CN112662646A - Method for immobilizing lipase by using layered column material and application - Google Patents
Method for immobilizing lipase by using layered column material and application Download PDFInfo
- Publication number
- CN112662646A CN112662646A CN202011108840.1A CN202011108840A CN112662646A CN 112662646 A CN112662646 A CN 112662646A CN 202011108840 A CN202011108840 A CN 202011108840A CN 112662646 A CN112662646 A CN 112662646A
- Authority
- CN
- China
- Prior art keywords
- lipase
- column material
- carrier
- linking agent
- modified layer
- 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
- 108090001060 Lipase Proteins 0.000 title claims abstract description 50
- 102000004882 Lipase Human genes 0.000 title claims abstract description 50
- 239000004367 Lipase Substances 0.000 title claims abstract description 49
- 235000019421 lipase Nutrition 0.000 title claims abstract description 49
- 239000000463 material Substances 0.000 title claims abstract description 40
- 238000000034 method Methods 0.000 title claims abstract description 19
- 230000003100 immobilizing effect Effects 0.000 title claims abstract description 5
- 238000005406 washing Methods 0.000 claims abstract description 10
- 230000001588 bifunctional effect Effects 0.000 claims abstract description 9
- 239000003431 cross linking reagent Substances 0.000 claims abstract description 9
- 229910052751 metal Inorganic materials 0.000 claims abstract description 9
- 239000002184 metal Substances 0.000 claims abstract description 9
- 150000003839 salts Chemical class 0.000 claims abstract description 9
- 229920001131 Pulp (paper) Polymers 0.000 claims abstract description 7
- 239000003607 modifier Substances 0.000 claims abstract description 7
- 230000008569 process Effects 0.000 claims abstract description 6
- 238000002156 mixing Methods 0.000 claims abstract description 5
- 239000007864 aqueous solution Substances 0.000 claims abstract description 3
- 230000008878 coupling Effects 0.000 claims abstract description 3
- 238000010168 coupling process Methods 0.000 claims abstract description 3
- 238000005859 coupling reaction Methods 0.000 claims abstract description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 12
- 229910052757 nitrogen Inorganic materials 0.000 claims description 6
- -1 fatty acid salts Chemical class 0.000 claims description 5
- 239000002202 Polyethylene glycol Substances 0.000 claims description 4
- 229920001223 polyethylene glycol Polymers 0.000 claims description 4
- AOBIOSPNXBMOAT-UHFFFAOYSA-N 2-[2-(oxiran-2-ylmethoxy)ethoxymethyl]oxirane Chemical compound C1OC1COCCOCC1CO1 AOBIOSPNXBMOAT-UHFFFAOYSA-N 0.000 claims description 3
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical group O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims description 3
- 235000014113 dietary fatty acids Nutrition 0.000 claims description 3
- 229930195729 fatty acid Natural products 0.000 claims description 3
- 239000000194 fatty acid Substances 0.000 claims description 3
- VOZRXNHHFUQHIL-UHFFFAOYSA-N glycidyl methacrylate Chemical compound CC(=C)C(=O)OCC1CO1 VOZRXNHHFUQHIL-UHFFFAOYSA-N 0.000 claims description 3
- CUGZWHZWSVUSBE-UHFFFAOYSA-N 2-(oxiran-2-ylmethoxy)ethanol Chemical compound OCCOCC1CO1 CUGZWHZWSVUSBE-UHFFFAOYSA-N 0.000 claims description 2
- KUAUJXBLDYVELT-UHFFFAOYSA-N 2-[[2,2-dimethyl-3-(oxiran-2-ylmethoxy)propoxy]methyl]oxirane Chemical compound C1OC1COCC(C)(C)COCC1CO1 KUAUJXBLDYVELT-UHFFFAOYSA-N 0.000 claims description 2
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical group [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 2
- 230000035484 reaction time Effects 0.000 claims description 2
- DVKJHBMWWAPEIU-UHFFFAOYSA-N toluene 2,4-diisocyanate Chemical compound CC1=CC=C(N=C=O)C=C1N=C=O DVKJHBMWWAPEIU-UHFFFAOYSA-N 0.000 claims description 2
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims 2
- PXHVJJICTQNCMI-UHFFFAOYSA-N Nickel Chemical compound [Ni] PXHVJJICTQNCMI-UHFFFAOYSA-N 0.000 claims 2
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims 1
- VYZAMTAEIAYCRO-UHFFFAOYSA-N Chromium Chemical compound [Cr] VYZAMTAEIAYCRO-UHFFFAOYSA-N 0.000 claims 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 claims 1
- FYYHWMGAXLPEAU-UHFFFAOYSA-N Magnesium Chemical compound [Mg] FYYHWMGAXLPEAU-UHFFFAOYSA-N 0.000 claims 1
- HCHKCACWOHOZIP-UHFFFAOYSA-N Zinc Chemical compound [Zn] HCHKCACWOHOZIP-UHFFFAOYSA-N 0.000 claims 1
- XUGUHTGSMPZQIW-UHFFFAOYSA-N [[4-(4-diazonioiminocyclohexa-2,5-dien-1-ylidene)cyclohexa-2,5-dien-1-ylidene]hydrazinylidene]azanide Chemical compound C1=CC(N=[N+]=[N-])=CC=C1C1=CC=C(N=[N+]=[N-])C=C1 XUGUHTGSMPZQIW-UHFFFAOYSA-N 0.000 claims 1
- 229910052782 aluminium Inorganic materials 0.000 claims 1
- XAGFODPZIPBFFR-UHFFFAOYSA-N aluminium Chemical compound [Al] XAGFODPZIPBFFR-UHFFFAOYSA-N 0.000 claims 1
- 229910052791 calcium Inorganic materials 0.000 claims 1
- 239000011575 calcium Substances 0.000 claims 1
- 229910052804 chromium Inorganic materials 0.000 claims 1
- 239000011651 chromium Substances 0.000 claims 1
- 229910017052 cobalt Inorganic materials 0.000 claims 1
- 239000010941 cobalt Substances 0.000 claims 1
- GUTLYIVDDKVIGB-UHFFFAOYSA-N cobalt atom Chemical compound [Co] GUTLYIVDDKVIGB-UHFFFAOYSA-N 0.000 claims 1
- 229910052802 copper Inorganic materials 0.000 claims 1
- 239000010949 copper Substances 0.000 claims 1
- 229910052742 iron Inorganic materials 0.000 claims 1
- 229910052749 magnesium Inorganic materials 0.000 claims 1
- 239000011777 magnesium Substances 0.000 claims 1
- WPBNNNQJVZRUHP-UHFFFAOYSA-L manganese(2+);methyl n-[[2-(methoxycarbonylcarbamothioylamino)phenyl]carbamothioyl]carbamate;n-[2-(sulfidocarbothioylamino)ethyl]carbamodithioate Chemical compound [Mn+2].[S-]C(=S)NCCNC([S-])=S.COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC WPBNNNQJVZRUHP-UHFFFAOYSA-L 0.000 claims 1
- 229910052759 nickel Inorganic materials 0.000 claims 1
- 230000002194 synthesizing effect Effects 0.000 claims 1
- 229910052725 zinc Inorganic materials 0.000 claims 1
- 239000011701 zinc Substances 0.000 claims 1
- 108090000790 Enzymes Proteins 0.000 abstract description 17
- 102000004190 Enzymes Human genes 0.000 abstract description 17
- 230000000694 effects Effects 0.000 abstract description 9
- 238000013461 design Methods 0.000 abstract description 4
- 239000012876 carrier material Substances 0.000 abstract description 3
- 239000011148 porous material Substances 0.000 abstract description 2
- 229940088598 enzyme Drugs 0.000 description 13
- 239000000243 solution Substances 0.000 description 10
- 238000007385 chemical modification Methods 0.000 description 7
- 238000001035 drying Methods 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 5
- OZAIFHULBGXAKX-UHFFFAOYSA-N 2-(2-cyanopropan-2-yldiazenyl)-2-methylpropanenitrile Chemical compound N#CC(C)(C)N=NC(C)(C)C#N OZAIFHULBGXAKX-UHFFFAOYSA-N 0.000 description 4
- 238000005516 engineering process Methods 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 230000006872 improvement Effects 0.000 description 4
- QIQXTHQIDYTFRH-UHFFFAOYSA-N octadecanoic acid Chemical compound CCCCCCCCCCCCCCCCCC(O)=O QIQXTHQIDYTFRH-UHFFFAOYSA-N 0.000 description 4
- 239000008055 phosphate buffer solution Substances 0.000 description 4
- 238000004537 pulping Methods 0.000 description 4
- AZDRQVAHHNSJOQ-UHFFFAOYSA-N alumane Chemical class [AlH3] AZDRQVAHHNSJOQ-UHFFFAOYSA-N 0.000 description 3
- 238000006243 chemical reaction Methods 0.000 description 3
- 238000009776 industrial production Methods 0.000 description 3
- 159000000003 magnesium salts Chemical class 0.000 description 3
- 238000004519 manufacturing process Methods 0.000 description 3
- 238000002360 preparation method Methods 0.000 description 3
- 108010079522 solysime Proteins 0.000 description 3
- 239000000758 substrate Substances 0.000 description 3
- RYHBNJHYFVUHQT-UHFFFAOYSA-N 1,4-Dioxane Chemical compound C1COCCO1 RYHBNJHYFVUHQT-UHFFFAOYSA-N 0.000 description 2
- 241000222175 Diutina rugosa Species 0.000 description 2
- 108010093096 Immobilized Enzymes Proteins 0.000 description 2
- 230000003213 activating effect Effects 0.000 description 2
- 238000007605 air drying Methods 0.000 description 2
- 239000003513 alkali Substances 0.000 description 2
- 238000006555 catalytic reaction Methods 0.000 description 2
- 230000007547 defect Effects 0.000 description 2
- 239000008367 deionised water Substances 0.000 description 2
- 229910021641 deionized water Inorganic materials 0.000 description 2
- 238000010438 heat treatment Methods 0.000 description 2
- 239000000178 monomer Substances 0.000 description 2
- 239000002086 nanomaterial Substances 0.000 description 2
- 239000003960 organic solvent Substances 0.000 description 2
- 229920000642 polymer Polymers 0.000 description 2
- 238000006116 polymerization reaction Methods 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 229920005989 resin Chemical class 0.000 description 2
- 239000011347 resin Chemical class 0.000 description 2
- 238000007789 sealing Methods 0.000 description 2
- 238000002791 soaking Methods 0.000 description 2
- 238000001179 sorption measurement Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- CJDRUOGAGYHKKD-XMTJACRCSA-N (+)-Ajmaline Natural products O[C@H]1[C@@H](CC)[C@@H]2[C@@H]3[C@H](O)[C@@]45[C@@H](N(C)c6c4cccc6)[C@@H](N1[C@H]3C5)C2 CJDRUOGAGYHKKD-XMTJACRCSA-N 0.000 description 1
- JCZPMGDSEAFWDY-SQOUGZDYSA-N (2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanamide Chemical compound NC(=O)[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO JCZPMGDSEAFWDY-SQOUGZDYSA-N 0.000 description 1
- 108010031797 Candida antarctica lipase B Proteins 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- 229930182558 Sterol Natural products 0.000 description 1
- 241000223258 Thermomyces lanuginosus Species 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 150000007513 acids Chemical class 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 230000004888 barrier function Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 159000000007 calcium salts Chemical class 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 150000001844 chromium Chemical class 0.000 description 1
- 238000004140 cleaning Methods 0.000 description 1
- 150000001868 cobalt Chemical class 0.000 description 1
- 239000002131 composite material Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 150000001879 copper Chemical class 0.000 description 1
- 238000004132 cross linking Methods 0.000 description 1
- 239000013078 crystal Substances 0.000 description 1
- 230000008021 deposition Effects 0.000 description 1
- 238000011161 development Methods 0.000 description 1
- 229940079919 digestives enzyme preparation Drugs 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000006911 enzymatic reaction Methods 0.000 description 1
- 150000004665 fatty acids Chemical class 0.000 description 1
- 150000002191 fatty alcohols Chemical class 0.000 description 1
- 150000004676 glycans Chemical class 0.000 description 1
- 238000009396 hybridization Methods 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 150000002505 iron Chemical class 0.000 description 1
- 150000002696 manganese Chemical class 0.000 description 1
- 229910000000 metal hydroxide Inorganic materials 0.000 description 1
- 150000004692 metal hydroxides Chemical class 0.000 description 1
- 239000002105 nanoparticle Substances 0.000 description 1
- 150000002815 nickel Chemical class 0.000 description 1
- 229920001282 polysaccharide Polymers 0.000 description 1
- 239000005017 polysaccharide Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 235000003702 sterols Nutrition 0.000 description 1
- 239000000126 substance Substances 0.000 description 1
- 238000001308 synthesis method Methods 0.000 description 1
- 150000003505 terpenes Chemical class 0.000 description 1
- 150000003626 triacylglycerols Chemical class 0.000 description 1
- 239000001993 wax Substances 0.000 description 1
- 150000003751 zinc Chemical class 0.000 description 1
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention discloses a method for immobilizing lipase by using a layered column material and application thereof. Firstly, mixing and reacting a plurality of metal salt aqueous solutions with an organic modifier to prepare an organic modified layer columnar material, covalently connecting the organic modified layer columnar material with a bifunctional cross-linking agent, and then covalently coupling lipase to the bifunctional cross-linking agent to obtain the immobilized lipase. The immobilized lipase of the laminated column material is directly added into a pulp forming pool, a pre-papermaking pool or a pulp washing tank in the papermaking process, and stickies in paper pulp are removed. The invention synthesizes a molecular reactor carrier with a supermolecular structure through molecular design and assembly. The carrier has excellent performances of larger pore space, multiple reactive active sites, better rigidity and the like, can be recycled, and the enzyme activity is not less than 80% of the original total enzyme activity after repeated utilization, so that the carrier is a novel carrier material with wide application value.
Description
Technical Field
The invention belongs to the technical field of papermaking additives, and particularly relates to a layered column material immobilized lipase and application thereof in papermaking production.
Background
Some fat-soluble extractives are present in pulping and papermaking processes, such as: alkane compounds, fatty alcohols, fatty acids, resin acids, sterol compounds, terpenoids, triglycerides, waxes and the like, which are insoluble in water and are removed after falling off, form "paper defects" if deposited in the paper pulp carried into the paper, and require time-consuming cleaning if deposited on the paper machine to cause mechanical failure. The use of lipase to control pitch deposition in pulp and paper mills is a very effective control technique. In recent years, the problem of pitch barrier is successfully solved by using lipase to treat paper pulp through research at home and abroad, and the technology is practically applied to industrial production at present. However, the enzyme is easy to inactivate at high temperature, and the specific enzyme has a limited pH adaptation range, so that the industrial popularization range is far from sufficient.
The microbial lipase has various types, wide sources and short period, has wider pH value, action temperature range, high stability and activity than animal and plant lipases, and has specificity to substrates, such as chemoselectivity, stereoselectivity, site selectivity and the like, so that the microbial lipase is convenient for industrial production and obtains enzyme preparations with higher purity, and becomes a main source for industrial production of lipase. However, the physicochemical properties of lipase itself, such as non-high temperature resistance, narrow pH application range, etc., limit its large-scale industrial application, and the enzymatic properties of the existing lipase can be greatly changed by performing immobilization treatment or chemical modification treatment on the existing lipase, so as to obtain a novel lipase preparation.
Enzyme immobilization technology has evolved in two directions: new materials and the use of the new process. Among various novel immobilization materials, the immobilization effect of the nanomaterial is particularly remarkable, for example, Humicola lanuginosa lipase is embedded in nanoparticles formed by sol-gel by Persson, and the specific activity of the lipase is improved by 320 times. Chemical modification techniques allow for virtually endless design and engineering of the side chains of lipase molecules. Common techniques for chemical modification of lipase molecules include: chemical modification of lipase active site or side chain group except active site with small molecular compound (such as aminoglucose), covalent cross-linking of lipase molecule with bifunctional or multifunctional cross-linking agent (such as glutaraldehyde), formation of cross-linked enzyme crystal, and chemical modification of monofunctional reagent (such as polyethylene glycol and its derivatives). The chemical modification of the lipase molecule can obviously enhance the stability of the lipase molecule and improve (or change) the activity of the lipase molecule, for example, after Siddiqui modifies Candida antarctica lipase B by adopting polysaccharide, the half-life of the lipase at 70 ℃ is improved from 18 min to 168 min.
Immobilized lipases also have the following limitations: the enzyme activity is lost; is suitable for small molecular substrates, and macromolecular substrates can not react basically; is not suitable for a multi-enzyme reaction system.
Disclosure of Invention
The invention is based on two key technologies of nano materials and chemical modification, and the microbial lipase is immobilized to obtain a novel lipase enzyme preparation which is applied to a resin control link in the paper industry to decompose stickies in paper pulp, improve the quality of finished paper and promote the clean production of a paper machine. Therefore, the invention adopts the following technical scheme:
a method for immobilizing lipase by using a layered column material comprises the following steps:
(1) mixing and reacting a plurality of metal salt aqueous solutions with an organic modifier, wherein the molar ratio of the metal salt to the organic modifier is between 1:4 and 4: 1;
(2) reasonably adjusting and controlling the pH value of the system, introducing nitrogen, and reacting for a plurality of times to obtain the organic modified layer columnar material;
(3) and covalently connecting the organic modified layered material with a bifunctional cross-linking agent, and then covalently coupling lipase to the bifunctional cross-linking agent to obtain the immobilized lipase.
The further improvement is that the material of the layer column in the step 1 belongs to soluble salt, and belongs to at least two of magnesium salt, iron salt, zinc salt, nickel salt, aluminum salt, copper salt, calcium salt, cobalt salt, manganese salt and chromium salt.
In a further improvement, the organic modifier in the step 1 is: the RCOOM type fatty acid salts have the carbon chain length of between C5 and C22 and the unsaturation degree omega of less than or equal to 3.
In a further improvement, the pH value in the synthesis method of the organic modified layer columnar material in the step 2 is 6-12, and the reaction time is 4-24 h.
In a further improvement, the bifunctional or multifunctional crosslinking agent in step 3 is glutaraldehyde, toluene-2, 4-diisocyanate, glycidyl methacrylate, diazadicyclobenzidine, N' -ethylene bismaleimide, polyethylene glycol diglycidyl ether, ethylene glycol glycidyl ether or neopentyl glycol diglycidyl ether.
The application of the immobilized lipase of the chromatographic column material is that the immobilized lipase of the chromatographic column material is directly added into a pulping pool, a pre-papermaking pool or a pulp washing tank in the papermaking process, and stickies in paper pulp are removed.
The invention has the following beneficial effects:
1. the invention synthesizes a molecular reactor carrier with a supermolecular structure through molecular design and assembly. The carrier has the excellent performances of larger pore space, multiple reactive active sites, better rigidity and the like, and is a novel carrier material with wide application value. The design of a carrier material with excellent synthetic performance becomes the main research content in the field of current immobilized enzymes, and is also the direction of the development of the future immobilized enzyme technology.
2. The invention carries out hybridization reaction on biological enzyme and layered composite metal hydroxide to obtain a novel biological enzyme preparation. Overcomes the defects of sensitivity, instability and easy inactivation of the enzyme, and ensures that the enzyme has stronger adaptability and tolerance to the use environment, thereby greatly widening the application range of the biological enzyme.
3. The immobilized lipase of the layer column material can be recycled, the enzyme activity is not lower than 80% of the original total enzyme activity after repeated use, the optimal catalytic temperature and the temperature range of the catalytic reaction are improved compared with that of free lipase, and the optimal pH range of the enzyme catalytic reaction is wider than that of the free lipase.
Detailed Description
The present invention is further illustrated by the following examples, but the scope of the present invention is not limited to the following examples, and is not limited thereto.
Example 1
Preparing an organic modified layer columnar material: preparing a solution from a metal magnesium salt, an aluminum salt and stearate, wherein the molar ratio of the metal salt to the stearate is 2: 1. Introducing nitrogen, slowly adding an alkali solution dropwise, adjusting the pH value to 8, and reacting for 12 hours. Separating, washing and drying for later use.
Preparing immobilized lipase of a layer column material:
(1) activating a carrier: adding 1, 4-dioxane organic solvent and 20ml of 1 mass percent glycidyl methacrylate solution into a 500ml reaction flask, mixing, adding azobisisobutyronitrile accounting for 2 mass percent of the monomer, adding a dry chromatographic column material, fully soaking until the adsorption is balanced, introducing nitrogen into the flask, sealing, and heating to 70 ℃ for polymerization reaction for 6 hours. Filtering out the block macroporous material by using a screen, absorbing the polymer solution adhered to the surface of the material by using filter paper, washing the material by using deionized water for 3 times, and drying the material for 4 hours in a 50 ℃ forced air drying oven to obtain the activated carrier for later use.
(2) And (3) lipase immobilization: adding 0.1g of activated carrier into 10ml of candida rugosa lipase solution which is 0.5mg/ml and prepared by phosphate buffer solution with pH7.5 and 0.05mol/L, oscillating for 8h in a constant-temperature water bath shaking table with the rotating speed of 50r/min at 25 ℃, filtering the activated carrier, washing for many times by phosphate buffer solution with the same concentration and pH until no enzyme protein is detected in the supernatant, and then drying for 12h in vacuum to obtain the immobilized lipase.
Example 2
Preparing an organic modified layer columnar material: preparing a solution from a metal magnesium salt, an aluminum salt and stearate, wherein the molar ratio of the metal salt to the stearate is 3: 1. Introducing nitrogen, slowly dropwise adding an alkali solution, adjusting the pH value to be 9, and reacting for 6 hours. Separating, washing and drying for later use.
Preparing immobilized lipase of a layer column material:
(1) activating a carrier: adding 1, 4-dioxane organic solvent and 20ml of polyethylene glycol diglycidyl ether solution with the mass fraction of 1.5% into a 500ml reaction flask, mixing, adding azobisisobutyronitrile accounting for 2% of the mass of the monomer, adding a dried chromatographic material, fully soaking until the adsorption is balanced, introducing nitrogen into the flask, sealing, and heating to 80 ℃ for polymerization reaction for 5 hours. Filtering out the block macroporous material by using a screen, absorbing the polymer solution adhered to the surface of the material by using filter paper, washing the material by using deionized water for 3 times, and drying the material for 4 hours in a 50 ℃ forced air drying oven to obtain the activated carrier for later use.
(2) And (3) lipase immobilization: adding 0.1g of activated carrier into 10ml of 1.0mg/ml candida rugosa lipase solution prepared by phosphate buffer solution with pH7.5 and 0.05mol/L, oscillating for 8 hours in a constant-temperature water bath shaking table with the rotating speed of 50r/min at 25 ℃, filtering the activated carrier, washing for multiple times by phosphate buffer solution with the same concentration and pH until no enzyme protein is detected in the supernatant, and then drying for 12 hours in vacuum to obtain the immobilized lipase.
EXAMPLE 3 use of immobilized Lipase with layered column Material
When the multi-layer column material immobilized lipase provided by the invention is used, different dosing points can be selected according to specific production conditions, and generally the dosing points are recommended to be added in a pulping tank, a pre-pulping tank or a washing tank and other parts; generally, a continuous or intermittent dosing mode is adopted, and the dosing is performed once every 4-12 hours, namely 2-6 times per day; the amount of the added medicine is 100-1000 g per ton of paper per day according to the paper making process condition and the pollution degree of a paper making system, and sticky substances in paper pulp are removed.
Claims (6)
1. A method for immobilizing lipase by using a layered column material is characterized by comprising the following steps:
(1) mixing and reacting a plurality of metal salt aqueous solutions with an organic modifier, wherein the molar ratio of the metal salt to the organic modifier is between 1:4 and 4: 1;
(2) reasonably adjusting and controlling the pH value of the system, introducing nitrogen, and reacting for a plurality of times to obtain the organic modified layer columnar material;
(3) and covalently connecting the organic modified layer columnar material with a bifunctional cross-linking agent, and then covalently coupling lipase to the bifunctional cross-linking agent to obtain the immobilized lipase.
2. The method of claim 1, wherein the layer column material is soluble salts of at least two of magnesium, iron, zinc, nickel, aluminum, copper, calcium, cobalt, manganese, and chromium.
3. The method of claim 1, wherein the organic modifier is: the RCOOM type fatty acid salts have the carbon chain length of between C5 and C22 and the unsaturation degree omega of less than or equal to 3.
4. The method of claim 1, wherein the pH of the method for synthesizing the organic modified layer column material is 6-12, and the reaction time is 4-24 h.
5. The method of claim 1, wherein the bifunctional or multifunctional cross-linking agent is glutaraldehyde, toluene-2, 4-diisocyanate, glycidyl methacrylate, bis-diazobenzidine, N' -ethylenebismaleimide, polyethylene glycol diglycidyl ether, ethylene glycol glycidyl ether, or neopentyl glycol diglycidyl ether.
6. The use of the immobilized lipase for stratified column material as defined in claim 1, wherein the immobilized lipase for stratified column material is directly added to a machine chest, a pre-machine chest or a washing tank in a paper making process to remove stickies from paper pulp.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011108840.1A CN112662646A (en) | 2020-10-16 | 2020-10-16 | Method for immobilizing lipase by using layered column material and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202011108840.1A CN112662646A (en) | 2020-10-16 | 2020-10-16 | Method for immobilizing lipase by using layered column material and application |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN112662646A true CN112662646A (en) | 2021-04-16 |
Family
ID=75402839
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202011108840.1A Pending CN112662646A (en) | 2020-10-16 | 2020-10-16 | Method for immobilizing lipase by using layered column material and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112662646A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115184299A (en) * | 2022-06-01 | 2022-10-14 | 广东惠尔泰生物科技有限公司 | Method for detecting content of immobilized lipase enzyme protein based on near infrared spectrum |
-
2020
- 2020-10-16 CN CN202011108840.1A patent/CN112662646A/en active Pending
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115184299A (en) * | 2022-06-01 | 2022-10-14 | 广东惠尔泰生物科技有限公司 | Method for detecting content of immobilized lipase enzyme protein based on near infrared spectrum |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Mo et al. | Porous biochar/chitosan composites for high performance cellulase immobilization by glutaraldehyde | |
| Chen et al. | Immobilization of lipase AYS on UiO-66-NH2 metal-organic framework nanoparticles as a recyclable biocatalyst for ester hydrolysis and kinetic resolution | |
| Liu et al. | A novel approach to efficient degradation of indole using co-immobilized horseradish peroxidase-syringaldehyde as biocatalyst | |
| Tan et al. | Designing multifunctional biocatalytic cascade system by multi-enzyme co-immobilization on biopolymers and nanostructured materials | |
| Manikandan et al. | Role of catalyst supports in biocatalysis | |
| Gu et al. | A novel support of sponge-like cellulose composite polymer for immobilizing laccase and its application in nitrogenous organics biodegradation | |
| CN112662646A (en) | Method for immobilizing lipase by using layered column material and application | |
| Malhotra et al. | Application of invertase immobilized on chitosan using glutaraldehyde or tris (Hydroxymethyl) phosphine as cross-linking agent to produce bioethanol | |
| Spasojević et al. | The enzyme immobilization: carriers and immobilization methods | |
| CN113307359B (en) | Composite carrier material for biological fluidized bed and preparation method thereof | |
| de Castro et al. | Lipase immobilization on biodegradable film with sericin | |
| Vieira et al. | A Perspective Review on the Application of Polyacrylonitrile‐Based Supports for Laccase Immobilization | |
| JPH0787974A (en) | Immobilized lipase | |
| CN107164360B (en) | Sodium alginate grafted natural cyclodextrin immobilized cell and application thereof | |
| Chen et al. | Synthesis of chitin/graphene oxide composite aerogel beads for lipase immobilization | |
| WO2024002326A1 (en) | Preparation method for and use of double-enzyme-inorganic hybrid nanoflower microspheres | |
| Li et al. | Reversible, selective immobilization of nuclease P1 from a crude enzyme solution on a weak base anion resin activated by polyethylenimine | |
| JPS6255084A (en) | Method for immobilizing living body and insoluble living body composite obtained therefrom | |
| CN104711248A (en) | Substrate toxicity eliminating method | |
| CN112126108A (en) | Preparation of high-strength and high-adsorption-property nano-cellulose aerogel material | |
| CN106148319B (en) | Method for preparing immobilized enzyme based on reaction adsorption method | |
| Vaz et al. | Ion exchange chromatography for enzyme immobilization | |
| EP0186523A2 (en) | Insolubilized biological material composites | |
| KR101717818B1 (en) | Fe2O3 yolk shell nano structure and enzyme immobilization using the same | |
| CN110294824B (en) | Preparation method and application of efficient cationic polymer |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20210416 |
|
| WD01 | Invention patent application deemed withdrawn after publication |