CN113151241B - Method for immobilizing cellulase by two-dimensional nanosheets - Google Patents
Method for immobilizing cellulase by two-dimensional nanosheets Download PDFInfo
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- 108010059892 Cellulase Proteins 0.000 title claims abstract description 71
- 239000002135 nanosheet Substances 0.000 title claims abstract description 65
- 229940106157 cellulase Drugs 0.000 title claims abstract description 59
- 238000000034 method Methods 0.000 title claims abstract description 17
- 230000003100 immobilizing effect Effects 0.000 title claims abstract description 8
- 102000004190 Enzymes Human genes 0.000 claims abstract description 43
- 108090000790 Enzymes Proteins 0.000 claims abstract description 43
- 229940088598 enzyme Drugs 0.000 claims abstract description 42
- 239000007788 liquid Substances 0.000 claims abstract description 7
- 230000000694 effects Effects 0.000 claims description 35
- 239000008363 phosphate buffer Substances 0.000 claims description 15
- 239000012266 salt solution Substances 0.000 claims description 15
- 108010047754 beta-Glucosidase Proteins 0.000 claims description 14
- 102000006995 beta-Glucosidase Human genes 0.000 claims description 14
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 14
- 239000000243 solution Substances 0.000 claims description 13
- 238000006243 chemical reaction Methods 0.000 claims description 9
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical group COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 7
- HJCNSOVRAZFJLK-UHFFFAOYSA-N C1=CC(C(=O)O)=CC=C1C1=CC2=CC([N]3)=CC=C3C=C(C=C3)NC3=CC([N]3)=CC=C3C=C1N2 Chemical compound C1=CC(C(=O)O)=CC=C1C1=CC2=CC([N]3)=CC=C3C=C(C=C3)NC3=CC([N]3)=CC=C3C=C1N2 HJCNSOVRAZFJLK-UHFFFAOYSA-N 0.000 claims description 7
- 235000019253 formic acid Nutrition 0.000 claims description 7
- 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
- 241001465754 Metazoa Species 0.000 claims description 5
- 229920002678 cellulose Polymers 0.000 claims description 5
- 239000001913 cellulose Substances 0.000 claims description 5
- 239000002253 acid Substances 0.000 claims description 4
- 239000008055 phosphate buffer solution Substances 0.000 claims description 4
- 238000002360 preparation method Methods 0.000 claims description 4
- 230000035484 reaction time Effects 0.000 claims description 4
- 229920000875 Dissolving pulp Polymers 0.000 claims description 3
- 241000196324 Embryophyta Species 0.000 claims description 3
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 3
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 claims description 3
- 239000008367 deionised water Substances 0.000 claims description 3
- 229910021641 deionized water Inorganic materials 0.000 claims description 3
- 238000004090 dissolution Methods 0.000 claims description 3
- 238000004108 freeze drying Methods 0.000 claims description 3
- 125000000524 functional group Chemical group 0.000 claims description 3
- 244000005700 microbiome Species 0.000 claims description 3
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- 239000000843 powder Substances 0.000 claims description 3
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- 239000006228 supernatant Substances 0.000 claims description 3
- 238000005406 washing Methods 0.000 claims description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 3
- 238000000967 suction filtration Methods 0.000 claims 1
- 108010093096 Immobilized Enzymes Proteins 0.000 abstract description 24
- 239000003446 ligand Substances 0.000 abstract description 10
- 230000003197 catalytic effect Effects 0.000 abstract description 6
- 238000005286 illumination Methods 0.000 abstract description 3
- 238000011068 loading method Methods 0.000 abstract description 3
- 229910021645 metal ion Inorganic materials 0.000 abstract description 3
- 150000004032 porphyrins Chemical class 0.000 abstract description 3
- 238000001338 self-assembly Methods 0.000 abstract description 3
- 102000005575 Cellulases Human genes 0.000 description 9
- 108010084185 Cellulases Proteins 0.000 description 9
- 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 description 6
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 6
- 230000003287 optical effect Effects 0.000 description 6
- 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 4
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 4
- 230000009471 action Effects 0.000 description 4
- WPYMKLBDIGXBTP-UHFFFAOYSA-N benzoic acid Chemical compound OC(=O)C1=CC=CC=C1 WPYMKLBDIGXBTP-UHFFFAOYSA-N 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 230000002255 enzymatic effect Effects 0.000 description 4
- 238000006460 hydrolysis reaction Methods 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 3
- 230000007062 hydrolysis Effects 0.000 description 3
- 230000008569 process Effects 0.000 description 3
- 239000000047 product Substances 0.000 description 3
- 239000005711 Benzoic acid Substances 0.000 description 2
- 101710112457 Exoglucanase Proteins 0.000 description 2
- 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
- 241000233866 Fungi Species 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 2
- 235000010233 benzoic acid Nutrition 0.000 description 2
- 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 2
- 239000004305 biphenyl Substances 0.000 description 2
- 230000008859 change Effects 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 238000011161 development Methods 0.000 description 2
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- 230000006872 improvement Effects 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 229920001542 oligosaccharide Polymers 0.000 description 2
- 150000002482 oligosaccharides Chemical class 0.000 description 2
- 230000002035 prolonged effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 150000004043 trisaccharides Chemical class 0.000 description 2
- 239000011800 void material Substances 0.000 description 2
- 108010065511 Amylases Proteins 0.000 description 1
- 102000013142 Amylases Human genes 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 108010008885 Cellulose 1,4-beta-Cellobiosidase Proteins 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- 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
- 229920001410 Microfiber Polymers 0.000 description 1
- 241000228143 Penicillium Species 0.000 description 1
- 102000035195 Peptidases Human genes 0.000 description 1
- 108091005804 Peptidases Proteins 0.000 description 1
- 239000004365 Protease Substances 0.000 description 1
- BOTDANWDWHJENH-UHFFFAOYSA-N Tetraethyl orthosilicate Chemical compound CCO[Si](OCC)(OCC)OCC BOTDANWDWHJENH-UHFFFAOYSA-N 0.000 description 1
- 238000005054 agglomeration Methods 0.000 description 1
- 230000002776 aggregation Effects 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 235000019418 amylase Nutrition 0.000 description 1
- 229940025131 amylases Drugs 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000011942 biocatalyst Substances 0.000 description 1
- 239000000872 buffer Substances 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 238000003028 enzyme activity measurement method Methods 0.000 description 1
- 238000000605 extraction Methods 0.000 description 1
- 230000002349 favourable effect Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000002538 fungal effect Effects 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 108010002430 hemicellulase Proteins 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
- 229930013686 lignan Natural products 0.000 description 1
- 150000005692 lignans Chemical class 0.000 description 1
- 235000009408 lignans Nutrition 0.000 description 1
- 238000012423 maintenance Methods 0.000 description 1
- 238000005259 measurement Methods 0.000 description 1
- 239000012621 metal-organic framework Substances 0.000 description 1
- 239000003658 microfiber Substances 0.000 description 1
- 239000003960 organic solvent Substances 0.000 description 1
- 230000001681 protective effect Effects 0.000 description 1
- 239000002994 raw material Substances 0.000 description 1
- 239000011541 reaction mixture Substances 0.000 description 1
- 238000004064 recycling Methods 0.000 description 1
- 230000000630 rising effect Effects 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 238000003980 solgel method Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 239000000758 substrate Substances 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 230000002194 synthesizing effect Effects 0.000 description 1
Classifications
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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
-
- 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)
-
- 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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- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
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- Health & Medical Sciences (AREA)
- Zoology (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biotechnology (AREA)
- Biomedical Technology (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention discloses a method for immobilizing cellulase by two-dimensional nano-sheets, which comprises the steps of 1) preparing a two-dimensional nano-sheet solution, 2) preparing cellulase enzyme liquid, 3) immobilizing cellulase and the like. The two-dimensional nano sheet is prepared by self-assembly of metal ions and porphyrin ligands, and the thickness of the nano sheet is reduced to below 20nm in the third dimension, so that the prepared nano sheet has higher specific surface area and the enzyme loading capacity is obviously improved. The nano-sheet has good stability and can be repeatedly used. Meanwhile, as the self-assembled ligand uses the ligand with photosensitive property, the nano-sheet 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 two-dimensional nano sheets.
Background
Cellulases are widely found in organisms in nature. Bacteria, fungi, animals, etc., can produce cellulases. The cellulases generally used in production are derived from fungi, typically lignans, aspergillus and penicillium. Cellulase-producing strains are susceptible to degradation, resulting in reduced enzyme production capacity. Cellulases are widely used in both the food industry and the environmental industry. When alcohol fermentation is carried out, the addition of cellulase can increase the utilization rate of raw materials and improve the quality of wine. Because cellulases are difficult to purify, hemicellulases and other related enzymes, such as amylases, proteases, and the like, are also commonly included in practical applications.
Cellulases are of a wide variety and are widely available. Cellulases of different origins vary widely in their structure and function. The cellulase used in animal husbandry and feed industry is mainly fungal cellulase because of its high yield and high activity. As a green biocatalyst, the cellulase 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 that the cellulase is not stable enough under the conditions of high temperature, organic solvent, strong acid or strong alkali and the like, is easy to be denatured and inactivated and has poor tolerance to the environment; is easy to be degraded, is easy to generate agglomeration phenomenon in the reaction process, and is difficult to fully contact with a substrate. The free cellulase has high price, can only catalyze one reaction generally, and cannot be recovered and recycled from a reaction system, so that the production cost of the product is greatly increased, and the wide application of the product in industrial practice is limited.
Beta-glucosidase acts mainly on beta- (1, 4) glycosidic bonds, and also on beta- (1, 1), (1, 2), (1, 3), (1, 6) glycosidic bonds. It is capable of hydrolytically binding to the terminal non-reducing beta-D-glucosidic bond, releasing beta-D-glucose and the corresponding ligand. When the cellulose is catalyzed and hydrolyzed, endoglucanase acts on the noncrystalline region of the microfiber to expose a plurality of tail ends for exoenzyme action, the exoglucanase (cellobiohydrolase) is sequentially decomposed from the non-reducing tail ends to generate cellobiose, the partially degraded cellulose is further decomposed to generate cellobiose, trisaccharide and other oligosaccharides by the synergistic action of the endoglucanase and the exoenzyme, and finally, the cellobiose, the trisaccharide and the other oligosaccharides are decomposed to glucose by the action of beta-glucosidase. In the process, beta-glucosidase plays a key role, but the content of beta-glucosidase in the cellulase component is low, the activity is low, and the maximum roles of endoglucanase and exoglucanase in the enzyme system are restricted, so that cellobiose in hydrolysis 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 required to be additionally added in the cellulase hydrolysis process.
And the enzyme immobilization technology greatly expands the application range of the enzyme. The immobilized enzyme is immobilized in a certain space by a proper method, and can be recovered and reused while maintaining the characteristics of the immobilized enzyme. The advent of immobilized enzymes has addressed many of the limitations of free enzyme practice. Therefore, research on the immobilization technology of cellulase is urgent. The immobilization of the enzyme may allow the enzyme to be easily separated and recovered from the reaction mixture for use in the next reaction. In addition, the immobilization of enzymes can also improve the thermal stability and chemical stability and resist the extreme changing conditions of the outside, so the development of new cellulase immobilization systems is the center of current interest.
Chinese patent CN106867990a discloses a method for preparing immobilized enzyme of fructose aqueous solution, in which fructose is used as pore-forming agent, immobilized beta-glucosidase is prepared by hydrolysis reaction of tetraethoxysilane by sol-gel method. However, the extraction has low enzyme loading and low enzyme recycling rate. Therefore, the development of two-dimensional nanosheets has important significance for the immobilization technology of enzymes, especially cellulases, and the application of the nanosheets in the immobilization of beta-glucosidase, but not limited to the industrial production of the product.
Disclosure of Invention
The invention aims to: aiming at the defects and shortcomings of the prior art, the invention provides a two-dimensional nano-sheet immobilized cellulase method, which prepares the two-dimensional nano-sheet through self-assembly of metal ions and porphyrin ligands, and the thickness of the nano-sheet is reduced to below 20nm in the third dimension, so that the prepared nano-sheet has higher specific surface area and the enzyme loading capacity is obviously improved. The nano-sheet has good stability and can be repeatedly used. Meanwhile, as the self-assembled ligand uses the ligand with photosensitive property, the nano-sheet improves the catalytic efficiency of the immobilized enzyme under illumination.
The technical scheme is as follows: the invention relates to a method for immobilizing cellulase by two-dimensional nano sheets, which comprises the following steps:
1) Preparing a two-dimensional nano sheet solution: lutetium (III) acetate tetrahydrate and tetra (4-carboxyphenyl) porphyrin (H) are prepared by using acid containing carboxyl functional group as regulator 2 TCPP) self-assembling to obtain two-dimensional nano-sheets, and dissolving the two-dimensional nano-sheets in phosphate buffer salt solution to prepare phosphate buffer salt solution containing the two-dimensional nano-sheets, namely two-dimensional nano-sheet solution.
2) Preparing cellulase enzyme liquid: dissolving cellulose powder in phosphate buffer salt solution, uniformly stirring at 2-7 ℃, centrifuging at a rotating speed of 12000-15000 r/min for 3-5 min, transferring supernatant into a volumetric flask, and fixing the volume by using the phosphate buffer salt solution to obtain enzymatic synthesis beta-glucosidase cellulose, namely cellulase enzyme solution;
among them, it is preferable that the mixture is stirred uniformly at 5℃and centrifuged at 12000r/min for 3min.
3) Immobilization of cellulase: mixing the two-dimensional nano-sheet solution with cellulase enzyme liquid, filtering, adding deionized water for dissolution, washing off redundant cellulase by using phosphate buffer solution, fixing the volume, and freeze-drying to obtain the two-dimensional nano-sheet immobilized cellulase.
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 medium-tetra (4-carboxyphenyl) porphyrin to the lutetium (III) acetate tetrahydrate in the step (1) is 1:1-4, and the regulator and the medium-tetra (4-carboxyphenyl) porphyrin (H) 2 TCPP) is 0.6-1.5:1.
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 nano-sheet 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 enzymatic synthesis of the beta-glucosidase cellulase in the step (2) has the activity of 500U/g and the enzyme adding amount of 30-35 mg/g. The enzyme addition amount is preferably 30mg/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 12 hours, the immobilization temperature is preferably 27 ℃, and the immobilization pH is preferably 7.0.
Description of principle: according to the preparation method, the two-dimensional nano sheet is prepared by self-assembly of the metal ions and the porphyrin ligand, and the thickness of the nano sheet is reduced to below 20nm in the third dimension, so that the prepared nano sheet has a higher specific surface area, and the enzyme load capacity is obviously improved. The nano-sheet has good stability and can be repeatedly used. Meanwhile, as the self-assembled ligand uses the ligand with photosensitive property, the nano-sheet improves the catalytic efficiency of the immobilized enzyme under illumination.
The beneficial effects are that: compared with the prior art, the invention has the following remarkable advantages: the two-dimensional nano-sheet prepared by the invention has high specific surface area and high void ratio, which is beneficial to improving the enzyme carrying capacity of immobilized enzyme and improving the catalytic efficiency of immobilized enzyme; the two-dimensional nano-sheet is an immobilized carrier of enzyme, is easy to recycle from a reaction system, can be reused, and greatly improves the utilization rate of the enzyme and reduces the production cost; the two-dimensional nano-sheet has good biocompatibility, and can provide a good microenvironment for the immobilized enzyme protein on the surface of the two-dimensional nano-sheet; the service life and efficiency of the enzyme are greatly improved, and the optical purity of the beta-glucosidase reaches more than 99% after the immobilized enzyme is repeatedly used for 10 times.
Drawings
Fig. 1 is a schematic structural view of a two-dimensional nano-sheet according to the present invention.
Detailed Description
The technical scheme of the invention is further described below with reference to the accompanying drawings and the specific embodiments.
As shown in FIG. 1, the two-dimensional nano-sheet of the invention has a length of 0.2 μm to 10 μm and a thickness of 2nm to 20nm. The projection electron microscope shows that the immobilized enzyme has high specific surface area and high void ratio, and is favorable for improving the enzyme carrying amount 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 nano sheet solution: the method comprises the steps of self-assembling lutetium (III) acetate tetrahydrate and tetra (4-carboxyphenyl) porphyrin (H2 TCPP) to obtain two-dimensional nano-sheets by taking acid containing carboxyl functional groups as a regulator, and dissolving the two-dimensional nano-sheets in a phosphate buffer salt solution to prepare a phosphate buffer salt solution containing the two-dimensional nano-sheets, namely a two-dimensional nano-sheet solution.
2) Preparing cellulase enzyme liquid: dissolving cellulose powder in phosphate buffer salt solution, uniformly stirring at 2-7 ℃, centrifuging at a rotating speed of 12000-15000 r/min for 3-5 min, transferring supernatant into a volumetric flask, and fixing the volume by using the phosphate buffer salt solution to obtain enzymatic synthesis beta-glucosidase cellulose, namely cellulase enzyme solution;
wherein, after being stirred uniformly at 5 ℃, the mixture is centrifuged for 3min at a rotation speed of 12000 r/min.
3) Immobilization of cellulase: mixing the two-dimensional nano-sheet solution with cellulase enzyme liquid, filtering, adding deionized water for dissolution, washing off redundant cellulase by using phosphate buffer solution, fixing the volume, and freeze-drying to obtain the two-dimensional nano-sheet immobilized cellulase.
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 medium-tetra (4-carboxyphenyl) porphyrin to the lutetium (III) acetate tetrahydrate in the step (1) is 1:1-4, and the molar ratio of the regulator to the medium-tetra (4-carboxyphenyl) porphyrin (H2 TCPP) is 0.6-1.5:1; lutetium (III) acetate tetrahydrate was selected to be 22mg, medium-tetra (4-carboxyphenyl) porphyrin was selected to be 10mg, and formic acid was selected to be 9mg. 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 nano-sheet in the preparation step (1) is 120-200 ℃, and the reaction time is 8-24 hours; the reaction temperature was selected to be 160℃and the reaction time was 16 hours. The cellulase powder in the 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 30 mg/g-35 mg/g; the enzyme addition amount was selected to be 30mg/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 12 hours, the immobilization temperature is preferably 27 ℃, and the immobilization pH is preferably 7.0. The research of the inventor shows that:
1. effect of immobilization time on immobilization
The immobilization time is too short, the immobilization is incomplete, the activity of immobilized enzyme is too low, the immobilization time is too long, the activity of enzyme is reduced, and the activity of immobilized enzyme is indirectly reduced. Therefore, when the immobilization time of the two-dimensional nanosheets is less than 12 hours, the activity of immobilized enzymes increases with the increase of the immobilization time. And the enzyme activity starts to decline after the immobilization time of the enzyme and the carrier is longer than 12 hours, probably because the immobilization time is prolonged, the enzyme activity is declined, and the improvement of the immobilized enzyme activity is unfavorable. In conclusion, the optimal immobilization time of the immobilized carrier two-dimensional nano-sheet to the cellulase is 12 hours.
2. Effect of immobilization temperature on immobilization
The selection of a suitable immobilization temperature is critical to the activity of the immobilized enzyme. Under the low-temperature condition, the enzyme activity is kept, but the molecular exchange speed is slow, so that the immobilization time is prolonged; and the temperature is increased, which is unfavorable for maintaining the enzyme activity, 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, which is possibly the increase of the temperature, so that the adsorption of the enzyme is facilitated; the temperature exceeds 27 ℃, the activity of immobilized enzyme begins to decline, and the optimal immobilization temperature of immobilized carrier two-dimensional nanosheets on cellulase is 27 ℃ probably because the activity of enzyme declines due to the rising of temperature.
3. Effect of pH on immobilization
Too high a pH directly affects the activity of the enzyme, and too low a pH is unfavorable for the improvement of the activity of the immobilized enzyme. Cellulase activity was highest in buffer at ph=7.0. Therefore, the optimal immobilization pH of the immobilized carrier two-dimensional nano-sheet to the cellulase is 7.0.
From the above, the present invention provides an optimal immobilization condition in a cellulase immobilization method: the immobilization time was 12h, the immobilization temperature was 27℃and the immobilization pH was 7.0.
4. Thermal stability of immobilized cellulases and free enzymes
The activity change of immobilized cellulase and free cellulase after the action at 20 ℃, 30 ℃, 40 ℃, 50 ℃, 60 ℃, 70 ℃ and 80 ℃ is measured by researching the activity change of immobilized cellulase and free cellulase after the action at different temperatures, and the highest enzyme activity data is taken as 100 percent enzyme activity, and the result shows that: the activity of the free cellulase remained at 26.7% after heating at 80℃for 3 hours, and the enzyme activity of the immobilized cellulase remained at 82.4%. The activity of the two-dimensional nano-sheet immobilized cellulase is greatly improved compared with that of free enzyme, and the thermal stability of the enzyme is enhanced after immobilization, because the immobilized carrier provides an additional protective framework for the maintenance of enzyme molecules.
5. Reusability and optical purity of immobilized cellulase
The immobilized cellulase was subjected to enzyme activity measurement under cellulase measurement conditions (35 ℃ C., 10 min), washed twice with phosphate buffer solution having pH=7, and the reusability and optical purity of the immobilized enzyme required for synthesizing β -glucosidase were measured, and the procedure was repeated 10 times. The remaining immobilized enzyme activities and optical purity were calculated each time. The result shows that after the immobilized carrier two-dimensional nano-sheet immobilized cellulase is repeatedly used for 6 times, the activity of the immobilized carrier two-dimensional nano-sheet immobilized cellulase is kept to 92%, and the optical purity is kept to be more than 97%. The immobilized cellulase is tightly attached to the carrier during the repeated use process, thereby maintaining the activity. After repeated use for 10 times, the activity of the cellulase modified by the two-dimensional nano sheet is kept at 82.6%, and the optical purity is kept above 96%.
Claims (3)
1. A method for immobilizing cellulase by two-dimensional nanosheets is characterized by comprising the following steps: comprising the following steps:
1) Preparing a two-dimensional nano sheet solution: the preparation method comprises the steps of self-assembling lutetium (III) acetate tetrahydrate and medium-tetra (4-carboxyphenyl) porphyrin by taking acid containing carboxyl functional groups as a regulator to obtain two-dimensional nano-sheets, dissolving the two-dimensional nano-sheets in a phosphate buffer salt solution to prepare a phosphate buffer salt solution containing the two-dimensional nano-sheets, namely a two-dimensional nano-sheet solution, wherein the reaction temperature is 160 ℃ and the reaction time is 16 hours during the preparation; the pH value of the phosphate buffer salt solution is 6.8-7.4; the regulator is formic acid; the lutetium (III) acetate tetrahydrate is 22mg, the medium-tetra (4-carboxyphenyl) porphyrin is 10mg, and the formic acid is 9mg;
2) Preparing cellulase enzyme liquid: dissolving cellulose powder in phosphate buffer salt solution, uniformly stirring at 2-7 ℃, centrifuging at a rotating speed of 12000-15000 r/min for 3-5 min, transferring supernatant into a volumetric flask, and fixing the volume by using phosphate buffer salt solution to obtain cellulose enzyme solution; the cellulase is beta-glucosidase;
3) Immobilization of cellulase: mixing the two-dimensional nano-sheet solution with cellulase enzyme liquid, immobilizing cellulase by the two-dimensional nano-sheet, adding deionized water for dissolution after suction filtration, washing off redundant cellulase by using phosphate buffer solution, fixing the volume, and freeze-drying to obtain immobilized cellulase by the two-dimensional nano-sheet; the immobilization time is 12 hours, the immobilization temperature is 27 ℃, and the immobilization pH is 7.0.
2. The method of two-dimensional nanosheet immobilization of cellulase according to claim 1, wherein: the cellulase powder in the step (2) is one extracted from animals, plants or microorganisms.
3. The method of two-dimensional nanosheet immobilization of cellulase according to claim 1, wherein: the activity of the cellulase in the step (2) is 500U/g, and the enzyme adding amount is 30 mg/g-35 mg/g.
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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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