CN113151241A - Method for immobilizing cellulase by using two-dimensional nanosheets - Google Patents
Method for immobilizing cellulase by using two-dimensional nanosheets Download PDFInfo
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- 108010059892 Cellulase Proteins 0.000 title claims abstract description 87
- 229940106157 cellulase Drugs 0.000 title claims abstract description 77
- 239000002135 nanosheet Substances 0.000 title claims abstract description 62
- 238000000034 method Methods 0.000 title claims abstract description 22
- 230000003100 immobilizing effect Effects 0.000 title claims abstract description 12
- 102000004190 Enzymes Human genes 0.000 claims abstract description 44
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- 229940088598 enzyme Drugs 0.000 claims abstract description 43
- 238000001338 self-assembly Methods 0.000 claims abstract description 4
- 230000000694 effects Effects 0.000 claims description 35
- 239000000243 solution Substances 0.000 claims description 26
- 108010047754 beta-Glucosidase Proteins 0.000 claims description 15
- 102000006995 beta-Glucosidase Human genes 0.000 claims description 15
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 12
- HHDUMDVQUCBCEY-UHFFFAOYSA-N 4-[10,15,20-tris(4-carboxyphenyl)-21,23-dihydroporphyrin-5-yl]benzoic acid Chemical compound OC(=O)c1ccc(cc1)-c1c2ccc(n2)c(-c2ccc(cc2)C(O)=O)c2ccc([nH]2)c(-c2ccc(cc2)C(O)=O)c2ccc(n2)c(-c2ccc(cc2)C(O)=O)c2ccc1[nH]2 HHDUMDVQUCBCEY-UHFFFAOYSA-N 0.000 claims description 10
- 239000008055 phosphate buffer solution Substances 0.000 claims description 10
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- 238000006243 chemical reaction Methods 0.000 claims description 9
- SBPNVTUPNZVOOR-UHFFFAOYSA-K lutetium(3+) triacetate tetrahydrate Chemical compound O.O.O.O.[Lu+3].CC([O-])=O.CC([O-])=O.CC([O-])=O SBPNVTUPNZVOOR-UHFFFAOYSA-K 0.000 claims description 7
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 6
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- 230000015572 biosynthetic process Effects 0.000 claims description 6
- LOKCTEFSRHRXRJ-UHFFFAOYSA-I dipotassium trisodium dihydrogen phosphate hydrogen phosphate dichloride Chemical compound P(=O)(O)(O)[O-].[K+].P(=O)(O)([O-])[O-].[Na+].[Na+].[Cl-].[K+].[Cl-].[Na+] LOKCTEFSRHRXRJ-UHFFFAOYSA-I 0.000 claims description 6
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- 239000003446 ligand Substances 0.000 abstract description 10
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- 238000005286 illumination Methods 0.000 abstract description 3
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- GUBGYTABKSRVRQ-CUHNMECISA-N D-Cellobiose Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)OC(O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-CUHNMECISA-N 0.000 description 3
- 101710112457 Exoglucanase Proteins 0.000 description 3
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- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 3
- 229920002678 cellulose Polymers 0.000 description 3
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- 230000007423 decrease Effects 0.000 description 3
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- 229930091371 Fructose Natural products 0.000 description 2
- 239000005715 Fructose Substances 0.000 description 2
- RFSUNEUAIZKAJO-ARQDHWQXSA-N Fructose Chemical compound OC[C@H]1O[C@](O)(CO)[C@@H](O)[C@@H]1O RFSUNEUAIZKAJO-ARQDHWQXSA-N 0.000 description 2
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- 241000228212 Aspergillus Species 0.000 description 1
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- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
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- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
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- BOTDANWDWHJENH-UHFFFAOYSA-N Tetraethyl orthosilicate Chemical compound CCO[Si](OCC)(OCC)OCC BOTDANWDWHJENH-UHFFFAOYSA-N 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
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- 239000012621 metal-organic framework Substances 0.000 description 1
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- 150000004043 trisaccharides Chemical class 0.000 description 1
- 239000002023 wood Substances 0.000 description 1
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- 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
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- 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
- C12N9/14—Hydrolases (3)
- C12N9/24—Hydrolases (3) acting on glycosyl compounds (3.2)
- C12N9/2402—Hydrolases (3) acting on glycosyl compounds (3.2) hydrolysing O- and S- glycosyl compounds (3.2.1)
- C12N9/2405—Glucanases
- C12N9/2434—Glucanases acting on beta-1,4-glucosidic bonds
- C12N9/2445—Beta-glucosidase (3.2.1.21)
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y302/00—Hydrolases acting on glycosyl compounds, i.e. glycosylases (3.2)
- C12Y302/01—Glycosidases, i.e. enzymes hydrolysing O- and S-glycosyl compounds (3.2.1)
- C12Y302/01021—Beta-glucosidase (3.2.1.21)
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- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
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- General Health & Medical Sciences (AREA)
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- Biomedical Technology (AREA)
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Abstract
The invention discloses a method for immobilizing cellulase by using two-dimensional nanosheets, which comprises the steps of 1) preparing a two-dimensional nanosheet solution, 2) preparing a cellulase enzyme solution, 3) immobilizing the cellulase and the like. The two-dimensional nanosheet is prepared by self-assembly of metal ions and porphyrin ligands, and the thickness of the nanosheet is reduced to be less than 20nm in the third dimension, so that the prepared nanosheet has a high specific surface area, and the loading capacity of enzyme is obviously improved. And the nano sheet has good stability and can be repeatedly used. Meanwhile, as the self-assembled ligand uses the ligand with photosensitive property, the nanosheet improves the catalytic efficiency of the immobilized enzyme under illumination.
Description
Technical Field
The invention relates to the field of metal organic frameworks, in particular to a method for immobilizing cellulase by using two-dimensional nanosheets.
Background
Cellulases are widely present in organisms in nature. Cellulase is produced in bacteria, fungi, animals, etc. Cellulases generally used in the production are derived from fungi, typically wood enzymes, Aspergillus and Penicillium. Cellulase producing strains are susceptible to degradation resulting in reduced enzyme production capacity. The cellulase has wide application in food industry and environmental industry. During alcohol fermentation, the addition of cellulase can increase the utilization rate of raw materials and improve the quality of wine. Because cellulase is difficult to purify, it is generally used in practice to contain hemicellulase and other related enzymes, such as amylase, protease, etc.
The cellulase has various varieties and wide sources. Cellulases from different sources vary widely in structure and function. The cellulase used in animal husbandry and feed industry is mainly fungal cellulase due to its high yield and high activity. The cellulase serving as a green biocatalyst has a series of advantages of high catalytic activity, less side reaction, low pollution, high operation stability and the like, however, the cellulase has some limitations in practical industrial application, such as insufficient stability under the conditions of high temperature, organic solvent, strong acid or alkali and the like, easy inactivation and poor tolerance to the environment; is easy to degrade, is easy to agglomerate in the reaction process, and is difficult to fully contact with a substrate. Free cellulase has higher price, can catalyze only one reaction generally, and cannot be recovered and recycled from a reaction system, so that the production cost of the product is greatly improved, and the wide application of the free cellulase in industrial practice is limited.
Beta-glucosidase acts mainly on beta- (1, 4) glycosidic bonds, and also acts on beta- (1, 1), (1, 2), (1, 3), (1, 6) glycosidic bonds. It can be hydrolyzed and combined with a terminal non-reducing beta-D-glucose glycosidic bond, and simultaneously releases beta-D-glucose and corresponding ligand. When the cellulase is used for catalyzing and hydrolyzing cellulose, firstly, endoglucanase acts on a non-crystallization area of microfibril to expose a plurality of ends for acting of exonuclease, exoglucanase (cellobiohydrolase) is sequentially decomposed from non-reduction ends to generate cellobiose, part of degraded cellulose is further decomposed by the synergy of the endoglucanase and the exoglucanase to generate oligosaccharides such as cellobiose, trisaccharide and the like, and finally, the cellulose is decomposed into glucose by the action of beta-glucosidase. In the process, beta-glucosidase plays a key role, the content of the beta-glucosidase in the cellulase components is low, the activity is low, and the maximum functions of endoglucanase and exoglucanase in an enzyme system are restricted, so that cellobiose in a hydrolyzed sugar solution is accumulated, the sugar amount available for subsequent fermentation is reduced, the bottleneck of cellulase hydrolysis is formed, and the beta-glucosidase is usually required to be additionally added in the cellulase hydrolysis process.
The enzyme immobilization technology greatly expands the application range of the enzyme. The immobilized enzyme is prepared by immobilizing enzyme in a certain space by a proper method, and can be recovered and recycled while maintaining the characteristics of the immobilized enzyme. The advent of immobilized enzymes has solved many of the limitations of practical applications of free enzymes. Therefore, the research on the immobilization technology of cellulase is very urgent. The immobilization of the enzyme allows the enzyme to be easily separated and recovered from the reaction mixture for use in the next reaction. In addition, the enzyme immobilization can also improve the thermal stability and chemical stability and resist external extreme change conditions, so that the development of a new cellulase immobilization system becomes the focus of current attention.
Chinese patent CN106867990A discloses a preparation method of fructose aqueous solution immobilized enzyme, which adopts fructose as pore-forming agent and prepares immobilized beta-glucosidase by tetraethoxysilane hydrolysis reaction sol-gel method. However, this extraction has a low enzyme load and a low enzyme recovery efficiency. Therefore, the development of the two-dimensional nanosheet has important significance for the immobilization technology of enzymes, particularly cellulase, and the application of the two-dimensional nanosheet in the immobilization of beta-glucosidase, but not limited to the industrial production of the product.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the defects of the prior art, the invention provides a method for preparing two-dimensional nanosheet immobilized cellulase, the two-dimensional nanosheet is prepared by self-assembly of metal ions and porphyrin ligands, and the thickness of the nanosheet is reduced to be less than 20nm in the third dimension, so that the prepared nanosheet has a higher specific surface area, and the loading capacity of enzyme is obviously improved. And the nano sheet has good stability and can be repeatedly used. Meanwhile, as the self-assembled ligand uses the ligand with photosensitive property, the nanosheet improves the catalytic efficiency of the immobilized enzyme under illumination.
The technical scheme is as follows: the invention discloses a method for immobilizing cellulase by using two-dimensional nanosheets, which comprises the following steps:
1) preparing a two-dimensional nanosheet solution: using acid containing carboxyl functional group as regulator, lutetium (III) acetate tetrahydrate and meso-tetra (4-carboxyphenyl) porphyrin (H)2TCPP) to obtain two-dimensional nanosheets, and dissolving the two-dimensional nanosheets in phosphate buffered saline solution to prepare phosphate buffered saline solution containing the two-dimensional nanosheets, namely two-dimensional nanosheet solution.
2) Preparing a cellulase solution: dissolving cellulase powder in phosphate buffer solution, stirring uniformly at 2-7 ℃, centrifuging for 3-5 min at the rotating speed of 12000-15000 r/min, transferring supernatant into a volumetric flask, and performing constant volume by using the phosphate buffer solution to obtain enzymatic synthesis beta-glucosidase cellulase, namely cellulase enzyme solution;
wherein, the mixture is preferably stirred uniformly at 5 ℃ and then centrifuged at 12000r/min for 3 min.
3) And (3) immobilizing cellulase: mixing the two-dimensional nanosheet solution with a cellulase enzyme solution, carrying out suction filtration, adding deionized water for dissolution, washing off redundant cellulase by using a phosphate buffer solution, carrying out constant volume, and carrying out freeze drying to obtain the cellulase immobilized by the two-dimensional nanosheets.
Wherein the pH value of the phosphate buffer salt solution is 6.8-7.4. Preferably the pH is 7.0.
Wherein the mass ratio of the meso-tetra (4-carboxyphenyl) porphyrin and the lutetium acetate tetrahydrate (III) in the step (1) is 1: 1-4, and the regulator and the meso-tetra (4-carboxyphenyl) porphyrin (H)2TCPP) in a molar ratio of 0.6 to 1.5: 1.
Wherein, the regulator in the step (1) is one or more than two of formic acid, 4-diphenyl dicarboxylic acid, acetic acid, benzoic acid and trifluoroacetic acid. Formic acid is preferred.
Wherein the reaction temperature for preparing the two-dimensional nanosheet in the step (1) is 120-200 ℃, and the reaction time is 8-24 h.
Wherein, the cellulase powder in the step (2) is one extracted from animals, plants or microorganisms.
Wherein the activity of the enzymatic synthesis beta-glucosidase cellulase in the step (2) is 500U/g, and the enzyme adding amount is 30 mg/g-35 mg/g. The amount of enzyme added is preferably 30 mg/g.
Wherein the immobilization time of the step (3) is 12-16 h, the immobilization temperature is 25-28 ℃, and the immobilization pH is 6.8-7.4. The immobilization time is preferably 12h, the immobilization temperature is preferably 27 ℃, and the immobilization pH is preferably 7.0.
Description of the principle: according to the preparation method, the two-dimensional nanosheet is prepared by self-assembly of metal ions and porphyrin ligands, and the thickness of the nanosheet is reduced to be less than 20nm in the third dimension, so that the prepared nanosheet has a high specific surface area, and the loading capacity of enzyme is obviously improved. And the nano sheet has good stability and can be repeatedly used. Meanwhile, as the self-assembled ligand uses the ligand with photosensitive property, the nanosheet improves the catalytic efficiency of the immobilized enzyme under illumination.
Has the advantages that: compared with the prior art, the invention has the following remarkable advantages: the two-dimensional nanosheet prepared by the method has the advantages that the two-dimensional nanosheet has high specific surface area and high porosity from a projection electron microscope, and is beneficial to improving the enzyme carrying amount of the immobilized enzyme and improving the catalytic efficiency of the immobilized enzyme; the two-dimensional nanosheet is an immobilized carrier of the enzyme, is easy to recover from a reaction system, can be repeatedly used, greatly improves the utilization rate of the enzyme and reduces the production cost; the two-dimensional nanosheet has good biocompatibility, and can provide a good microenvironment for surface immobilized enzyme protein; the service life and efficiency of the enzyme are greatly improved, and the optical purity of the beta-glucosidase reaches more than 99 percent after the immobilized enzyme is repeatedly used for 10 times.
Drawings
Fig. 1 is a schematic structural view of a two-dimensional nanoplate of the present invention.
Detailed Description
The technical solution of the present invention is further described with reference to the accompanying drawings and the detailed description.
As shown in figure 1, the length of the two-dimensional nano sheet is 0.2-10 μm, and the thickness of the nano sheet is 2-20 nm. The high-specific-surface-area high-porosity catalyst has high specific surface area and high porosity, and is favorable for improving the enzyme-carrying capacity of the immobilized enzyme and the catalytic efficiency of the immobilized enzyme.
The technical scheme of the invention is as follows:
1) preparing a two-dimensional nanosheet solution: the preparation method comprises the steps of taking acid containing carboxyl functional groups as a regulator, self-assembling lutetium (III) acetate tetrahydrate and meso-tetra (4-carboxyphenyl) porphyrin (H2TCPP) to obtain two-dimensional nanosheets, and dissolving the two-dimensional nanosheets in phosphate buffered saline solution to prepare phosphate buffered saline solution containing the two-dimensional nanosheets, namely the two-dimensional nanosheet solution.
2) Preparing a cellulase solution: dissolving cellulase powder in phosphate buffer solution, stirring uniformly at 2-7 ℃, centrifuging for 3-5 min at the rotating speed of 12000-15000 r/min, transferring supernatant into a volumetric flask, and performing constant volume by using the phosphate buffer solution to obtain enzymatic synthesis beta-glucosidase cellulase, namely cellulase enzyme solution;
wherein, the mixture is selected to be stirred evenly at 5 ℃ and then centrifuged for 3min at the rotating speed of 12000 r/min.
3) And (3) immobilizing cellulase: mixing the two-dimensional nanosheet solution with a cellulase enzyme solution, carrying out suction filtration, adding deionized water for dissolution, washing off redundant cellulase by using a phosphate buffer solution, carrying out constant volume, and carrying out freeze drying to obtain the cellulase immobilized by the two-dimensional nanosheets.
Wherein the pH value of the phosphate buffer salt solution is 6.8-7.4; the pH was chosen to be 7.0. The mass ratio of the meso-tetra (4-carboxyphenyl) porphyrin and the lutetium acetate tetrahydrate (III) in the step (1) is 1: 1-4, and the molar ratio of the regulator to the meso-tetra (4-carboxyphenyl) porphyrin (H2TCPP) is 0.6-1.5: 1; 22mg of lutetium (III) acetate tetrahydrate, 10mg of meso-tetra (4-carboxyphenyl) porphyrin and 9mg of formic acid were selected. The regulator in the step (1) is one or more than two of formic acid, 4-diphenyl dicarboxylic acid, acetic acid, benzoic acid and trifluoroacetic acid; formic acid is selected. The reaction temperature of the two-dimensional nanosheet in the preparation step (1) is 120-200 ℃, and the reaction time is 8-24 h; the reaction temperature was chosen to be 160 ℃ and the reaction time 16 h. The cellulase powder of step (2) is one extracted from animals, plants or microorganisms. The enzymatic synthesis of the beta-glucosidase cellulase in the step (2) has the activity of 500U/g and the enzyme adding amount of 30mg/g to 35 mg/g; the enzyme dosage was selected to be 30 mg/g.
Wherein the immobilization time in the step (3) is 12-16 h, the immobilization temperature is 25-28 ℃, and the immobilization pH is 6.8-7.4. The immobilization time is preferably 12h, the immobilization temperature is preferably 27 ℃, and the immobilization pH is preferably 7.0. Through the research of the inventor, the following results are found:
1. effect of immobilization time on immobilization
The activity of the immobilized enzyme is too low due to too short immobilization time and incomplete immobilization, and the activity of the immobilized enzyme is reduced due to too long immobilization time, so that the activity of the immobilized enzyme is indirectly reduced. Therefore, when the immobilization time of the two-dimensional nanosheet is less than 12 hours, the activity of the immobilized enzyme increases with the increase of the immobilization time. And the enzyme activity begins to decrease after the immobilization time of the enzyme and the carrier is longer than 12 hours, probably because the enzyme activity decreases due to the prolonged immobilization time, but the enzyme activity is not beneficial to the improvement of the immobilized enzyme activity. In conclusion, the optimal immobilization time of the immobilized carrier two-dimensional nanosheet on the cellulase is 12 h.
2. Effect of immobilization temperature on immobilization
The selection of proper immobilization temperature is crucial to the activity of immobilized enzyme. Under the condition of low temperature, the enzyme activity is favorably maintained, but the speed of molecular exchange is slowed down, so that the immobilization time is prolonged; and the temperature is increased, so that the enzyme activity is not kept, but the immobilization time is relatively reduced, and the immobilization efficiency is improved. When the immobilization temperature is between 5 ℃ and 27 ℃, the activity of the immobilized enzyme is increased along with the increase of the temperature, possibly the temperature is increased, and the adsorption of the enzyme is facilitated; when the temperature exceeds 27 ℃, the activity of the immobilized enzyme begins to decrease, possibly due to the temperature rise, the activity of the enzyme is reduced, and the optimal immobilization temperature of the immobilized carrier two-dimensional nanosheet to the cellulase is 27 ℃.
3. Effect of pH on immobilization
The pH value directly influences the activity of the enzyme, and the activity of the immobilized enzyme is not improved when the pH value is too high or too low. Cellulase was most active in pH 7.0 buffer. Therefore, the optimum immobilization pH of the immobilized carrier two-dimensional nanosheet to the cellulase is 7.0.
In view of the above, the present invention provides an optimal immobilization condition in a method for immobilizing cellulase: the immobilization time is 12h, the immobilization temperature is 27 ℃, and the immobilization pH is 7.0.
4. Thermal stability of immobilized cellulase and free enzyme
Research on the action of the immobilized cellulase and the free cellulase at 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃ and 80 ℃ for 3h, and the activity change after the action at different temperatures is measured, wherein the highest enzyme activity data is 100 percent of the enzyme activity, and the result shows that: the activity of free cellulase is 26.7 percent remained after being heated for 3 hours at 80 ℃, and the activity of immobilized cellulase is kept at 82.4 percent. The activity of the two-dimensional nanosheet immobilized cellulase is greatly improved compared with that of a free enzyme, and the thermal stability of the enzyme is enhanced after immobilization because the immobilized carrier provides an additional protective framework for the retention of enzyme molecules.
5. Reusability and optical purity of immobilized cellulase
The enzyme activity of the immobilized cellulase is measured according to the measuring conditions of the cellulase (35 ℃, 10min), the immobilized cellulase is washed twice by phosphate buffer solution with the pH value of 7, the reusability and the optical purity of the immobilized enzyme required for synthesizing the beta-glucosidase are measured, and the process is repeated for 10 times. And calculating the enzyme activity and the optical purity of the immobilized enzyme remained each time. The result shows that after the cellulase is repeatedly used for 6 times, the immobilized carrier two-dimensional nanosheet immobilized cellulase keeps 92% of the activity thereof, and the optical purity is kept above 97%. During the repeated use process, the immobilized cellulase is closely attached to the carrier, so that the activity is maintained. After 10 times of repeated use, the activity of the two-dimensional nanosheet modified cellulase is maintained at 82.6%, and the optical purity is maintained above 96%.
Claims (8)
1. A method for immobilizing cellulase by two-dimensional nanosheets is characterized by comprising the following steps: the method comprises the following steps:
1) preparing a two-dimensional nanosheet solution: using acid containing carboxyl functional group as regulator, lutetium (III) acetate tetrahydrate and meso-tetra (4-carboxyphenyl) porphyrin(H2TCPP) to obtain two-dimensional nanosheets by self-assembly, and dissolving the two-dimensional nanosheets in phosphate buffered saline solution to prepare phosphate buffered saline solution containing the two-dimensional nanosheets, namely two-dimensional nanosheet solution;
2) preparing a cellulase solution: dissolving cellulase powder in phosphate buffer solution, stirring uniformly at 2-7 ℃, centrifuging for 3-5 min at the rotating speed of 12000-15000 r/min, transferring supernatant into a volumetric flask, and performing constant volume by using the phosphate buffer solution to obtain enzymatic synthesis beta-glucosidase cellulase, namely cellulase enzyme solution;
3) and (3) immobilizing cellulase: mixing the two-dimensional nanosheet solution with a cellulase enzyme solution, carrying out suction filtration, adding deionized water for dissolution, washing off redundant cellulase by using a phosphate buffer solution, carrying out constant volume, and carrying out freeze drying to obtain the cellulase immobilized by the two-dimensional nanosheets.
2. A method of two-dimensional nanoplatelet immobilized cellulase as defined in claim 1, characterized in that: the pH value of the phosphate buffer salt solution is 6.8-7.4.
3. A method of two-dimensional nanoplatelet immobilized cellulase as defined in claim 1, characterized in that: the mass ratio of the meso-tetra (4-carboxyphenyl) porphyrin and the lutetium acetate tetrahydrate (III) in the step (1) is 1: 1-4, and the regulator and the meso-tetra (4-carboxyphenyl) porphyrin (H)2TCPP) in a molar ratio of 0.6 to 1.5: 1.
4. A method of two-dimensional nanoplatelet immobilized cellulase as defined in claim 1, characterized in that: the regulator in the step (1) is one or more than two of formic acid, 4-diphenyl dicarboxylic acid, acetic acid, benzoic acid and trifluoroacetic acid.
5. A method of two-dimensional nanoplatelet immobilized cellulase as defined in claim 1, characterized in that: the reaction temperature for preparing the two-dimensional nanosheet in the step (1) is 120-200 ℃, and the reaction time is 8-24 h.
6. A method of two-dimensional nanoplatelet immobilized cellulase as defined in claim 1, characterized in that: the cellulase powder in the step (2) is one extracted from animals, plants or microorganisms.
7. A method of two-dimensional nanoplatelet immobilized cellulase as defined in claim 1, characterized in that: the enzymatic synthesis of the beta-glucosidase cellulase in the step (2) has the activity of 500U/g and the enzyme adding amount of 30 mg/g-35 mg/g.
8. A method of two-dimensional nanoplatelet immobilized cellulase as defined in claim 1, characterized in that: the immobilization time of the step (3) is 12-16 h, the immobilization temperature is 25-28 ℃, and the immobilization pH is 6.8-7.4.
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|---|---|---|---|---|
| US3556945A (en) * | 1968-02-05 | 1971-01-19 | Corning Glass Works | Enzyme stabilization |
| CA1277621C (en) * | 1986-06-26 | 1990-12-11 | Fumiko Yaku | Process for producing immobilized beta-glucosidase |
| CN109082420A (en) * | 2018-08-21 | 2018-12-25 | 江苏大学 | Metal-organic framework material immobilized β-glucosidase and its preparation method and application |
| CN111044587A (en) * | 2019-11-25 | 2020-04-21 | 西安医学院 | 2D porphyrin MOF nano material for electrochemical sensor and preparation method thereof |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US3556945A (en) * | 1968-02-05 | 1971-01-19 | Corning Glass Works | Enzyme stabilization |
| CA1277621C (en) * | 1986-06-26 | 1990-12-11 | Fumiko Yaku | Process for producing immobilized beta-glucosidase |
| CN109082420A (en) * | 2018-08-21 | 2018-12-25 | 江苏大学 | Metal-organic framework material immobilized β-glucosidase and its preparation method and application |
| CN111044587A (en) * | 2019-11-25 | 2020-04-21 | 西安医学院 | 2D porphyrin MOF nano material for electrochemical sensor and preparation method thereof |
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