CN113881661A - Method for immobilizing enzymes based on carboxymethyl starch modified magnetic nanoparticles - Google Patents
Method for immobilizing enzymes based on carboxymethyl starch modified magnetic nanoparticles Download PDFInfo
- Publication number
- CN113881661A CN113881661A CN202111154397.6A CN202111154397A CN113881661A CN 113881661 A CN113881661 A CN 113881661A CN 202111154397 A CN202111154397 A CN 202111154397A CN 113881661 A CN113881661 A CN 113881661A
- Authority
- CN
- China
- Prior art keywords
- carboxymethyl starch
- magnetic
- enzyme
- nanoparticles
- starch
- 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
- 239000008107 starch Substances 0.000 title claims abstract description 102
- 229920002472 Starch Polymers 0.000 title claims abstract description 101
- 235000019698 starch Nutrition 0.000 title claims abstract description 101
- 102000004190 Enzymes Human genes 0.000 title claims abstract description 43
- 108090000790 Enzymes Proteins 0.000 title claims abstract description 43
- 125000002057 carboxymethyl group Chemical group [H]OC(=O)C([H])([H])[*] 0.000 title claims abstract description 34
- 239000002122 magnetic nanoparticle Substances 0.000 title claims abstract description 25
- 238000000034 method Methods 0.000 title claims abstract description 23
- 230000003100 immobilizing effect Effects 0.000 title claims abstract description 8
- 229910052708 sodium Inorganic materials 0.000 claims abstract description 61
- 239000011734 sodium Substances 0.000 claims abstract description 61
- 239000002105 nanoparticle Substances 0.000 claims abstract description 60
- 239000000243 solution Substances 0.000 claims abstract description 43
- 108010093096 Immobilized Enzymes Proteins 0.000 claims abstract description 31
- 239000002244 precipitate Substances 0.000 claims abstract description 31
- 238000003756 stirring Methods 0.000 claims abstract description 28
- 238000005406 washing Methods 0.000 claims abstract description 27
- 238000004108 freeze drying Methods 0.000 claims abstract description 16
- 238000001816 cooling Methods 0.000 claims abstract description 13
- FDRCDNZGSXJAFP-UHFFFAOYSA-M sodium chloroacetate Chemical compound [Na+].[O-]C(=O)CCl FDRCDNZGSXJAFP-UHFFFAOYSA-M 0.000 claims abstract description 13
- 229920001685 Amylomaize Polymers 0.000 claims abstract description 12
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims abstract description 10
- 238000004132 cross linking Methods 0.000 claims abstract description 7
- 239000008363 phosphate buffer Substances 0.000 claims abstract description 6
- 239000007864 aqueous solution Substances 0.000 claims abstract description 5
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 39
- 229940088598 enzyme Drugs 0.000 claims description 37
- 238000002360 preparation method Methods 0.000 claims description 35
- 238000006243 chemical reaction Methods 0.000 claims description 25
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 24
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 14
- 239000008055 phosphate buffer solution Substances 0.000 claims description 13
- 229910052757 nitrogen Inorganic materials 0.000 claims description 12
- 239000008367 deionised water Substances 0.000 claims description 11
- 229910021641 deionized water Inorganic materials 0.000 claims description 11
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 10
- 239000004367 Lipase Substances 0.000 claims description 10
- 102000004882 Lipase Human genes 0.000 claims description 9
- 108090001060 Lipase Proteins 0.000 claims description 9
- 235000019421 lipase Nutrition 0.000 claims description 9
- 230000004048 modification Effects 0.000 claims description 8
- 238000012986 modification Methods 0.000 claims description 8
- 101100383916 Drosophila melanogaster kdn gene Proteins 0.000 claims description 6
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- 108091005804 Peptidases Proteins 0.000 claims description 4
- 239000004365 Protease Substances 0.000 claims description 4
- 102100037486 Reverse transcriptase/ribonuclease H Human genes 0.000 claims description 4
- 235000019419 proteases Nutrition 0.000 claims description 4
- 238000007670 refining Methods 0.000 claims description 4
- 108010059892 Cellulase Proteins 0.000 claims description 3
- 229940106157 cellulase Drugs 0.000 claims description 3
- 239000004382 Amylase Substances 0.000 claims description 2
- 108010065511 Amylases Proteins 0.000 claims description 2
- 102000013142 Amylases Human genes 0.000 claims description 2
- 108010004032 Bromelains Proteins 0.000 claims description 2
- 108010059820 Polygalacturonase Proteins 0.000 claims description 2
- 235000019418 amylase Nutrition 0.000 claims description 2
- 108010093305 exopolygalacturonase Proteins 0.000 claims description 2
- 238000001556 precipitation Methods 0.000 claims 4
- 101000984201 Thermomyces lanuginosus Lipase Proteins 0.000 claims 1
- 108010089934 carbohydrase Proteins 0.000 claims 1
- 239000006193 liquid solution Substances 0.000 claims 1
- 239000002245 particle Substances 0.000 abstract description 12
- 229920000881 Modified starch Polymers 0.000 abstract description 3
- 239000004368 Modified starch Substances 0.000 abstract description 3
- 235000019426 modified starch Nutrition 0.000 abstract description 3
- 239000003960 organic solvent Substances 0.000 abstract description 3
- 230000000694 effects Effects 0.000 abstract description 2
- 239000000872 buffer Substances 0.000 abstract 1
- SZVJSHCCFOBDDC-UHFFFAOYSA-N iron(II,III) oxide Inorganic materials O=[Fe]O[Fe]O[Fe]=O SZVJSHCCFOBDDC-UHFFFAOYSA-N 0.000 description 82
- 238000004128 high performance liquid chromatography Methods 0.000 description 12
- 230000003197 catalytic effect Effects 0.000 description 11
- 239000000758 substrate Substances 0.000 description 9
- HSTZMXCBWJGKHG-UHFFFAOYSA-N (E)-piceid Natural products OC1C(O)C(O)C(CO)OC1OC1=CC(O)=CC(C=CC=2C=CC(O)=CC=2)=C1 HSTZMXCBWJGKHG-UHFFFAOYSA-N 0.000 description 6
- 239000005018 casein Substances 0.000 description 6
- BECPQYXYKAMYBN-UHFFFAOYSA-N casein, tech. Chemical compound NCCCCC(C(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(CC(C)C)N=C(O)C(CCC(O)=O)N=C(O)C(CC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(C(C)O)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=N)N=C(O)C(CCC(O)=O)N=C(O)C(CCC(O)=O)N=C(O)C(COP(O)(O)=O)N=C(O)C(CCC(O)=N)N=C(O)C(N)CC1=CC=CC=C1 BECPQYXYKAMYBN-UHFFFAOYSA-N 0.000 description 6
- 235000021240 caseins Nutrition 0.000 description 6
- 230000005389 magnetism Effects 0.000 description 6
- 229960003764 polydatin Drugs 0.000 description 6
- HSTZMXCBWJGKHG-CUYWLFDKSA-N trans-piceid Polymers O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC(O)=CC(\C=C\C=2C=CC(O)=CC=2)=C1 HSTZMXCBWJGKHG-CUYWLFDKSA-N 0.000 description 6
- 239000000413 hydrolysate Substances 0.000 description 5
- 230000007062 hydrolysis Effects 0.000 description 5
- 238000006460 hydrolysis reaction Methods 0.000 description 5
- 238000002156 mixing Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 239000006228 supernatant Substances 0.000 description 5
- 238000004458 analytical method Methods 0.000 description 4
- 238000009210 therapy by ultrasound Methods 0.000 description 4
- 239000002253 acid Substances 0.000 description 3
- 238000005917 acylation reaction Methods 0.000 description 3
- 230000008859 change Effects 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 239000006185 dispersion Substances 0.000 description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 3
- 230000000977 initiatory effect Effects 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000004064 recycling Methods 0.000 description 3
- 238000005070 sampling Methods 0.000 description 3
- 238000005303 weighing Methods 0.000 description 3
- DGAQECJNVWCQMB-PUAWFVPOSA-M Ilexoside XXIX Chemical compound C[C@@H]1CC[C@@]2(CC[C@@]3(C(=CC[C@H]4[C@]3(CC[C@@H]5[C@@]4(CC[C@@H](C5(C)C)OS(=O)(=O)[O-])C)C)[C@@H]2[C@]1(C)O)C)C(=O)O[C@H]6[C@@H]([C@H]([C@@H]([C@H](O6)CO)O)O)O.[Na+] DGAQECJNVWCQMB-PUAWFVPOSA-M 0.000 description 2
- 238000002835 absorbance Methods 0.000 description 2
- 238000010521 absorption reaction Methods 0.000 description 2
- 230000009471 action Effects 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 230000015271 coagulation Effects 0.000 description 2
- 238000005345 coagulation Methods 0.000 description 2
- 239000011248 coating agent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- -1 enol ethylene ester Chemical class 0.000 description 2
- 150000002772 monosaccharides Chemical class 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000035945 sensitivity Effects 0.000 description 2
- 229920001567 vinyl ester resin Polymers 0.000 description 2
- JWUJQDFVADABEY-UHFFFAOYSA-N 2-methyltetrahydrofuran Chemical compound CC1CCCO1 JWUJQDFVADABEY-UHFFFAOYSA-N 0.000 description 1
- PUQSUZTXKPLAPR-KSSYENDESA-N 4-(beta-D-Glucopyranosyloxy) benzyl alcohol Natural products O([C@H]1[C@H](O)[C@@H](O)[C@@H](O)[C@@H](CO)O1)c1ccc(CO)cc1 PUQSUZTXKPLAPR-KSSYENDESA-N 0.000 description 1
- 229920000856 Amylose Polymers 0.000 description 1
- 239000005977 Ethylene Substances 0.000 description 1
- 229910017135 Fe—O Inorganic materials 0.000 description 1
- 238000005033 Fourier transform infrared spectroscopy Methods 0.000 description 1
- PUQSUZTXKPLAPR-UJPOAAIJSA-N Gastrodin Chemical compound O[C@@H]1[C@@H](O)[C@H](O)[C@@H](CO)O[C@H]1OC1=CC=C(CO)C=C1 PUQSUZTXKPLAPR-UJPOAAIJSA-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
- 229910021578 Iron(III) chloride Inorganic materials 0.000 description 1
- 238000005481 NMR spectroscopy Methods 0.000 description 1
- 238000003917 TEM image Methods 0.000 description 1
- 241000223258 Thermomyces lanuginosus Species 0.000 description 1
- 230000010933 acylation Effects 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 239000008346 aqueous phase Substances 0.000 description 1
- 238000012658 bimolecular nucleophilic substitution Methods 0.000 description 1
- 239000011942 biocatalyst Substances 0.000 description 1
- 239000012620 biological material Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 125000004432 carbon atom Chemical group C* 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 238000006555 catalytic reaction Methods 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 238000003889 chemical engineering Methods 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000004090 dissolution Methods 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 239000003792 electrolyte Substances 0.000 description 1
- 238000005538 encapsulation Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 230000009144 enzymatic modification Effects 0.000 description 1
- 238000006266 etherification reaction Methods 0.000 description 1
- 238000000855 fermentation Methods 0.000 description 1
- 230000004151 fermentation Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 229930193974 gastrodin Natural products 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 125000002791 glucosyl group Chemical group C1([C@H](O)[C@@H](O)[C@H](O)[C@H](O1)CO)* 0.000 description 1
- PUQSUZTXKPLAPR-NZEXEKPDSA-N helicidol Natural products O([C@H]1[C@H](O)[C@H](O)[C@@H](O)[C@@H](CO)O1)c1ccc(CO)cc1 PUQSUZTXKPLAPR-NZEXEKPDSA-N 0.000 description 1
- 238000000338 in vitro Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- RBTARNINKXHZNM-UHFFFAOYSA-K iron trichloride Chemical compound Cl[Fe](Cl)Cl RBTARNINKXHZNM-UHFFFAOYSA-K 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 229910052603 melanterite Inorganic materials 0.000 description 1
- 239000012528 membrane Substances 0.000 description 1
- 239000003607 modifier Substances 0.000 description 1
- 239000002114 nanocomposite Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 229920001343 polytetrafluoroethylene Polymers 0.000 description 1
- 239000004810 polytetrafluoroethylene Substances 0.000 description 1
- 239000000843 powder Substances 0.000 description 1
- 230000001376 precipitating effect Effects 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000004007 reversed phase HPLC Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- 238000007711 solidification Methods 0.000 description 1
- 230000008023 solidification Effects 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
- 238000009736 wetting Methods 0.000 description 1
Images
Classifications
-
- 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
- C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
- C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
- C08B31/00—Preparation of derivatives of starch
- C08B31/08—Ethers
- C08B31/12—Ethers having alkyl or cycloalkyl radicals substituted by heteroatoms, e.g. hydroxyalkyl or carboxyalkyl starch
-
- 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
- C12N11/18—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
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
-
- 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)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/63—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from plants
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/22—Cysteine endopeptidases (3.4.22)
- C12Y304/22004—Bromelain (3.4.22.4)
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)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Microbiology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Molecular Biology (AREA)
- Medicinal Chemistry (AREA)
- Inorganic Chemistry (AREA)
- Materials Engineering (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Polymers & Plastics (AREA)
- Botany (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
本发明涉及固定化酶领域，公开了一种基于羧甲基淀粉修饰的磁性纳米粒子固定化酶的方法，将高直链淀粉在碱性条件下与氯乙酸钠反应，得羧甲基淀粉钠；将羧甲基淀粉钠加入磁性Fe3O4纳米粒子水溶液中，持续搅拌，经洗涤沉淀后冷冻干燥得磁性羧甲基淀粉纳米粒子；将磁性变性淀粉纳米粒子加入磷酸缓冲液A和戊二醛溶液，交联后，经洗涤获得沉淀，后加入磷酸缓冲液B及游离酶液，于固定温度下进行搅拌，冷却至室温，经缓冲液洗涤后获取沉淀，冷冻干燥得磁性淀粉纳米粒子固定化酶。通过本发明制备的磁性淀粉纳米粒子固定化酶粒径小、酶活高，分散性好，相比游离酶其具有更优良的热稳定性、pH稳定性及有机溶剂耐受性。
The invention relates to the field of immobilized enzymes, and discloses a method for immobilizing enzymes based on carboxymethyl starch modified magnetic nanoparticles. The high amylose starch is reacted with sodium chloroacetate under alkaline conditions to obtain sodium carboxymethyl starch; Sodium carboxymethyl starch was added to the magnetic Fe 3 O 4 nanoparticle aqueous solution, stirred continuously, washed and precipitated and then freeze-dried to obtain magnetic carboxymethyl starch nanoparticles; the magnetic modified starch nanoparticles were added to phosphate buffer A and glutaraldehyde solution , after cross-linking, washing to obtain a precipitate, then adding phosphate buffer B and free enzyme solution, stirring at a fixed temperature, cooling to room temperature, washing with a buffer to obtain a precipitate, and freeze-drying to obtain a magnetic starch nanoparticle immobilized enzyme . The magnetic starch nanoparticle immobilized enzyme prepared by the invention has small particle size, high enzyme activity and good dispersibility, and has better thermal stability, pH stability and organic solvent tolerance than free enzyme.
Description
Technical Field
The invention relates to the field of nano composite materials, in particular to a method for preparing magnetic nanoparticle immobilized enzyme based on carboxymethyl starch modification.
Background
The enzyme is used as a biocatalyst with high specificity and high-efficiency catalytic performance, has high catalytic activity even in vitro, and is widely applied to industries such as food, chemical engineering, medicine and the like. Therefore, the utilization of technical means to improve the stability and tolerance of the enzyme and realize the recycling of the enzyme is a hot spot of the present research.
The immobilized enzyme is an enzyme modification means for immobilizing volatile active denatured free enzyme on a solid carrier or wrapping the volatile active denatured free enzyme by the carrier so as to improve the stability and adaptability of the enzyme. The immobilized enzyme technology enables the enzyme to be easily separated and recovered, improves the use efficiency of the enzyme, has stronger tolerance to organic solvents compared with free enzyme, and can realize non-aqueous phase enzyme catalysis. The material of the carrier can endow the immobilized enzyme with more properties. Magnetic Fe3O4The nano particles have the advantages of small size, large specific surface area, small mass transfer resistance and high catalytic efficiency, and can recycle the magnetic immobilized enzyme under the condition of applying a magnetic field to the outside.
The magnetic nano particles have high surface energy, so that the magnetic nano particles are easy to agglomerate, the surfaces of the magnetic nano particles are protected and modified, and the magnetic nano particles are prevented from being excessively agglomerated and oxidized, and simultaneously, the magnetic nano particles also enable the surfaces of the magnetic nano particles to be protected and modifiedThe surface of which has more reactive groups to attach to the enzyme. The sodium carboxymethyl starch contains abundant hydroxyl groups and carboxymethyl groups in the molecular structure, the hydroxyl groups enable the sodium carboxymethyl starch to have good hydrophilicity and modifiability, the carboxymethyl groups reduce the retrogradation of the magnetic nanoparticles, improve the dispersion stability of the magnetic nanoparticles, and modify and wrap Fe3O4Good biomaterials of nanoparticles. Modified starch as Fe3O4The surface modifier is applied to the preparation of materials in the fields of biomedicine, nuclear magnetic resonance and the like.
Disclosure of Invention
The purpose of the invention is as follows: aiming at the technical defects of free enzyme in industrial application, such as sensitivity of a primary structure and a spatial structure to the environment, easiness in damage, difficulty in recycling, narrow action range, sensitivity to a trace inhibitor and the like, the invention provides a method for immobilizing the enzyme by using magnetic nanoparticles based on carboxymethyl starch modification.
The technical scheme is as follows: the invention provides a method for preparing magnetic nanoparticle immobilized enzyme based on carboxymethyl starch modification, which comprises the following steps: preparation of sodium carboxymethyl starch: taking sodium chloroacetate and high amylose starch in proportion into an ethanol solution, adding a sodium hydroxide solution to adjust to be alkaline, continuously stirring for 0.5-10.0 h at 40-70 ℃, adding a hydrochloric acid solution to adjust the pH value to 7-8, cooling to room temperature to obtain a precipitate, washing the precipitate for multiple times, and freeze-drying to obtain sodium carboxymethyl starch; preparing magnetic sodium carboxymethyl starch nanoparticles: to magnetic Fe3O4Adding the sodium carboxymethyl starch into the nano particle water solution, continuously stirring for 0.5-5.0 h at 50-105 ℃, cooling to room temperature to obtain a precipitate, washing the precipitate for multiple times, and freeze-drying to obtain magnetic sodium carboxymethyl starch nano particles; preparing magnetic carboxymethyl starch sodium nano particle immobilized enzyme: adding the magnetic sodium carboxymethyl starch nanoparticles into a phosphate buffer solution A and a glutaraldehyde solution, crosslinking for 0.5-4.0 h at room temperature, washing for multiple times to obtain a precipitate, adding a phosphate buffer solution B and a free enzyme solution, and solidifying at 20-55 DEG CAnd (3) solidifying for 2.5-15.0 h, stirring continuously, cooling the system to room temperature after solidification to obtain a precipitate, and freeze-drying to obtain the magnetic carboxymethyl starch sodium nanoparticle immobilized enzyme.
Preferably, in the preparation process of the sodium carboxymethyl starch, the mass ratio of the sodium chloroacetate to the high amylose is 2: 1-1: 1; the mass ratio of the sodium chloroacetate to the sodium hydroxide solution is 1: 2-1: 1.5.
Preferably, in the preparation process of the magnetic sodium carboxymethyl starch nanoparticles: the magnetic Fe3O4The mass ratio of the nano particles to the sodium carboxymethyl starch is 6: 1-3: 2.
Preferably, the feed-liquid ratio of the magnetic sodium carboxymethyl starch nanoparticles to the phosphate buffer solution A is 1: 5-1: 20 g/mL.
Preferably, the feed-liquid ratio of the free enzyme solution to the phosphate buffer solution B is 1: 10-1: 35 g/mL.
Further, the magnetic Fe3O4The preparation method of the nano-particles comprises the following steps: introducing nitrogen into a container filled with deionized water for 20-60 min, and respectively adding Fe according to the mol ratio of 1: 2-1: 0.52+And Fe3+Continuously stirring at 40-100 ℃ until the solution is dissolved, dropwise adding NaOH solution with nitrogen introduced for 20-60 min into the container, continuously stirring, stopping dropwise adding when the pH reaches 9-12, continuously introducing nitrogen and stirring for a period of time, cooling the reaction system to room temperature after the system is stabilized, washing for multiple times to obtain precipitate, and freeze-drying the precipitate to obtain the magnetic Fe3O4Nanoparticles.
Preferably, the Fe2+Derived from FeSO4·7H2O, said Fe3+Derived from FeCl3·6H2O。
Preferably, the molar concentration of the NaOH solution is 1.0-3.0 mol/L.
Preferably, the enzyme in the free enzyme solution is protease, lipase or KDN refining enzyme, and the protease is bromelain Bromelins, BR; the lipase is thermomyces lanuginosus lipaseThermomyces lanuginosus Lipase, TLL; the KDN refining enzyme is a plurality of pectinase components and xylanEnzyme, amylase, cellulase, lipase, etc.
Preferably, the pH values of the phosphate buffer solution A and the phosphate buffer solution B are both 6.0-9.0; the concentration of the glutaraldehyde is 0.5-4.0% v/v.
Preferably, in the preparation process of the immobilized enzyme, the stirring speed is 150-450 r/min.
Preferably, in the preparation process of the magnetic sodium carboxymethyl starch nanoparticles, the washing mode of the multiple washing precipitates is washing with deionized water; in the preparation process of the magnetic sodium carboxymethyl starch nanoparticle immobilized enzyme, the washing mode of obtaining the precipitate by multiple times of washing is as follows: washing was performed alternately with deionized water and phosphate buffer of the corresponding pH.
The method for preparing the magnetic nanoparticle immobilized enzyme based on carboxymethyl starch modification specifically comprises the following steps:
(1) preparation of sodium carboxymethyl starch: weighing 20-100 mL of 80-100% ethanol solution, adding sodium chloroacetate and high amylose starch into a three-neck flask according to the mass ratio of 2: 1-1: 1, dropwise adding 1.0-3.0 mol/L sodium hydroxide solution in the reaction process, stirring at high speed continuously, stopping dropwise adding when a certain pH value is reached, continuously stirring for 0.5-10.0 h in an oil bath kettle at 40-70 ℃, cooling to room temperature to obtain precipitate, washing the precipitate for multiple times, and freeze-drying to obtain carboxymethyl starch particles.
(2) Magnetic Fe3O4Preparing nano particles: introducing nitrogen into a container filled with deionized water for 20-60 min, and respectively adding Fe according to the mol ratio of 1: 2-1: 0.52+And Fe3+Continuously stirring at 40-100 ℃ until the solution is dissolved, dropwise adding NaOH solution with nitrogen introduced for 20-60 min into the container, continuously stirring, stopping dropwise adding when the pH reaches 9-12, continuously introducing nitrogen and stirring for a period of time, cooling the reaction system to room temperature after the system is stabilized, washing for multiple times to obtain precipitate, and freeze-drying the precipitate to obtain the magnetic Fe3O4Nanoparticles.
(3) Preparing magnetic carboxymethyl starch nanoparticles: taking 0.4-2.0 g of the prepared sodium carboxymethyl starch to be put into a 500 ml three-neck flask, and adding 10100ml of deionized water, performing ultrasonic treatment for 10-30 min to fully dissolve the deionized water, and adding Fe with a certain mass ratio to the sodium carboxymethyl starch3O4Continuously stirring for 0.5-5.0 h at the rotating speed of 100-600 r/min in an oil bath pan at the temperature of 50-105 ℃, cooling to room temperature, obtaining precipitates by utilizing the magnetism of the precipitates, washing for many times until supernatant is transparent, and storing for later use in a sealing way at the temperature of 4 ℃ after freeze drying.
(4) Preparing magnetic carboxymethyl starch nano particle immobilized enzyme: weighing 0.5-3.0 g of magnetic carboxymethyl starch nanoparticles, adding 5-30 mL of phosphate buffer solution A with certain pH and glutaraldehyde with certain concentration, crosslinking for 0.5-5 h at room temperature at 100-500 r/min, then obtaining a precipitate by utilizing the magnetism of the magnetic carboxymethyl starch nanoparticles, and washing the precipitate for multiple times by using deionized water until the supernatant is transparent. And then adding the free enzyme solution into a reaction bottle according to a certain material-to-solution ratio, adding 5-30 mL of phosphate buffer solution B with a certain pH, immobilizing for 0.5-24 h at 4-60 ℃, stirring continuously, cooling the system to room temperature after immobilization is finished, obtaining precipitates by utilizing magnetism of the system, washing for many times until the upper layer liquid is transparent, and storing for later use in a sealed manner at 4 ℃ after freeze drying.
Has the advantages that:
the sodium carboxymethyl starch is etherified modified starch, can be directly dissolved in cold water, has lower gelatinization temperature compared with original starch (such as soluble starch), belongs to anionic electrolyte, can be directly adsorbed and connected with enzyme or ferroferric oxide under the control of ph, and has better dispersion performance of magnetic sodium carboxymethyl starch nanoparticles. Sodium carboxymethyl starch is prepared through bimolecular nucleophilic substitution reaction of sodium chloroacetate on the hydroxyl radicals in the sixth, second and third carbon atoms with etherifying capacity on glucose residue in starch, and has the features of high hydrophilicity, high electronegativity, weak coagulation property, etc. owing to the cationic exchange characteristic of carboxymethyl starch, sodium carboxymethyl starch may be well combined with Fe3O4The nanometer particle combination and the electronegativity of sodium carboxymethyl starch can well improve the magnetic Fe3O4Nanoparticle coagulation phenomenon.
The invention uses etherified sodium carboxymethyl starch as a magnetic Fe3O4The nano particles are subjected to surface modification to obtain a nano-particle with a large amount of surfaceThe immobilized BR, the immobilized KDN and the immobilized TLL prepared by the method have good dispersibility, high enzyme activity recovery rate, good thermal stability and pH stability, and stronger tolerance to organic solvents. Mixing Fe3O4@ CMS @ BR is used for catalyzing casein hydrolysis, and the obtained hydrolysis rate can reach 81.5 percent; mixing Fe3O4The @ CMS @ TLL is used for catalyzing acylation reaction of polydatin and crotonoenoic acid vinyl ester, the conversion rate can reach 98.4 percent at most, and the catalyst has better catalytic activity; mixing Fe3O4The @ CMS @ KDN is used for catalyzing enzymolysis and saccharification of lignocellulose, and the total sugar yield of the obtained hydrolysate can reach 66%.
Drawings
FIG. 1 is Fe in embodiments 1 to 43O4、Fe3O4@CMS、Fe3O4@ CMS @ KDN and Fe3O4Transmission electron micrograph of @ CMS @ TLL;
FIG. 2 is a FT-IR chart of the magnetic carboxymethyl starch and the magnetic carboxymethyl starch immobilized enzyme in embodiments 1 to 4;
FIG. 3 shows Fe in embodiments 1 to 43O4The dispersion (A) and the external magnetic field adsorption (B) of @ CMS @ TLL in the aqueous solution;
FIG. 4 is an HPLC analysis chart of a casein sample in embodiments 1 to 4;
FIG. 5 shows the treatment of casein with Fe in embodiments 1 to 43O4HPLC analysis profile after @ CMS @ BR hydrolysis;
fig. 6 is an HPLC analysis profile of the polydatin standard substance according to embodiments 1 to 4;
FIG. 7 shows that polydatin is treated with Fe in embodiments 1 to 43O4HPLC analysis profile after acylation of @ CMS @ TLL;
FIG. 8 is an HPLC analysis chart of monosaccharide in hydrolysate of lignocellulose in embodiments 1 to 4;
FIG. 9 shows the conversion of lignocellulose to Fe in embodiments 1 to 43O4HPLC analysis map of hydrolysate monosaccharide after @ CMS @ KDN enzymolysis.
Detailed Description
The present invention will be described in detail with reference to the accompanying drawings.
Preparation of sodium carboxymethyl starch:
measuring 30 ml of 80% ethanol solution, putting the ethanol solution into a three-neck flask, putting 1.0 g of high amylose starch into the three-neck flask, fully dissolving the high amylose starch, adding 2 g of sodium chloroacetate, performing ultrasonic treatment for 3 min, after the high amylose starch is completely dissolved, dropwise adding sodium hydroxide solution with the molar concentration of 3.0 mol/L into the high amylose starch solution, continuously stirring the mixture at a high speed, stopping dropwise adding when the pH value reaches 10, continuously stirring the mixture in an oil bath kettle at 45 ℃ for 4h, cooling the mixture to room temperature after the reaction is finished, washing and precipitating the mixture for multiple times, and freeze-drying the mixture to obtain the carboxymethyl starch particles.
Magnetic Fe3O4Preparing nano particles:
50 mL of deionized water is put into a three-mouth bottle, nitrogen with the purity of more than 99.9 percent is introduced for 20 min, and Fe is added according to the molar ratio of 1:22+And Fe3+Stirring was continued until dissolved in an oil bath at a temperature of 50 ℃. After complete dissolution, dropwise adding 1.0 mol/L NaOH solution which is introduced with nitrogen for 20 min into a three-necked flask, stirring at high speed continuously, stopping dropwise adding when the pH of the solution reaches 10, continuously introducing nitrogen, stirring for 1 h, after the system is stable, obtaining precipitate by utilizing the magnetism of the solution, washing the precipitate for multiple times until the supernatant is transparent, and hermetically storing magnetic Fe at 4 ℃ after freeze drying3O4The nano particles are ready for use.
Magnetic sodium carboxymethyl starch nanoparticle Fe3O4Preparation of @ CMS:
dissolving 0.5 g of the above prepared sodium carboxymethyl starch particles in 25 ml of deionized water, performing ultrasonic treatment for 10 min to dissolve completely, and weighing 1 g of the above magnetic Fe3O4Placing the nano particles in a three-neck flask, transferring a sodium carboxymethyl starch solution into the three-neck flask, adjusting the pH to 5 by using a hydrochloric acid solution, continuously stirring for 1.0 h in a 65 ℃ oil bath kettle at a rotating speed of 200 r/min, cooling to room temperature, obtaining precipitates by using magnetism of the precipitates, washing for many times until supernatant is transparent, and storing Fe in a sealed manner at 4 ℃ after freeze drying3O4@ CMS for use.
Magnetic carboxymethyl starchSodium powder nano particle immobilized enzyme Fe3O4@ CMS @ BR (or Fe)3O4@ CMS @ TLL or Fe3O4@ CMS @ KDN) preparation:
0.5 g of magnetic sodium carboxymethyl starch nanoparticles are weighed, 10 mL of phosphate buffer with pH 5.5 and 1.0 mL of glutaraldehyde with concentration of 2.0% v/v are added, and after crosslinking is carried out for 1.0 h at room temperature at 200 r/min, precipitates are obtained by utilizing magnetism of the magnetic sodium carboxymethyl starch nanoparticles, and the magnetic sodium carboxymethyl starch nanoparticles are washed by deionized water for multiple times until supernatant is transparent. Adding 5.0 mL of BR enzyme solution (or KDN enzyme solution or TLL enzyme solution) into a reaction flask, adding 10 mL of phosphate buffer solution with pH of 5.5, immobilizing at 25 deg.C for 2 h while stirring, cooling to room temperature after immobilization, magnetically obtaining precipitate, washing for multiple times until the upper layer is transparent, freeze-drying, and hermetically storing Fe at 4 deg.C3O4@ CMS @ BR (or Fe)3O4@ CMS @ TLL or Fe3O4@ CMS @ KDN) for use.
Embodiment 2:
this embodiment is substantially the same as embodiment 1, and differs only in that: in the preparation process of the sodium carboxymethyl starch, the mass ratio of the high amylose starch to the sodium chloroacetate is 1:1.5, and the preparation temperature is 50 ℃.
In the presence of magnetic Fe3O4In the preparation of the nanoparticles, Fe2+With Fe3+At a molar ratio of 1:1.5, and a preparation temperature of 60 ℃.
In magnetic sodium carboxymethyl starch nano particle Fe3O4In the preparation process of @ CMS, sodium carboxymethyl starch and Fe3O4The mass ratio of (A) to (B) is 1:3, the preparation temperature is 90 ℃, and the immobilization pH is 7.
Magnetic sodium carboxymethyl starch nanoparticle immobilized enzyme Fe3O4@ CMS @ BR (or Fe)3O4@ CMS @ TLL or Fe3O4@ CMS @ KDN), the glutaraldehyde concentration is 3.0% v/v, the addition amount is 2.0 ml, the crosslinking time is 2 h, the immobilization temperature is 40 ℃, and the immobilization pH is 7.
Otherwise, this embodiment is identical to embodiment 1, and will not be described herein.
Embodiment 3:
this embodiment is substantially the same as embodiment 1, and differs only in that: in the preparation process of the sodium carboxymethyl starch, the mass ratio of the high amylose starch to the sodium chloroacetate is 1:1, and the preparation temperature is 40 ℃.
In the presence of magnetic Fe3O4In the preparation of the nanoparticles, Fe2+With Fe3+At a molar ratio of 1:1, and a preparation temperature of 70 ℃.
In magnetic sodium carboxymethyl starch nano particle Fe3O4In the preparation process of @ CMS, sodium carboxymethyl starch and Fe3O4The mass ratio of (A) to (B) is 1:4, the preparation temperature is 50 ℃, and the pH value of the immobilization environment is 10.
Magnetic sodium carboxymethyl starch nanoparticle immobilized enzyme Fe3O4@ CMS @ BR (or Fe)3O4@ CMS @ TLL or Fe3O4@ CMS @ KDN), the glutaraldehyde concentration is 2.5% v/v, the addition amount is 2.5 ml, the crosslinking time is 1 h, the immobilization temperature is 40 ℃, and the immobilization pH is 8.
Otherwise, this embodiment is identical to embodiment 1, and will not be described herein.
Embodiment 4:
this embodiment is substantially the same as embodiment 1, and differs only in that: in the preparation process of the sodium carboxymethyl starch, the mass ratio of the high amylose starch to the sodium chloroacetate is 1:3, and the preparation temperature is 50 ℃.
In the presence of magnetic Fe3O4In the preparation of the nanoparticles, Fe2+With Fe3+At a molar ratio of 1:0.5, at a preparation temperature of 80 ℃.
In magnetic sodium carboxymethyl starch nano particle Fe3O4In the preparation process of @ CMS, sodium carboxymethyl starch and Fe3O4The mass ratio of (A) to (B) is 1: 5.
Magnetic sodium carboxymethyl starch nanoparticle immobilized enzyme Fe3O4@ CMS @ BR (or Fe)3O4@ CMS @ TLL or Fe3O4@ CMS @ KDN), glutaraldehyde concentration of 2% v/v, addition amount of 1.5ml, and crosslinkingThe time is 4h, and the immobilization temperature is 30 ℃.
Otherwise, this embodiment is identical to embodiment 1, and will not be described herein.
For Fe prepared in the above embodiments 1 to 43O4、Fe3O4@CMS、Fe3O4@CMS@BR、Fe3O4@ CMS @ TLL and Fe3O4Transmission electron microscopy analysis of @ CMS @ KDN, Fe3O4The results are shown in FIG. 1 (A): fe3O4Is in a spherical shape with regular shape and smooth and flat surface, Fe3O4The particle size is about 7.8 +/-1.3 nm. Fe3O4The @ CMS results are shown in FIG. 1 (B): fe3O4The particle size of @ CMS is about 18.2 + -2.8 nm, and after coating with sodium carboxymethyl starch, the surface changes from being relatively flat to having rough cracks and rugged state, which may be caused by the surface becoming rough after etherification of sodium carboxymethyl starch, and the change in surface state indicates successful coating of sodium carboxymethyl starch. Fe3O4@CMS@BR、Fe3O4@ CMS @ TLL and Fe3O4The results of @ CMS @ KDN are shown in FIGS. 1 (C) and 1 (E): fe3O4@ CMS @ BR having a particle size of about 26.4. + -. 2.2 nm, Fe3O4@ CMS @ KDN has a particle size of about 20.3 + -3.2 nm, Fe3O4The particle size of @ CMS @ TLL is about 18.7 +/-2.1 nm, the shape of the particles is changed from a state of surface crack unevenness to a relatively smooth spherical shape, and the particle outline is relatively blurred, which intuitively shows the successful immobilization of the enzyme.
For Fe prepared in the above embodiments 1 to 43O4、Fe3O4@CMS、Fe3O4@CMS@BR、Fe3O4@ CMS @ TLL and Fe3O4Infrared analysis was carried out at @ CMS @ KDN, and as a result, as shown in FIG. 2, it can be seen that the characteristic absorption band of Fe-O stretching vibration is located at 581 cm-1Of Fe3O4@ CMS at 3380 cm-1A characteristic peak of starch appears, which is an absorption peak belonging to hydroxyl groups, and simultaneously, Fe3O4@ CMS in2930 cm-11619 cm-1, 1482 cm-1 and 1020cm-1The characteristic peak of starch, 1619 cm, appears on the left and right-1The absorbance of (B) is due to C-O-C stretching vibration in starch, 1020cm-1The absorbance of (A) was due to the C-C and C-O stretching patterns of the starch backbone, which all indicate successful loading of the starch. At 690--1In the beam range, Fe3O4@CMS@BR、Fe3O4@ CMS @ TLL and Fe3O4@ CMS @ KDN at 1602 cm-1The area of the peak was significantly reduced due to encapsulation by cellulase or lipase, and immobilization of BR, TLL or KDN was also demonstrated.
For Fe prepared in the above embodiments 1 to 43O4The results of the analysis of the dispersibility of @ CMS are shown in FIG. 3A, and Fe3O4The @ CMS has good dispersibility in aqueous solution, and effectively solves the problem of Fe3O4B shows Fe3O4@ CMS is rapidly separated in solution under the action of an external magnetic field, indicating that Fe3O4@ CMS good recycling efficiency.
The magnetic sodium carboxymethyl starch nanoparticles prepared in the above embodiments 1 to 4 were immobilized on the enzyme Fe3O4The catalytic activity of @ CMS @ BR in catalyzing casein hydrolysis was verified as follows:
adding buffer solution with a certain volume into a reaction bottle, adding casein into the reaction bottle, fully dissolving the substrate, and taking 20μL, HPLC analysis, addition of Fe3O4@ CMS @ BR, reacting for a certain time, and carrying out HPLC analysis on hydrolysate after passing through a membrane under the same conditions. The conversion was calculated as peak area.
The results are shown in FIGS. 4 and 5: the hydrolysis rate of casein was 81.35. + -. 2.15%, indicating that Fe prepared by the present invention3O4@ CMS @ BR has good catalytic activity.
For Fe prepared in the above embodiments 1 to 43O4The catalytic activity of @ CMS @ TLL in catalyzing the acylation reaction of polydatin crotonyl enol ethylene ester is verified, and the process is as follows:
adding a certain volume of 2-methyltetrahydrofuran into a reaction bottle with a polytetrafluoroethylene cover, adding polydatin into the reaction bottle, performing ultrasonic treatment for 20 s to fully dissolve the substrate until the substrate is invisible to naked eyes, and sampling for 30 daysμL, HPLC analysis. Adding crotonoenoic acid vinyl ester and Fe3O4@ CMS @ TLL, shaking, mixing uniformly, reacting at 45 ℃ at 200 r/min, sampling after a period of time, and analyzing by HPLC. And (5) obtaining the conversion rate of the gastrodin according to the change of the peak area.
The catalytic activity of the magnetic starch immobilized lipase is verified, HPLC analysis is shown in figures 6 and 7, the conversion rate of polydatin can reach 98.04 +/-1.46%, and the Fe prepared by the invention can be known3O4@ CMS @ TLL has good catalytic activity.
The magnetic carboxymethyl starch nanoparticle prepared in the above embodiments 1 to 4 is immobilized with enzyme Fe3O4The catalytic activity of @ CMS @ KDN in catalyzing the lignocellulose enzymolysis is verified, and the process is as follows:
adding a substrate into a 500 mL reaction bottle, adding a 5% sodium hydroxide solution, reacting at 50 ℃, completing the reaction after a period of time, carrying out acid washing until a water washing solution is neutral, and drying to obtain the substrate. Adding 2% (w/v) of the treated substrate into a 25 mL reaction flask, adding a certain volume of citric acid buffer solution into the reaction flask, fully wetting the substrate, and adding the prepared Fe3O4The method comprises the following steps of carrying out enzymolysis reaction on lignocellulose with @ CMS @ KDN, vibrating, uniformly mixing, reacting at 45 ℃ at 150 r/min, sampling after a period of time, and carrying out RP-HPLC analysis; the same amount of substrate was taken for acidolysis and the hydrolysate was subjected to HPLC analysis. And (5) obtaining the sugar yield according to the change of the peak area.
The results are shown in fig. 8 and 9: the yield of the substrate enzymolysis fermentation enzymolysis sugar is 78.85 +/-2.90%, the yield of the glucose can reach 68..12 +/-1.24%, and the Fe prepared by the method is known3O4@ CMS @ KDN has good catalytic activity.
After the reaction is finished, the Fe is treated by an external magnetic field3O4@CMS@BR、Fe3O4@ CMS @ TRY and Fe3O4@ CMS @ TLL, freeze-drying, and recoveringThe next reaction, after repeating the reaction 10 times, Fe3O4@ CMS @ BR keeping 52.16. + -. 1.74% of the initial activity, Fe3O4@ CMS @ TLL maintains 54.57. + -. 1.58% of the initial activity, Fe3O4The @ CMS @ KDN keeps 49.76 +/-1.58% of the initial activity, and the magnetic carboxymethyl starch immobilized enzyme prepared by the method has good reusability.
The above embodiments are merely illustrative of the technical concepts and features of the present invention, and the purpose of the embodiments is to enable those skilled in the art to understand the contents of the present invention and implement the present invention, and not to limit the protection scope of the present invention. All equivalent changes and modifications made according to the spirit of the present invention should be covered within the protection scope of the present invention.
Claims (10)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111154397.6A CN113881661A (en) | 2021-09-29 | 2021-09-29 | Method for immobilizing enzymes based on carboxymethyl starch modified magnetic nanoparticles |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202111154397.6A CN113881661A (en) | 2021-09-29 | 2021-09-29 | Method for immobilizing enzymes based on carboxymethyl starch modified magnetic nanoparticles |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113881661A true CN113881661A (en) | 2022-01-04 |
Family
ID=79008440
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202111154397.6A Pending CN113881661A (en) | 2021-09-29 | 2021-09-29 | Method for immobilizing enzymes based on carboxymethyl starch modified magnetic nanoparticles |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113881661A (en) |
Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999062079A1 (en) * | 1998-05-26 | 1999-12-02 | Bar-Ilan University | Nucleation and growth of magnetic metal oxide nanoparticles and its use |
| US20080206146A1 (en) * | 2005-03-21 | 2008-08-28 | Massoud Akhtari | Functionalized Magnetic Nanoparticles and Methods of Use Thereof |
| CN101503683A (en) * | 2009-03-13 | 2009-08-12 | 华东理工大学 | Magnetic nano particle immobilized Serratieae lipase, preparation and use |
| CN102337258A (en) * | 2011-10-10 | 2012-02-01 | 青岛农业大学 | Pectinase immobilizing carrier, preparation thereof, and method for immobilizing pectinase |
| CN102517348A (en) * | 2011-12-14 | 2012-06-27 | 浙江大学 | Method for preparing 1,3-diglyceride from surface active magnetic nanoparticle immobilized lipase |
| CN104099317A (en) * | 2014-07-18 | 2014-10-15 | 江南大学 | Method for fixing pullulanase with chitosan magnetic nanoparticles |
| CN107384978A (en) * | 2017-07-15 | 2017-11-24 | 安徽工程大学 | A kind of method that magnetic immobilized multi-enzyme system one kettle way prepares gluconic acid |
| CN107988198A (en) * | 2018-01-14 | 2018-05-04 | 浙江工商大学 | Magnetic flexible carrier Fe3O4The preparation method and applications of-PDA-DAS |
| CN111593046A (en) * | 2020-04-16 | 2020-08-28 | 东北农业大学 | Immobilized magnetoenzyme preparation method of OPL grease |
| CN113789320A (en) * | 2021-09-29 | 2021-12-14 | 淮阴工学院 | Preparation method of immobilized enzyme with magnetic starch as carrier |
-
2021
- 2021-09-29 CN CN202111154397.6A patent/CN113881661A/en active Pending
Patent Citations (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO1999062079A1 (en) * | 1998-05-26 | 1999-12-02 | Bar-Ilan University | Nucleation and growth of magnetic metal oxide nanoparticles and its use |
| US20080206146A1 (en) * | 2005-03-21 | 2008-08-28 | Massoud Akhtari | Functionalized Magnetic Nanoparticles and Methods of Use Thereof |
| CN101503683A (en) * | 2009-03-13 | 2009-08-12 | 华东理工大学 | Magnetic nano particle immobilized Serratieae lipase, preparation and use |
| CN102337258A (en) * | 2011-10-10 | 2012-02-01 | 青岛农业大学 | Pectinase immobilizing carrier, preparation thereof, and method for immobilizing pectinase |
| CN102517348A (en) * | 2011-12-14 | 2012-06-27 | 浙江大学 | Method for preparing 1,3-diglyceride from surface active magnetic nanoparticle immobilized lipase |
| CN104099317A (en) * | 2014-07-18 | 2014-10-15 | 江南大学 | Method for fixing pullulanase with chitosan magnetic nanoparticles |
| CN107384978A (en) * | 2017-07-15 | 2017-11-24 | 安徽工程大学 | A kind of method that magnetic immobilized multi-enzyme system one kettle way prepares gluconic acid |
| CN107988198A (en) * | 2018-01-14 | 2018-05-04 | 浙江工商大学 | Magnetic flexible carrier Fe3O4The preparation method and applications of-PDA-DAS |
| CN111593046A (en) * | 2020-04-16 | 2020-08-28 | 东北农业大学 | Immobilized magnetoenzyme preparation method of OPL grease |
| CN113789320A (en) * | 2021-09-29 | 2021-12-14 | 淮阴工学院 | Preparation method of immobilized enzyme with magnetic starch as carrier |
Non-Patent Citations (15)
| Title |
|---|
| KUN FANG等: "Starch-based magnetic nanocomposite as an efficient absorbent for anticancer drug removal from aqueous solution", 《INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES》, vol. 184, pages 509, XP086701347, DOI: 10.1016/j.ijbiomac.2021.06.103 * |
| NANTA P等: "Synthesis of Superparamagnetic Nanoparticles Coated with Carboxymethyl Starch", 《 NANOTECHNOLOGY 2011: BIO SENSORS, INSTRUMENTS, MEDICAL, ENVIRONMENT AND ENERGY, NSTI-NANOTECH 2011》, pages 141 * |
| NASRIN S等: "Enhanced stability and catalytic activity of immobilized α-amylase on modified Fe3O4 nanoparticles", 《CHEMICAL ENGINEERING JOURNAL》, vol. 240 * |
| PENG ZG等: "Preparation and protein immobilization of magnetic dialdehyde starch nanoparticles changes", 《COLLOIDS AND SURFACES B》, vol. 35 * |
| S. MOHAPATRA等: "Carboxymethyl Assam Bora rice starch coated SPIONs: Synthesis, characterization and in vitro localization in a micro capillary for simulating a targeted drug delivery system", 《INT. J. BIOL. MACROMOL》, vol. 115 * |
| T.T. DUNG 等: "Structural and magnetic properties of starch-coated magnetite nanoparticles", 《JOURNAL OF EXPERIMENTAL NANOSCIENCE》, vol. 4, no. 3, pages 260 * |
| 刁怡涵: "淀粉基纳米载体固定化酶的制备及其应用研究", 《中国优秀硕士学位论文全文数据库（电子期刊）工程科技I辑》 * |
| 唐玲利等: "磁性纳米粒子的制备及对α-淀粉酶的固定化研究", 《中国卫生检验杂志》 * |
| 张春晓;任洁;马艳梅;刘扬;秦泗超;唐逸飞;: "磁性两性淀粉复合微球的制备及吸附性能研究", 化学工程师, no. 10 * |
| 张睿: "磁性纳米淀粉固定化酶的制备及其应用研究", 中国优秀硕士学位论文全文数据库（电子期刊）工程科技I辑》 * |
| 徐星星;朱宏;: "磁性纳米颗粒及其在生物医学领域中的应用", 磁性材料及器件, no. 05 * |
| 王艳丽: "磁性淀粉的制备、性质及应用研究", 《中国优秀硕士学位论文全文数据库（电子期刊）工程科技I辑》, no. 2, pages 15 * |
| 胡剑等: "改性高直链超顺磁性淀粉的制备及其结构研究", 《中国粮油学报》, vol. 20, no. 1 * |
| 茅燕勇;李文谦;王朝宇;时号;: "磁性纳米粒固定化脂肪酶的制备及稳定性研究", 食品科技, no. 09 * |
| 郭文淼;朱慧红;: "羧甲基淀粉包覆Fe_3O_4磁性纳米颗粒的制备与表征", 应用化工, no. 10 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Han et al. | Preparation and characterization of Fe3O4-NH2@ 4-arm-PEG-NH2, a novel magnetic four-arm polymer-nanoparticle composite for cellulase immobilization | |
| Reis et al. | Design of immobilized enzyme biocatalysts: Drawbacks and opportunities | |
| Soozanipour et al. | Preparation of a stable and robust nanobiocatalyst by efficiently immobilizing of pectinase onto cyanuric chloride-functionalized chitosan grafted magnetic nanoparticles | |
| Thangaraj et al. | Immobilization of lipases–a review. Part II: carrier materials | |
| Sheng et al. | Enhanced activity and stability of papain by covalent immobilization on porous magnetic nanoparticles | |
| Vaidya et al. | Biological metal organic framework (bio-MOF) of glucoamylase with enhanced stability | |
| Talekar et al. | Carrier free co-immobilization of alpha amylase, glucoamylase and pullulanase as combined cross-linked enzyme aggregates (combi-CLEAs): a tri-enzyme biocatalyst with one pot starch hydrolytic activity | |
| Li et al. | Fabrication of graphene oxide decorated with Fe 3 O 4@ SiO 2 for immobilization of cellulase | |
| Wang et al. | Enzyme hybrid nanoflowers and enzyme@ metal–organic frameworks composites: fascinating hybrid nanobiocatalysts | |
| Hosseini et al. | Lipase-immobilized chitosan-crosslinked magnetic nanoparticle as a biocatalyst for ring opening esterification of itaconic anhydride | |
| Zhang et al. | Co‐immobilization of cellulase and glucose oxidase on graphene oxide by covalent bonds: a biocatalytic system for one‐pot conversion of gluconic acid from carboxymethyl cellulose | |
| Seenuvasan et al. | Magnetic nanoparticles: a versatile carrier for enzymes in bio‐processing sectors | |
| Manasa et al. | Immobilization of cellulase enzyme on zinc ferrite nanoparticles in increasing enzymatic hydrolysis on ultrasound-assisted alkaline pretreated Crotalaria juncea biomass | |
| Liu et al. | Immobilization of thermophilic lipase in inorganic hybrid nanoflower through biomimetic mineralization | |
| Han et al. | Immobilization of cellulase on thermo-sensitive magnetic microspheres: improved stability and reproducibility | |
| Narisetty et al. | An overview of cellulase immobilization strategies for biofuel production | |
| CN104099317A (en) | Method for fixing pullulanase with chitosan magnetic nanoparticles | |
| Sui et al. | A facile route to preparation of immobilized cellulase on polyurea microspheres for improving catalytic activity and stability | |
| CN111672432A (en) | Preparation method of graphene oxide/chitosan composite aerogel for enzyme immobilization | |
| Lucena et al. | Synthesis and characterization of magnetic cross-linked enzyme aggregate and its evaluation of the alternating magnetic field (AMF) effects in the catalytic activity | |
| Wang et al. | Lactate dehydrogenase encapsulated in a metal-organic framework: A novel stable and reusable biocatalyst for the synthesis of D-phenyllactic acid | |
| Ortiz et al. | Immobilization and stabilization of enzymes using biomimetic silicification reactions | |
| Huang et al. | Cellulase immobilized onto amino-functionalized magnetic Fe3O4@ SiO2 nanoparticle for poplar deconstruction | |
| Kaur et al. | Fungal cellulolytic enzyme complex immobilized on chitosan-functionalised magnetic nanoparticles for paddy straw saccharification | |
| Almaghrabi et al. | A biocatalytic system obtained via immobilization of urease onto magnetic metal/alginate nanocomposite: Improving reusability and enhancing stability |
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 |