was weighed into a 250mL Erlenmeyer flask, 100mL aniline-simulated wastewater was added, and H was added2O2Adding 0.3 wt% of aniline simulation wastewater (200 mg of aniline liquid is accurately weighed, 200mg/L aniline solution is prepared by using 1L of distilled water and serves as aniline simulation wastewater) as an auxiliary oxidant, adjusting the pH value to 5, placing each group of conical flasks in a constant-temperature shaking table with the temperature of 35 ℃ and the rotating speed of 140rpm, carrying out oscillation reaction for 24 hours, and measuring the aniline removal rate to be 73.17% after the reaction is finished.
The preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various features described in the above embodiments may be combined in any suitable manner without departing from the scope of the invention. The invention is not described in detail in order to avoid unnecessary repetition.
In addition, any combination of the various embodiments of the present invention is also possible, and the same should be considered as the disclosure of the present invention as long as it does not depart from the spirit of the present invention.
Claims (10)
1. A co-immobilized enzyme, comprising: the co-immobilized enzyme is sodium alginate-CaCl2And (3) taking the gel and the active carbon as carriers, and immobilizing catalase and laccase.
2. The co-immobilized enzyme according to claim 1, wherein: the optimal temperature of the co-immobilized enzyme is 45 ℃;
optionally, the co-immobilized enzyme has high operational stability, and catalase and laccase still retain more than 61% and more than 74% of activity after 5 times of recycling at 25 ℃.
3. A co-immobilized enzyme according to claim 1 or 2, characterized in that: the co-immobilized enzyme has good storage stability, and the immobilized catalase and laccase still keep more than 65% and more than 61% of activity after being stored for 30 days at 4 ℃.
4. A process for the preparation of a co-immobilized enzyme according to any one of claims 1 to 3, characterized in that: the method comprises the following steps:
step 1: preparing a mixed buffer solution of catalase and laccase;
step 2: mixing the complex enzyme solution with active carbon, adding sodium alginate after complete adsorption, stirring, cooling, granulating with injector, and adding CaCl2In the aqueous solution of (A), smooth beads are generated, and the immobilized gel beads are in CaCl2Is hardened, and then washed with water and dried to obtain a co-immobilized enzyme.
5. The method for preparing a co-immobilized enzyme according to claim 4, wherein: the step 1 comprises the steps of 1a dissolving catalase freeze-dried powder by using a phosphate buffer solution to obtain a catalase solution, 1b dissolving laccase freeze-dried powder by using an acetic acid-sodium acetate buffer solution to obtain a laccase solution, and 1c mixing the catalase solution and the laccase solution.
6. The method for preparing a co-immobilized enzyme according to claim 5, wherein: the pH value of the phosphate buffer solution is 6-7, and the concentration is 0.05-0.1M;
optionally, the concentration of catalase in the catalase solution is 0.5-1.25 mg/mL;
optionally, the acetic acid-sodium acetate buffer has a pH of 4-5 and a concentration of 0.2-0.3M;
optionally, the concentration of laccase in the laccase solution is 0.5-1.25 mg/mL;
optionally, when the catalase solution and the laccase solution are mixed, the mass ratio of the catalase to the laccase is 1:2-2: 1.
7. The method for preparing a co-immobilized enzyme according to claim 4, wherein: in the step 2, the ratio of the addition amount of the activated carbon to the w: v ratio of the complex enzyme solution is 1:100, and the ratio of the addition amount of the sodium alginate solution to the volume of the complex enzyme solution is 1: 5;
optionally, the sodium alginate is 1-3% aqueous solution.
8. The method for preparing a co-immobilized enzyme according to claim 4, wherein: in the step 2, CaCl2The mass concentration of the aqueous solution is 1-3%.
9. The method for preparing a co-immobilized enzyme according to claim 4, wherein: in the step 2, the temperature is reduced to 0-4 ℃ in an ice water bath, and the immobilization time is 30-60 min.
10. Use of the co-immobilized enzyme of any one of claims 1-3 for aniline wastewater treatment.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910979731.8A CN110628756A (en) | 2019-10-15 | 2019-10-15 | Co-immobilized enzyme and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910979731.8A CN110628756A (en) | 2019-10-15 | 2019-10-15 | Co-immobilized enzyme and preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN110628756A true CN110628756A (en) | 2019-12-31 |
Family
ID=68974894
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910979731.8A Pending CN110628756A (en) | 2019-10-15 | 2019-10-15 | Co-immobilized enzyme and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110628756A (en) |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111729624A (en) * | 2020-07-08 | 2020-10-02 | 江苏科技大学 | Preparation and application method of biogel for environmental remediation |
| CN112047570A (en) * | 2020-09-03 | 2020-12-08 | 常德鑫芙蓉环保有限公司 | Treatment method of oily industrial wastewater |
| CN115521931A (en) * | 2022-10-10 | 2022-12-27 | 南开大学 | Enzyme load prediction and preparation method for immobilized laccase based on general linear and multilayer perceptron model |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104263766A (en) * | 2014-09-01 | 2015-01-07 | 郸城财鑫糖业有限责任公司 | Method for preparing sodium gluconate by using immobilized enzyme |
| CN105462953A (en) * | 2014-08-25 | 2016-04-06 | 姜丹宁 | Preparation of magnetic chitosan microsphere and its application as laccase immobilization carrier |
| CN105505914A (en) * | 2016-02-02 | 2016-04-20 | 江苏时空涂料有限公司 | Preparation method for magnetic sodium alginate-immobilized laccase |
| CN106434622A (en) * | 2016-08-26 | 2017-02-22 | 仲恺农业工程学院 | Preparation method of co-immobilized enzyme |
| CN107012138A (en) * | 2017-05-03 | 2017-08-04 | 福州晨翔环保工程有限公司 | Silicon/carbon-based complex solidifying enzyme environment-friendly materials and preparation method thereof |
| CN109234262A (en) * | 2018-09-17 | 2019-01-18 | 北京化工大学 | A kind of method that carrier granulating technique prepares immobilised enzymes |
-
2019
- 2019-10-15 CN CN201910979731.8A patent/CN110628756A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105462953A (en) * | 2014-08-25 | 2016-04-06 | 姜丹宁 | Preparation of magnetic chitosan microsphere and its application as laccase immobilization carrier |
| CN104263766A (en) * | 2014-09-01 | 2015-01-07 | 郸城财鑫糖业有限责任公司 | Method for preparing sodium gluconate by using immobilized enzyme |
| CN105505914A (en) * | 2016-02-02 | 2016-04-20 | 江苏时空涂料有限公司 | Preparation method for magnetic sodium alginate-immobilized laccase |
| CN106434622A (en) * | 2016-08-26 | 2017-02-22 | 仲恺农业工程学院 | Preparation method of co-immobilized enzyme |
| CN107012138A (en) * | 2017-05-03 | 2017-08-04 | 福州晨翔环保工程有限公司 | Silicon/carbon-based complex solidifying enzyme environment-friendly materials and preparation method thereof |
| CN109234262A (en) * | 2018-09-17 | 2019-01-18 | 北京化工大学 | A kind of method that carrier granulating technique prepares immobilised enzymes |
Non-Patent Citations (5)
| Title |
|---|
| XIAOFENG WEN等: "Immobilizing Laccase on Kaolinite and Its Application in Treatment of Malachite Green Effluent with the Coexistence of Cd (П)", 《CHEMOSPHERE》 * |
| 张树江等: "固定化漆酶对二氯酚的脱氯作用", 《化工学报》 * |
| 徐国英等: "海藻酸钠-壳聚糖固定化漆酶的研究", 《西南师范大学学报》 * |
| 王建辉等: "固定化酶在有机废水处理中的应用", 《吉林建筑大学学报》 * |
| 黄洁等: "酶的固定化及其在纺织染整工业中的应用", 《印染助剂》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111729624A (en) * | 2020-07-08 | 2020-10-02 | 江苏科技大学 | Preparation and application method of biogel for environmental remediation |
| CN111729624B (en) * | 2020-07-08 | 2021-04-02 | 江苏科技大学 | Preparation and application method of biogel for environmental remediation |
| CN112047570A (en) * | 2020-09-03 | 2020-12-08 | 常德鑫芙蓉环保有限公司 | Treatment method of oily industrial wastewater |
| CN115521931A (en) * | 2022-10-10 | 2022-12-27 | 南开大学 | Enzyme load prediction and preparation method for immobilized laccase based on general linear and multilayer perceptron model |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN110628756A (en) | Co-immobilized enzyme and preparation method and application thereof | |
| Cao et al. | Immobilization of Bacillus subtilis lipase on a Cu-BTC based hierarchically porous metal–organic framework material: a biocatalyst for esterification | |
| Öztürk et al. | Reversible adsorption of lipase on novel hydrophobic nanospheres | |
| CN107446915B (en) | Magnetic graphene oxide composite material immobilized horseradish peroxidase and preparation method and application thereof | |
| Kim et al. | Immobilization of Mucor javanicus lipase on effectively functionalized silica nanoparticles | |
| CN103012673B (en) | Biological enzyme immobilizing core-shell type superparamagnetic polymeric microsphere and preparing method thereof | |
| Ai et al. | Immobilization of horseradish peroxidase enzymes on hydrous-titanium and application for phenol removal | |
| Cui et al. | Enzyme shielding by mesoporous organosilica shell on Fe3O4@ silica yolk-shell nanospheres | |
| Cho et al. | Immobilization of enzymes on activated carbon: properties of immobilized glucoamylase, glucose oxidase, and gluconolactonase | |
| CN108855083B (en) | Method for removing sulfonamides in water by activating peracetic acid with modified zeolite | |
| Zhou et al. | Immobilization of β-glucosidase onto magnetic nanoparticles and evaluation of the enzymatic properties | |
| CN105647901B (en) | Process for immobilizing enzyme/microbe is prepared using the modified calcium alginate of graphene oxide | |
| CN112980827B (en) | Immobilized glucose oxidase of metal organic framework material and preparation method and application thereof | |
| CN109810969B (en) | Method for constructing artificial multienzyme system based on lanthanide nucleotide complex and DNA directional immobilization technology | |
| Li et al. | Recent progress in the development of immobilized penicillin G acylase for chemical and industrial applications: A mini‐review | |
| CN109576256B (en) | Method for encapsulating double enzymes by magnetic DNA hydrogel | |
| CN104404023A (en) | Preparation method of magnetic carrier immobilized lipase, and method for preparing biodiesel under catalysis of magnetic carrier immobilized lipase | |
| Ren et al. | Multiscale immobilized lipase for rapid separation and continuous catalysis | |
| CN111676213B (en) | laccase-nanoflower-gamma-Fe2O3Copper alginate gel bead and preparation method and application thereof | |
| CN106242052A (en) | A kind of ultrasonic wave added nanometer alumina modified polyvinyl alcohol sodium alginate embedded material and its preparation method and application | |
| CN107475239A (en) | A kind of process for fixation of horseradish peroxidase and its application | |
| Wu et al. | Synthesis of polymer-functionalized β-cyclodextrin, Mg2+ doped, coating magnetic Fe3O4 nanoparticle carriers for penicillin G acylase immobilization | |
| CN113559247A (en) | Polyphenol nano reactor and preparation method and application thereof | |
| CN111013588B (en) | Fenton-like catalyst and preparation method and application thereof | |
| CN111939911A (en) | β-FeOOH/SiO2Preparation method and application of composite catalyst |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20191231 |