CN1766103A - High stability immobilized enzyme preparation method - Google Patents
High stability immobilized enzyme preparation method Download PDFInfo
- Publication number
- CN1766103A CN1766103A CN 200510060838 CN200510060838A CN1766103A CN 1766103 A CN1766103 A CN 1766103A CN 200510060838 CN200510060838 CN 200510060838 CN 200510060838 A CN200510060838 A CN 200510060838A CN 1766103 A CN1766103 A CN 1766103A
- Authority
- CN
- China
- Prior art keywords
- enzyme
- high stability
- immobilized enzyme
- preparation
- stability immobilized
- 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.)
- Granted
Links
- 108010093096 Immobilized Enzymes Proteins 0.000 title claims abstract description 36
- 238000002360 preparation method Methods 0.000 title claims abstract description 15
- 102000004190 Enzymes Human genes 0.000 claims abstract description 71
- 108090000790 Enzymes Proteins 0.000 claims abstract description 71
- 238000006243 chemical reaction Methods 0.000 claims abstract description 17
- 239000000758 substrate Substances 0.000 claims abstract description 16
- 239000003960 organic solvent Substances 0.000 claims abstract description 15
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 13
- 239000004530 micro-emulsion Substances 0.000 claims abstract description 11
- 239000000178 monomer Substances 0.000 claims abstract description 10
- 238000010438 heat treatment Methods 0.000 claims abstract description 6
- 238000010526 radical polymerization reaction Methods 0.000 claims abstract description 3
- 229940088598 enzyme Drugs 0.000 claims description 69
- 230000000694 effects Effects 0.000 claims description 26
- 238000000034 method Methods 0.000 claims description 22
- 229920000642 polymer Polymers 0.000 claims description 15
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 12
- 239000007788 liquid Substances 0.000 claims description 12
- 239000003795 chemical substances by application Substances 0.000 claims description 10
- POULHZVOKOAJMA-UHFFFAOYSA-N dodecanoic acid Chemical compound CCCCCCCCCCCC(O)=O POULHZVOKOAJMA-UHFFFAOYSA-N 0.000 claims description 10
- 239000002202 Polyethylene glycol Substances 0.000 claims description 8
- 229920001223 polyethylene glycol Polymers 0.000 claims description 8
- 239000000843 powder Substances 0.000 claims description 8
- 102000004882 Lipase Human genes 0.000 claims description 7
- 108090001060 Lipase Proteins 0.000 claims description 7
- 239000004367 Lipase Substances 0.000 claims description 7
- IMNFDUFMRHMDMM-UHFFFAOYSA-N N-Heptane Chemical compound CCCCCCC IMNFDUFMRHMDMM-UHFFFAOYSA-N 0.000 claims description 7
- 239000013543 active substance Substances 0.000 claims description 7
- 238000013467 fragmentation Methods 0.000 claims description 7
- 238000006062 fragmentation reaction Methods 0.000 claims description 7
- 238000000227 grinding Methods 0.000 claims description 7
- 235000019421 lipase Nutrition 0.000 claims description 7
- 238000000465 moulding Methods 0.000 claims description 7
- 238000012216 screening Methods 0.000 claims description 7
- 238000004513 sizing Methods 0.000 claims description 7
- 238000001291 vacuum drying Methods 0.000 claims description 7
- 238000005303 weighing Methods 0.000 claims description 7
- 239000005639 Lauric acid Substances 0.000 claims description 5
- DAKWPKUUDNSNPN-UHFFFAOYSA-N Trimethylolpropane triacrylate Chemical compound C=CC(=O)OCC(CC)(COC(=O)C=C)COC(=O)C=C DAKWPKUUDNSNPN-UHFFFAOYSA-N 0.000 claims description 5
- STVZJERGLQHEKB-UHFFFAOYSA-N ethylene glycol dimethacrylate Chemical compound CC(=C)C(=O)OCCOC(=O)C(C)=C STVZJERGLQHEKB-UHFFFAOYSA-N 0.000 claims description 5
- 239000000203 mixture Substances 0.000 claims description 5
- 230000005855 radiation Effects 0.000 claims description 5
- GZBSIABKXVPBFY-UHFFFAOYSA-N 2,2-bis(hydroxymethyl)propane-1,3-diol;prop-2-enoic acid Chemical compound OC(=O)C=C.OC(=O)C=C.OC(=O)C=C.OC(=O)C=C.OCC(CO)(CO)CO GZBSIABKXVPBFY-UHFFFAOYSA-N 0.000 claims description 4
- BQZJOQXSCSZQPS-UHFFFAOYSA-N 2-methoxy-1,2-diphenylethanone Chemical compound C=1C=CC=CC=1C(OC)C(=O)C1=CC=CC=C1 BQZJOQXSCSZQPS-UHFFFAOYSA-N 0.000 claims description 4
- VVQNEPGJFQJSBK-UHFFFAOYSA-N Methyl methacrylate Chemical compound COC(=O)C(C)=C VVQNEPGJFQJSBK-UHFFFAOYSA-N 0.000 claims description 4
- 229910052799 carbon Inorganic materials 0.000 claims description 4
- 150000001875 compounds Chemical class 0.000 claims description 4
- 239000003999 initiator Substances 0.000 claims description 4
- WRIDQFICGBMAFQ-UHFFFAOYSA-N (E)-8-Octadecenoic acid Natural products CCCCCCCCCC=CCCCCCCC(O)=O WRIDQFICGBMAFQ-UHFFFAOYSA-N 0.000 claims description 3
- LQJBNNIYVWPHFW-UHFFFAOYSA-N 20:1omega9c fatty acid Natural products CCCCCCCCCCC=CCCCCCCCC(O)=O LQJBNNIYVWPHFW-UHFFFAOYSA-N 0.000 claims description 3
- QSBYPNXLFMSGKH-UHFFFAOYSA-N 9-Heptadecensaeure Natural products CCCCCCCC=CCCCCCCCC(O)=O QSBYPNXLFMSGKH-UHFFFAOYSA-N 0.000 claims description 3
- 108700023418 Amidases Proteins 0.000 claims description 3
- 239000004382 Amylase Substances 0.000 claims description 3
- 102000013142 Amylases Human genes 0.000 claims description 3
- 108010065511 Amylases Proteins 0.000 claims description 3
- 108010059892 Cellulase Proteins 0.000 claims description 3
- 241000282326 Felis catus Species 0.000 claims description 3
- CERQOIWHTDAKMF-UHFFFAOYSA-N Methacrylic acid Chemical compound CC(=C)C(O)=O CERQOIWHTDAKMF-UHFFFAOYSA-N 0.000 claims description 3
- 239000005642 Oleic acid Substances 0.000 claims description 3
- ZQPPMHVWECSIRJ-UHFFFAOYSA-N Oleic acid Natural products CCCCCCCCC=CCCCCCCCC(O)=O ZQPPMHVWECSIRJ-UHFFFAOYSA-N 0.000 claims description 3
- 102000035195 Peptidases Human genes 0.000 claims description 3
- 108091005804 Peptidases Proteins 0.000 claims description 3
- OKKRPWIIYQTPQF-UHFFFAOYSA-N Trimethylolpropane trimethacrylate Chemical compound CC(=C)C(=O)OCC(CC)(COC(=O)C(C)=C)COC(=O)C(C)=C OKKRPWIIYQTPQF-UHFFFAOYSA-N 0.000 claims description 3
- 239000002253 acid Substances 0.000 claims description 3
- 102000005922 amidase Human genes 0.000 claims description 3
- 235000019418 amylase Nutrition 0.000 claims description 3
- 239000003945 anionic surfactant Substances 0.000 claims description 3
- XZKRXPZXQLARHH-UHFFFAOYSA-N buta-1,3-dienylbenzene Chemical compound C=CC=CC1=CC=CC=C1 XZKRXPZXQLARHH-UHFFFAOYSA-N 0.000 claims description 3
- 229940106157 cellulase Drugs 0.000 claims description 3
- HPXRVTGHNJAIIH-UHFFFAOYSA-N cyclohexanol Chemical compound OC1CCCCC1 HPXRVTGHNJAIIH-UHFFFAOYSA-N 0.000 claims description 3
- 125000004386 diacrylate group Chemical group 0.000 claims description 3
- QXJSBBXBKPUZAA-UHFFFAOYSA-N isooleic acid Natural products CCCCCCCC=CCCCCCCCCC(O)=O QXJSBBXBKPUZAA-UHFFFAOYSA-N 0.000 claims description 3
- ZQPPMHVWECSIRJ-KTKRTIGZSA-N oleic acid Chemical compound CCCCCCCC\C=C/CCCCCCCC(O)=O ZQPPMHVWECSIRJ-KTKRTIGZSA-N 0.000 claims description 3
- VVWRJUBEIPHGQF-UHFFFAOYSA-N propan-2-yl n-propan-2-yloxycarbonyliminocarbamate Chemical compound CC(C)OC(=O)N=NC(=O)OC(C)C VVWRJUBEIPHGQF-UHFFFAOYSA-N 0.000 claims description 3
- 239000004342 Benzoyl peroxide Substances 0.000 claims description 2
- OMPJBNCRMGITSC-UHFFFAOYSA-N Benzoylperoxide Chemical compound C=1C=CC=CC=1C(=O)OOC(=O)C1=CC=CC=C1 OMPJBNCRMGITSC-UHFFFAOYSA-N 0.000 claims description 2
- RWCCWEUUXYIKHB-UHFFFAOYSA-N benzophenone Chemical compound C=1C=CC=CC=1C(=O)C1=CC=CC=C1 RWCCWEUUXYIKHB-UHFFFAOYSA-N 0.000 claims description 2
- 239000012965 benzophenone Substances 0.000 claims description 2
- 235000019400 benzoyl peroxide Nutrition 0.000 claims description 2
- 150000002632 lipids Chemical class 0.000 claims description 2
- JRKICGRDRMAZLK-UHFFFAOYSA-L persulfate group Chemical group S(=O)(=O)([O-])OOS(=O)(=O)[O-] JRKICGRDRMAZLK-UHFFFAOYSA-L 0.000 claims description 2
- 238000004132 cross linking Methods 0.000 abstract description 4
- 239000003431 cross linking reagent Substances 0.000 abstract 1
- 230000001678 irradiating effect Effects 0.000 abstract 1
- 239000004094 surface-active agent Substances 0.000 abstract 1
- 238000006116 polymerization reaction Methods 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 239000000126 substance Substances 0.000 description 6
- -1 highly basic Substances 0.000 description 5
- 238000003756 stirring Methods 0.000 description 5
- 239000007864 aqueous solution Substances 0.000 description 4
- 239000012467 final product Substances 0.000 description 4
- 238000004519 manufacturing process Methods 0.000 description 4
- 230000008569 process Effects 0.000 description 4
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 238000010521 absorption reaction Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- DAJSVUQLFFJUSX-UHFFFAOYSA-M sodium;dodecane-1-sulfonate Chemical compound [Na+].CCCCCCCCCCCCS([O-])(=O)=O DAJSVUQLFFJUSX-UHFFFAOYSA-M 0.000 description 3
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 description 2
- 239000003054 catalyst Substances 0.000 description 2
- 238000006911 enzymatic reaction Methods 0.000 description 2
- 238000005286 illumination Methods 0.000 description 2
- 230000000977 initiatory effect Effects 0.000 description 2
- 239000012074 organic phase Substances 0.000 description 2
- 229910052708 sodium Inorganic materials 0.000 description 2
- 239000011734 sodium Substances 0.000 description 2
- 239000000243 solution Substances 0.000 description 2
- 239000002594 sorbent Substances 0.000 description 2
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- HVVWZTWDBSEWIH-UHFFFAOYSA-N [2-(hydroxymethyl)-3-prop-2-enoyloxy-2-(prop-2-enoyloxymethyl)propyl] prop-2-enoate Chemical compound C=CC(=O)OCC(CO)(COC(=O)C=C)COC(=O)C=C HVVWZTWDBSEWIH-UHFFFAOYSA-N 0.000 description 1
- KXKVLQRXCPHEJC-UHFFFAOYSA-N acetic acid trimethyl ester Natural products COC(C)=O KXKVLQRXCPHEJC-UHFFFAOYSA-N 0.000 description 1
- 230000006978 adaptation Effects 0.000 description 1
- 230000000274 adsorptive effect Effects 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 239000008367 deionised water Substances 0.000 description 1
- 229910021641 deionized water Inorganic materials 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 230000005251 gamma ray Effects 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 230000003993 interaction Effects 0.000 description 1
- 238000005342 ion exchange Methods 0.000 description 1
- 239000002502 liposome Substances 0.000 description 1
- 230000010534 mechanism of action Effects 0.000 description 1
- 229910052757 nitrogen Inorganic materials 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 238000007086 side reaction Methods 0.000 description 1
- 238000001179 sorption measurement Methods 0.000 description 1
Landscapes
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The invention discloses a preparation method for high-stable immobilized enzyme, which comprises: using enzyme, water, surfactant, organic solvent, monomer or prepolymer, and crosslinking agent to construct microemulsion system; adding a little reaction substrate to induce enzyme active conformation; heating, ultraviolet irradiating or direct radiating to initiate free radical polymerization and realize the embedding fix in high-crosslinking carrier. This invention is simple, has high yield and well stability, and fits to wide range.
Description
Technical field
The present invention relates to a kind of preparation method of high stability immobilized enzyme.
Background technology
As everyone knows, enzyme is a kind of efficient single-minded biological catalyst.When many organic chemical reactionses that are difficult to carry out adopt enzyme to make catalyzer, under normal temperature, non-pressurized mild conditions, just can successfully carry out, and can avoid or reduce the side reaction generation.Therefore, since ancient times enzyme just by people be widely used in brewageing, food, medicine and other fields.Particularly in modern age, because biochemical development, the mechanism of action of enzyme is clear day by day, and new enzyme source is continually developed, and the application of enzyme enlivens more.But, enzyme is made up of protein, in most of the cases, all stable inadequately to heat, strong acid, highly basic, organic solvent etc., even under the optimum condition of enzyme reaction, also tend to very fast inactivation, in the aqueous solution so enzyme only limits to use usually, and reclaim and the repeated use difficulty, also not a kind of ideal catalyzer for modern industry.
And immobilized enzyme is to improve the enzyme service efficiency at present and realize one of the most effective means of operation serialization, be used for the example of the existing many successes of aqueous enzymatic reaction, but the application in organic phase also is in the exploratory stage at present.Especially in hydrophobic organic phase, how to make the constitutional features of enzyme carrier meet the optimum microenvironment that enzyme can be given full play to vigor, and make enzyme activity keep advantages of higher stability, and be a difficult problem of biological catalyst heterogenize always, also be the challenging problem of filed of functional.And based on the modern chemistry industry of petrochemical complex, the production process more than 70% all be unable to do without catalyzer, and based on the nonaqueous phase heterogeneous catalytic system.Therefore, if can not fundamentally solve the heterogenize problem of biocatalysis system, then biotechnology will not known where to begin to the impact of the traditional chemical industry that forms huge production ability and height serialization and automatization.
The process for fixation of enzyme can roughly be divided into 4 kinds of absorption method, covalent method, crosslinking and entrapping methods etc.Absorption method is meant the method by the secondary key interaction immobilized enzyme between carrier surface and enzyme surface, and the characteristics of looking sorbent material can be divided into physical adsorption and ion-exchange absorption again.Advantages such as that this method has is easy and simple to handle, mild condition and sorbent material can use repeatedly, but also exist a little less than the adsorptive power, the shortcoming that comes off of desorb easily.Covalent method is to make enzyme with the process for fixation of covalent bonds in carrier by linked reaction, so show satisfactory stability, help the continuous use of enzyme, but because of common linked reaction condition harshness, can cause that the zymoprotein higher structure changes, so the ratio of this class immobilized enzyme is lived lower.Crosslinking is to add that linking agent makes between enzyme and the carrier, crosslinking reaction takes place between enzyme and the enzyme and the immobilized enzyme that forms, because the reaction conditions fierceness, so enzyme work yield is generally lower.Entrapping method comprises grid embedding, microcapsule-type embedding and liposome embedded etc., does not participate in the Chemical bond reaction because of enzyme itself in the entrapping method, thus can obtain higher enzyme activity recovery, but the common physical strength of prepared immobilized enzyme is relatively poor.
Summary of the invention
The object of the present invention is to provide a kind of method of utilizing entrapping method to prepare high stability immobilized enzyme, advantages such as this method has that preparation technology is simple, enzyme is lived the yield height, good stability, the suitable environment scope is wide, cost of manufacture is low and prepared immobilized enzyme physical strength is big.
The technical solution used in the present invention is as follows: this method at first adopts enzyme, water, tensio-active agent, organic solvent, monomer or prepolymer and linking agent to make up microemulsion system, add reaction substrate then and induce the activity conformation of enzyme, cause radical polymerization by heating, ultraviolet lighting or direct radiation mode, realize the embedded immobilization of enzyme in highly cross-linked carrier.
Concrete steps of the present invention are as follows:
1. take by weighing the enzyme powder and be dissolved in that to be mixed with mass percent in the buffered soln be that 0.05%～20% enzyme liquid is standby;
2. adopt enzyme liquid, tensio-active agent, organic solvent, monomer and linking agent to make up microemulsion system;
3. adding mass percent and be 0.1%～10% reaction substrate in above-mentioned microemulsion system induces the conformation of enzyme;
4. add mass percent and be 0.2%～3% thermal initiator or light trigger, the mode that causes Raolical polymerizable by heating, ultraviolet lighting or direct radiation is respectively finished enzyme and is immobilization under the active condition then;
5. after treating the polymer cure moulding, behind fragmentation, grinding, sizing screening, obtain granular polymer, place the Soxhlet extractor with acetone or sherwood oil extracting water and substrate after, can obtain remaining with the immobilized enzyme of enzymic activity conformation through vacuum-drying.
Described enzyme: be lipase, acylase, amylase, cellulase or proteolytic enzyme.
Described tensio-active agent: be a kind of or any two kinds of mixtures of anion surfactant, cats product, nonionogenic tenside or amphoterics.
Described organic solvent: be normal heptane or hexalin.
Described monomer: for methacrylic acid, methyl methacrylate or contain the compound of 1 carbon-to-carbon double bond; Described linking agent: the compound or any two kinds of mixtures that contain 2 carbon-to-carbon double bonds for Vinylstyrene, polyethyleneglycol diacrylate, polyethylene glycol dimethacrylate, Viscoat 295, trimethylolpropane trimethacrylate, tetramethylol methane tetraacrylate, some molecular structures at least; Both content account for 10～98% of system quality.
The useful effect that the present invention has is: utilize entrapping method to prepare the method for high stability immobilized enzyme, advantages such as this method has that preparation technology is simple, enzyme is lived the yield height, good stability, the suitable environment scope is wide, cost of manufacture is low and prepared immobilized enzyme physical strength is big.
Embodiment
Detailed process is as follows:
(1) contains the structure of enzyme microemulsion system.At first, will need immobilized enzyme powder (as lipase, acylase, amylase, cellulase or proteolytic enzyme etc.) to be dissolved in the buffered soln standby; Then, get a container, add entry, tensio-active agent (can be anion surfactant, cats product, nonionogenic tenside and amphoterics any or its mixture), organic solvent (as normal heptane, hexalin etc.) in proportion, and monomer (as methacrylic acid, methyl methacrylate), linking agent (as Vinylstyrene, Viscoat 295, tetramethylol methane tetraacrylate etc.) etc., stir, add a certain amount of enzyme aqueous solution subsequently and leave standstill after transparent and promptly get microemulsion.
(2) enzyme is the immobilization of activity conformation state.The above-mentioned microemulsion that fills enzyme is placed container, adds a small amount of reaction substrate (lauric acid, oleic acid, sad or other lipid acid) conformation of enzyme is induced, record activity after, can carry out immobilization operation.Can be as the case may be, add thermal initiator (as Diisopropyl azodicarboxylate, benzoyl peroxide, persulphate etc.), or light trigger (as benzoin methyl ether, benzophenone etc.), cause the mode of Raolical polymerizable then respectively by heating, ultraviolet lighting or direct radiation, will be enzyme under the activity conformation state and be embedded in the highly cross-linked carrier and realize immobilization.After treating the polymer cure moulding, behind to a certain degree fragmentation, grinding, sizing screening, obtain granular polymer, after placing the Soxhlet extractor with methyl alcohol and acetate extracting water and substrate, can obtain remaining with the immobilized enzyme of enzymic activity conformation through vacuum-drying.
In the process of prepared immobilized enzyme provided by the invention, enzyme, water, organic solvent, monomer or prepolymer and linking agent etc. are constructed microemulsion system together, under this condition, add the activity conformation that reaction substrate induces enzyme, and carry out polyreaction rapidly and implement immobilization, can make enzyme after the immobilization keep the activity conformation of simultaneous adaptation water and corresponding organic solvent, expand the range of application of enzyme.And owing to added a large amount of monomer with crosslinked function and linking agent in the system, improved the structural stability of polymer support greatly, and then also improved the stability of enzymic activity conformation, also make the physical strength of immobilized enzyme be enhanced simultaneously.
Embodiment 1
At first, take by weighing lipase powder (or above any enzyme powder, below identical) and be dissolved in that to be mixed with 0.1%～2% enzyme liquid in the buffered soln standby, taking by weighing sodium laurylsulfonate (SDS) and deionized water, to be mixed with 20% the aqueous solution standby.Then, measure 20% SDS aqueous solution 10ml, add successively after normal heptane 5ml, polyethyleneglycol diacrylate 10g, trimethylolpropane trimethacrylate 10g, methyl methacrylate 23g and 4ml enzyme liquid stirs, leave standstill.When treating that solution reaches transparent, add 1%～3% reaction substrate lauric acid, add 1～2g Diisopropyl azodicarboxylate at last, to the system curing molding, continue reaction 2h and get final product 40～60 ℃ of polyreactions.Take out polymkeric substance after polymerization is finished, behind fragmentation, grinding, sizing screening, obtain granular polymer, place the Soxhlet extractor, promptly get the immobilized enzyme product through vacuum-drying with acetone or sherwood oil organic solvent extracting water and lauric acid.The activity yield that records enzyme is 109%.Under 40～50 ℃ of conditions, immobilized enzyme used 30 days continuously, and the activity of enzyme keeps former activated 95%.Used 7 days at 80 ℃, polymer support is indeformable, and enzymic activity is stabilized in about 80%～85%.
Embodiment 2
At first, take by weighing the lipase powder and be dissolved in that to be mixed with 1%～4% enzyme liquid in the buffered soln standby.Then, measure normal heptane 10ml, add two (2-ethylhexyl) amber sodium sulfonates (AOT), 8～10g, enzyme liquid 2～4ml, polyethylene glycol dimethacrylate 20g, Viscoat 295 10g be stirred to transparent, the reaction substrate of adding 1%～3% is sad, add light trigger benzoin methyl ether 0.10～0.30g, pour in the mould after stirring, under ultraviolet lighting, carry out light initiation polymerization to the system curing molding, continue illumination 3～8s and get final product.Take out polymkeric substance after polymerization is finished, behind fragmentation, grinding, sizing screening, obtain granular polymer, place the Soxhlet extractor with acetone or sherwood oil organic solvent extracting water and substrate after, promptly get the immobilized enzyme product through vacuum-drying.The activity yield that records enzyme is 182%.Under 40～50 ℃ of conditions, immobilized enzyme used 30 days continuously, and the activity of enzyme keeps former activated 97%.Used 7 days at 75 ℃, polymer support is indeformable, and enzymic activity is stabilized in 75～80%.
Embodiment 3
At first, take by weighing the lipase powder and be dissolved in that to be mixed with 0.05%～1% enzyme liquid in the buffered soln standby.Then, measure normal heptane 5ml, add Tween802～6g, enzyme liquid 2～4ml, polyethylene glycol dimethacrylate 20g, tetramethylol methane tetraacrylate (PETA) 20g be stirred to transparent, the oleic acid of adding 2%, pour into after stirring in ampere irradiation bottle, vacuumize or logical high pure nitrogen seals, place 60Co gamma-ray irradiation field to be irradiated to the system curing molding and get final product.Take out polymkeric substance after polymerization is finished, behind fragmentation, grinding, sizing screening, obtain granular polymer, place the Soxhlet extractor with acetone or sherwood oil organic solvent extracting water and substrate after, promptly get the immobilized enzyme product through vacuum-drying.The activity yield that records enzyme is 133%.Under 40～50 ℃ of conditions, immobilized enzyme used 30 days continuously, and the activity of enzyme keeps former activated 93%.Used 7 days at 75 ℃, polymer support is indeformable, and enzymic activity is stabilized in 72～80%.
Embodiment 4
At first, take by weighing the lipase powder and be dissolved in that to be mixed with 10～20% enzyme liquid in the buffered soln standby.Then, measure normal heptane 10ml, add two (2-ethylhexyl) amber sodium sulfonates (AOT), 8～10g, enzyme liquid 1～4ml, polyethylene glycol dimethacrylate 30g, Viscoat 295 20g be stirred to transparent, the lauric acid of adding 3%, add light trigger benzoin methyl ether 0.20～0.50g, pour in the mould after stirring, under ultraviolet lighting, carry out light initiation polymerization to the system curing molding, continue illumination 5～10s and get final product.Take out polymkeric substance after polymerization is finished, behind fragmentation, grinding, sizing screening, obtain granular polymer, place the Soxhlet extractor with acetone or sherwood oil organic solvent extracting water and substrate after, promptly get the immobilized enzyme product through vacuum-drying.The activity yield that records enzyme is 210%.Under 40～50 ℃ of conditions, immobilized enzyme used 30 days continuously, and the activity of enzyme keeps former activated 94%.Used 7 days at 75 ℃, polymer support is indeformable, and enzymic activity is stabilized in about 75～85%.
Claims (9)
1, the preparation method of high stability immobilized enzyme, it is characterized in that: this method at first adopts enzyme, water, tensio-active agent, organic solvent, monomer or prepolymer and linking agent to make up microemulsion system, add reaction substrate then and induce the activity conformation of enzyme, cause radical polymerization by heating, ultraviolet lighting or direct radiation mode, realize the embedded immobilization of enzyme in highly cross-linked carrier.
2, the preparation method of high stability immobilized enzyme according to claim 1 is characterized in that the concrete steps of this method are as follows:
1. take by weighing the enzyme powder and be dissolved in that to be mixed with mass percent in the buffered soln be that 0.05%～20% enzyme liquid is standby;
2. adopt enzyme liquid, tensio-active agent, organic solvent, monomer and linking agent to make up microemulsion system;
3. adding mass percent and be 0.1%～10% reaction substrate in above-mentioned microemulsion system induces the conformation of enzyme;
4. add mass percent and be 0.2%～3% thermal initiator or light trigger, the mode that causes Raolical polymerizable by heating, ultraviolet lighting or direct radiation is respectively finished enzyme and is immobilization under the active condition then;
5. after treating the polymer cure moulding, behind fragmentation, grinding, sizing screening, obtain granular polymer, place the Soxhlet extractor with acetone or sherwood oil extracting water and substrate after, can obtain remaining with the immobilized enzyme of enzymic activity conformation through vacuum-drying.
3, the preparation method of high stability immobilized enzyme according to claim 2 is characterized in that described enzyme: be lipase, acylase, amylase, cellulase or proteolytic enzyme.
4, the preparation method of high stability immobilized enzyme according to claim 2 is characterized in that described tensio-active agent: be a kind of or any two kinds of mixtures of anion surfactant, cats product, nonionogenic tenside or amphoterics.
5, the preparation method of high stability immobilized enzyme according to claim 2 is characterized in that described organic solvent: be normal heptane or hexalin.
6, the preparation method of high stability immobilized enzyme according to claim 2 is characterized in that described monomer: for methacrylic acid, methyl methacrylate or contain the compound of 1 carbon-to-carbon double bond; Linking agent is compound or any two kinds of mixtures that Vinylstyrene, polyethyleneglycol diacrylate, polyethylene glycol dimethacrylate, Viscoat 295, trimethylolpropane trimethacrylate, tetramethylol methane tetraacrylate, some molecular structures contain 2 carbon-to-carbon double bonds at least; Both content account for 10～98% of system quality.
7, the preparation method of high stability immobilized enzyme according to claim 2 is characterized in that described reaction substrate: be lauric acid, oleic acid, sad or other lipid acid.
8, the preparation method of high stability immobilized enzyme according to claim 2 is characterized in that described thermal initiator: be Diisopropyl azodicarboxylate, benzoyl peroxide or persulphate.
9, the preparation method of high stability immobilized enzyme according to claim 2 is characterized in that described light trigger: be benzoin methyl ether or benzophenone.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100608380A CN100340664C (en) | 2005-09-21 | 2005-09-21 | High stability immobilized enzyme preparation method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2005100608380A CN100340664C (en) | 2005-09-21 | 2005-09-21 | High stability immobilized enzyme preparation method |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN1766103A true CN1766103A (en) | 2006-05-03 |
| CN100340664C CN100340664C (en) | 2007-10-03 |
Family
ID=36742220
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CNB2005100608380A Expired - Fee Related CN100340664C (en) | 2005-09-21 | 2005-09-21 | High stability immobilized enzyme preparation method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN100340664C (en) |
Cited By (10)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101338308B (en) * | 2007-07-06 | 2012-02-01 | 赢创戈尔德施米特有限公司 | Enzyme preparations |
| CN102492684A (en) * | 2011-11-25 | 2012-06-13 | 浙江大学 | Frozen gel embedding method for enzyme |
| CN107164359A (en) * | 2017-06-30 | 2017-09-15 | 鲁东大学 | A kind of preparation method of the glucose oxidase nanogel with good thermal stability |
| CN107619824A (en) * | 2017-10-30 | 2018-01-23 | 北京化工大学 | One kind prepares immobilised enzymes method based on photocuring hydrogel |
| CN109266637A (en) * | 2018-09-29 | 2019-01-25 | 天津医科大学 | Using trimethylol-propane trimethacrylate integral post as the immobilized enzyme reactor of matrix |
| CN110358758A (en) * | 2019-07-15 | 2019-10-22 | 泰兴市东圣生物科技有限公司 | A kind of preparation method of freezing gel glutamine transaminage |
| CN110777133A (en) * | 2019-05-07 | 2020-02-11 | 宁波大学 | Co-crosslinking immobilization method of lysozyme |
| CN110777139A (en) * | 2019-05-07 | 2020-02-11 | 宁波大学 | Co-crosslinking immobilization method of nitrile hydratase |
| CN111321134A (en) * | 2020-02-27 | 2020-06-23 | 西南科技大学 | Immobilized multienzyme system and preparation method thereof |
| CN113403298A (en) * | 2021-06-09 | 2021-09-17 | 万华化学集团股份有限公司 | Immobilization method of free enzyme |
Family Cites Families (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| DE19931847A1 (en) * | 1999-07-09 | 2001-01-11 | Basf Ag | Immobilized lipase |
| KR100985423B1 (en) * | 2002-07-02 | 2010-10-05 | 카오카부시키가이샤 | A process for preparing an immobilized enzyme |
-
2005
- 2005-09-21 CN CNB2005100608380A patent/CN100340664C/en not_active Expired - Fee Related
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101338308B (en) * | 2007-07-06 | 2012-02-01 | 赢创戈尔德施米特有限公司 | Enzyme preparations |
| CN102492684A (en) * | 2011-11-25 | 2012-06-13 | 浙江大学 | Frozen gel embedding method for enzyme |
| CN107164359B (en) * | 2017-06-30 | 2020-03-17 | 鲁东大学 | Preparation method of glucose oxidase nanogel with good thermal stability |
| CN107164359A (en) * | 2017-06-30 | 2017-09-15 | 鲁东大学 | A kind of preparation method of the glucose oxidase nanogel with good thermal stability |
| CN107619824A (en) * | 2017-10-30 | 2018-01-23 | 北京化工大学 | One kind prepares immobilised enzymes method based on photocuring hydrogel |
| CN109266637A (en) * | 2018-09-29 | 2019-01-25 | 天津医科大学 | Using trimethylol-propane trimethacrylate integral post as the immobilized enzyme reactor of matrix |
| CN109266637B (en) * | 2018-09-29 | 2021-10-15 | 天津医科大学 | Immobilized enzyme reactor using trimethylolpropane trimethacrylate monolithic column as matrix |
| CN110777133A (en) * | 2019-05-07 | 2020-02-11 | 宁波大学 | Co-crosslinking immobilization method of lysozyme |
| CN110777139A (en) * | 2019-05-07 | 2020-02-11 | 宁波大学 | Co-crosslinking immobilization method of nitrile hydratase |
| CN110777133B (en) * | 2019-05-07 | 2022-09-06 | 宁波大学 | Co-crosslinking immobilization method of lysozyme |
| CN110777139B (en) * | 2019-05-07 | 2022-09-06 | 宁波大学 | Co-crosslinking immobilization method of nitrile hydratase |
| CN110358758A (en) * | 2019-07-15 | 2019-10-22 | 泰兴市东圣生物科技有限公司 | A kind of preparation method of freezing gel glutamine transaminage |
| CN111321134A (en) * | 2020-02-27 | 2020-06-23 | 西南科技大学 | Immobilized multienzyme system and preparation method thereof |
| CN113403298A (en) * | 2021-06-09 | 2021-09-17 | 万华化学集团股份有限公司 | Immobilization method of free enzyme |
| CN113403298B (en) * | 2021-06-09 | 2024-02-27 | 万华化学集团股份有限公司 | Immobilization method of free enzyme |
Also Published As
| Publication number | Publication date |
|---|---|
| CN100340664C (en) | 2007-10-03 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN100340664C (en) | High stability immobilized enzyme preparation method | |
| US5405618A (en) | Biomosaic polymer obtained by emulsion polymerization of hydrophobic monomers in the presence of bioactive materials | |
| CN106866993B (en) | Compound self-healing hydrogel of a kind of macromolecule based on nano thin-layer curing molybdenum sheet and preparation method thereof | |
| CN104195042A (en) | Preparation method and application of nano material monolithic column immobilized enzyme biological micro-reactor | |
| CN103224926B (en) | Method of preparing immobilized lipase | |
| Vijayaraghavan et al. | A new approach to study the decolorization of complex reactive dye bath effluent by biosorption technique | |
| CN102925425B (en) | Method for preparing immobilized enzyme on surface of polymer base material | |
| CN108187641B (en) | A kind of preparation method and applications of sodium alginate/polyvinyl alcohol@polyacrylamide core-shell structure gel ball | |
| CN106582810A (en) | Preparation method of graphene immobilized enzyme catalyst | |
| CN104387712A (en) | Nano composite carrier with superparamagnetism and preparation method thereof | |
| CN109082126A (en) | A kind of hydrogel multi-stage motor and preparation method thereof of glucose responding driving | |
| CN1962861B (en) | Combined immobilization method applied to biocatalytic conversion | |
| Tobin et al. | Immobilisation protocols and effects on cadmium uptake by Rhizopus arrhizus biosorbents | |
| CN103882004B (en) | A kind of nanogel coating charcoal base immobilized lipase of oleic acid molecular modification and preparation method thereof | |
| CN109913441A (en) | A kind of method of polymer microcapsule embedding immobilized enzyme | |
| CN101838640B (en) | Unimolecule embedding method for enzyme | |
| CN102827310B (en) | Photochemical initiation system and method for initiating photopolymerization or embedment of transparent system or nontransparent system by using same | |
| CN107986387B (en) | Ultrasound-assisted selective photocatalysis method and device based on magnetic molecular imprinting | |
| CN105238828A (en) | Method for improving hydrolysis efficiency of celluloses and reducing cost of cellulosic ethanol by utilizing molecular net cloth to embed beta (Beta)-glucosidase | |
| CN102229923A (en) | Lipase nano-sized polymer biocatalyst particle and preparation method thereof | |
| CN105085827A (en) | Preparation method of tanshinol surface molecularly-imprinted polymer | |
| CN102676494B (en) | Core-shell structure immobilized enzyme particle and preparation method thereof | |
| CN1496282A (en) | Method for producing gel-type cation exchangers | |
| CN111303347A (en) | Preparation method of core-shell type temperature-sensitive protein molecularly imprinted polymer microspheres | |
| CN104845960A (en) | Preparation method for surfactant producing microbial composite carrier |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C14 | Grant of patent or utility model | ||
| GR01 | Patent grant | ||
| C17 | Cessation of patent right | ||
| CF01 | Termination of patent right due to non-payment of annual fee |
Granted publication date: 20071003 Termination date: 20100921 |