CN103013952B - Functional ionic liquid modified lipase and modification method thereof - Google Patents
Functional ionic liquid modified lipase and modification method thereof Download PDFInfo
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- 239000004367 Lipase Substances 0.000 title claims abstract description 86
- 102000004882 Lipase Human genes 0.000 title claims abstract description 86
- 108090001060 Lipase Proteins 0.000 title claims abstract description 86
- 235000019421 lipase Nutrition 0.000 title claims abstract description 86
- 239000002608 ionic liquid Substances 0.000 title abstract description 32
- 238000002715 modification method Methods 0.000 title abstract 2
- 125000002924 primary amino group Chemical group [H]N([H])* 0.000 claims abstract description 8
- 125000000539 amino acid group Chemical group 0.000 claims abstract description 5
- 229940040461 lipase Drugs 0.000 claims description 78
- 239000007788 liquid Substances 0.000 claims description 62
- 102000019280 Pancreatic lipases Human genes 0.000 claims description 26
- 108050006759 Pancreatic lipases Proteins 0.000 claims description 26
- 238000006243 chemical reaction Methods 0.000 claims description 26
- 229940116369 pancreatic lipase Drugs 0.000 claims description 26
- 238000000034 method Methods 0.000 claims description 23
- FPQQSJJWHUJYPU-UHFFFAOYSA-N 3-(dimethylamino)propyliminomethylidene-ethylazanium;chloride Chemical compound Cl.CCN=C=NCCCN(C)C FPQQSJJWHUJYPU-UHFFFAOYSA-N 0.000 claims description 15
- UHVULTHJALNHMG-UHFFFAOYSA-N 3-(3-methyl-2h-imidazol-1-yl)propan-1-amine Chemical class CN1CN(CCCN)C=C1 UHVULTHJALNHMG-UHFFFAOYSA-N 0.000 claims description 14
- 241000589513 Burkholderia cepacia Species 0.000 claims description 10
- HOGDNTQCSIKEEV-UHFFFAOYSA-N n'-hydroxybutanediamide Chemical compound NC(=O)CCC(=O)NO HOGDNTQCSIKEEV-UHFFFAOYSA-N 0.000 claims description 9
- 239000002253 acid Substances 0.000 claims description 7
- 230000031709 bromination Effects 0.000 claims description 7
- 238000005893 bromination reaction Methods 0.000 claims description 7
- 238000005660 chlorination reaction Methods 0.000 claims description 7
- 229950003549 rizolipase Drugs 0.000 claims description 7
- 241000234282 Allium Species 0.000 claims description 6
- 235000002732 Allium cepa var. cepa Nutrition 0.000 claims description 6
- 241000222120 Candida <Saccharomycetales> Species 0.000 claims description 6
- 241000321538 Candidia Species 0.000 claims description 6
- 241000607142 Salmonella Species 0.000 claims description 6
- 239000004744 fabric Substances 0.000 claims description 6
- 125000004573 morpholin-4-yl group Chemical group N1(CCOCC1)* 0.000 claims description 6
- 102000004190 Enzymes Human genes 0.000 abstract description 59
- 108090000790 Enzymes Proteins 0.000 abstract description 59
- 230000000694 effects Effects 0.000 abstract description 21
- -1 NHS Substances 0.000 abstract description 12
- 230000003197 catalytic effect Effects 0.000 abstract description 5
- 238000005728 strengthening Methods 0.000 abstract description 2
- 229940088598 enzyme Drugs 0.000 description 57
- 150000002500 ions Chemical class 0.000 description 57
- 150000002460 imidazoles Chemical class 0.000 description 17
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 12
- 230000004913 activation Effects 0.000 description 12
- 230000004048 modification Effects 0.000 description 11
- 238000012986 modification Methods 0.000 description 11
- 238000006555 catalytic reaction Methods 0.000 description 9
- 239000003607 modifier Substances 0.000 description 9
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 8
- 230000035484 reaction time Effects 0.000 description 8
- 238000000108 ultra-filtration Methods 0.000 description 6
- 239000002904 solvent Substances 0.000 description 5
- 230000001133 acceleration Effects 0.000 description 4
- 239000003960 organic solvent Substances 0.000 description 4
- URAYPUMNDPQOKB-UHFFFAOYSA-N triacetin Chemical compound CC(=O)OCC(OC(C)=O)COC(C)=O URAYPUMNDPQOKB-UHFFFAOYSA-N 0.000 description 4
- 230000008859 change Effects 0.000 description 3
- 238000006352 cycloaddition reaction Methods 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
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- 230000008569 process Effects 0.000 description 3
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- 238000005406 washing Methods 0.000 description 3
- QRZMXADUXZADTF-UHFFFAOYSA-N 4-aminoimidazole Chemical compound NC1=CNC=N1 QRZMXADUXZADTF-UHFFFAOYSA-N 0.000 description 2
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 2
- 102000018120 Recombinases Human genes 0.000 description 2
- 108010091086 Recombinases Proteins 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 150000001875 compounds Chemical class 0.000 description 2
- 238000004821 distillation Methods 0.000 description 2
- 150000002148 esters Chemical class 0.000 description 2
- 230000007062 hydrolysis Effects 0.000 description 2
- 238000006460 hydrolysis reaction Methods 0.000 description 2
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- 239000000126 substance Substances 0.000 description 2
- 238000003786 synthesis reaction Methods 0.000 description 2
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 2
- 229920002307 Dextran Polymers 0.000 description 1
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 1
- 108090000698 Formate Dehydrogenases Proteins 0.000 description 1
- 238000006736 Huisgen cycloaddition reaction Methods 0.000 description 1
- 108010020056 Hydrogenase Proteins 0.000 description 1
- 229920003171 Poly (ethylene oxide) Polymers 0.000 description 1
- NSOXQYCFHDMMGV-UHFFFAOYSA-N Tetrakis(2-hydroxypropyl)ethylenediamine Chemical compound CC(O)CN(CC(C)O)CCN(CC(C)O)CC(C)O NSOXQYCFHDMMGV-UHFFFAOYSA-N 0.000 description 1
- 238000002479 acid--base titration Methods 0.000 description 1
- 239000012190 activator Substances 0.000 description 1
- 238000006136 alcoholysis reaction Methods 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
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- 239000012496 blank sample Substances 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 125000002915 carbonyl group Chemical group [*:2]C([*:1])=O 0.000 description 1
- 150000001768 cations Chemical class 0.000 description 1
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- 230000009144 enzymatic modification Effects 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 125000002485 formyl group Chemical class [H]C(*)=O 0.000 description 1
- 238000005194 fractionation Methods 0.000 description 1
- 229910052739 hydrogen Inorganic materials 0.000 description 1
- 239000001257 hydrogen Substances 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- WGCNASOHLSPBMP-UHFFFAOYSA-N hydroxyacetaldehyde Natural products OCC=O WGCNASOHLSPBMP-UHFFFAOYSA-N 0.000 description 1
- 239000003262 industrial enzyme Substances 0.000 description 1
- 238000009776 industrial production Methods 0.000 description 1
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- 239000000758 substrate Substances 0.000 description 1
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- Preparation Of Compounds By Using Micro-Organisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a lipase modified by functionalized ionic liquid and a modification method thereof. Dissolving amino-containing functionalized ionic liquid, NHS, EDC & HCL and lipase in MES according to the molar ratio of 100-300:1:1-3:1, and mixing and reacting at 0-4 ℃ for 3-10h to obtain the lipase modified by the functionalized ionic liquid. The invention introduces functional ionic liquid with amino on the surface of lipase, and covalently combines with lipase molecule amino acid residue, thereby influencing the space structure of enzyme activity center and strengthening the catalytic performance of lipase.
Description
Division explanation
The present invention is to be on January 16th, 2012 applying date, and application number is 201210013025.6, and name is called a kind of lipase of modified by functionalized ion liquid and dividing an application of modifying method thereof.
Technical field
The invention belongs to biocatalysis technology field, relate to a kind of lipase and modifying method thereof of modified by functionalized ion liquid.
Background technology
Enzyme is a kind of special catalyzer, the multiple reaction of energy catalysis under gentle condition, the biotechnology industry that the enzyme of take is core is one of the industry that receives much concern of the world today (Selection and evolution of enzymes from partially randomized non-catalytic scaffold.Nature, 2007,448,828-831.).Industrial enzyme market, the five-year whole world, by the speed rapid growth with average annual 6.3%, is only the enzyme product market share to 2013 and will be reached 7,000,000,000 dollars, and market potential is huge.Lipase (EC3.1.1.3) is a kind of conventional, important enzyme in industrial production application, can be on water-oil interface the organic synthesis such as synthetic, the transesterify of the hydrolysis of catalysis ester or alcoholysis, ester and stereoisomerism fractionation, at very extensive (the Lipases for biotechnology.Curr.Opin.Biotechnol. of the traditional industry field application such as food, feed, washing, bioenergy (biofuel), medicine chiral separation, 2002,13,390-397.).
The current main stream approach about enzyme transformation comprises that (1) adopts transgenation or orthogenesis transformation lipase, obtains high catalytic performance; (2) immobilization technology; (3) chemically modified, generally comprises: the modification of small molecules (aldehyde, lipid acid), macromole (dextran), bi-functional cross-linking agent (quadrol) etc., wherein polyoxyethylene glycol (PEG) is most popular a kind of modifier.
Ionic liquid can change by changing the structure of zwitterion the character such as the wetting ability, hydrophobicity, stickiness, fusing point, density of ionic liquid, the synthetic ionic liquid with applicable certain reaction or separated specific demand of design, therefore, ionic liquid is also claimed " designability solvent "; Simultaneously, ionic liquid is owing to having environment amenable characteristic " green " solvent that is otherwise known as, (Catalysis in Ionic Liquids.Chem.Rev. is used widely in the fields such as, catalysis synthetic in organic synthesis, sepn process, material, 2007,107,2615-2665.).
But ionic liquid is all to use as reaction medium substantially in enzymic catalytic reaction at present, its usage quantity is large, and cost is high.In the last few years, patent JP2009207492, JP2009203454, WO2008090156, CN101225427 etc. utilize " green " solvent and " designability solvent " character of functionalized ion liquid, simplified the process of product separation, stability or the activity of enzyme have been improved, but it is excessive to have run into equally ionic liquid consumption, be not suitable for the problem of suitability for industrialized production.
Although can strengthen easy, fast catalytic performance, the change zymologic property of enzyme by the method for chemically modified, but can find out from this development trend in several years, the correlative study of chemically modified is tending towards stagnating, its major cause is that correlative study rests on traditional chemical modifier always, can not meet the multifarious demand of enzyme molecular modification modifier.
Studies have reported that (Ionic Liquid-Inspired Cations Covalently Bound to Formate Dehydrogenase Improve its Stability and Activity in Ionic Liquids.ChemCatChem., 2011,3,875-882.) use carbonyl dimidazoles as activator, to connect the functionalized ion liquid of hydrogenlyase and hydroxyl, but the enzyme modification reaction times reaches 24h, and it is larger to the activity influence of enzyme to use methyl-sulphoxide to make solvent.The Zou Bin of this seminar etc. are used modified by functionalized ion liquid carrier, find suitable fixing of carrier enzyme enzyme is lived and stability raising.Whole process comprises: the preparation of carrier, and ionic liquid is synthetic, Ionic Liquid Modified carrier, and then to being fixed of enzyme, the cycle of reaction is long, and complicated operation, ionic liquid is to combine by electrostatic force and hydrogen bond with the combination of enzyme.In contrast to the modification it is characterized in that carrier, feature of the present invention is the modification based on enzyme molecule itself, is a kind of diverse mode, simple to operate, mode is flexible, the cycle is short, and its object is all to improve the Activity and stabill of enzyme.
Summary of the invention
The object of the invention is the above-mentioned deficiency for prior art, a kind of lipase of modified by functionalized ion liquid is provided.
Another object of the present invention is to provide the method for this modified by functionalized ion liquid lipase.
A lipase for modified by functionalized ion liquid, the lipase of described modified by functionalized ion liquid is by following any one method, the organo-functional group of functionalized ion liquid and the amino-acid residue of lipase to be obtained by covalent attachment:
Method I: carboxylic functionalized ion liquid, N-hydroxy-succinamide (NHS) are dissolved in to morpholino b acid (MES) with 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDCHCL) by the mol ratio of 1:1:1-3; under 25 ~ 35 ℃ of conditions, react 1-2h and activate, by activation after functionalized ion liquid and lipase in molar ratio 200-1000:1 at 0-4 ℃ of hybrid reaction 2-8h;
Method II: by containing amino functionalized ion liquid, N-hydroxy-succinamide (NHS), 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride (EDCHCL) and lipase in molar ratio 100-300:1:1-3:1 be dissolved in morpholino b acid (MES), at 0-4 ℃ of hybrid reaction 3-10h.
The lipase of described modified by functionalized ion liquid, the negatively charged ion that it is characterized in that described functionalized ion liquid is to have different hydrophilic or electronegative polarity ion, positively charged ion is the imidazole-like ionic containing different organo-functional groups, and organo-functional group is carboxyl or amino.
The lipase of described modified by functionalized ion liquid, is characterized in that described functionalized ion liquid is selected from 1-methyl-3-propyloic imidazoles villaumite, 1-ethyl-3-propyloic imidazoles villaumite, chlorination 1-methyl-3-(3-aminopropyl) imidazoles, 1-methyl-3-propyloic imidazoles hexafluorophosphate, bromination 1-methyl-3-(3-aminopropyl) imidazoles or 1-butyl-3-propyloic tetrafluoroborate.
The preferred porcine pancreatic lipase of described lipase, rizolipase, pseudomonas cepacia lipase, fold lipase from candida sp, antarctic candidia lipase or onion cloth gram Salmonella lipase.
A method for modified by functionalized ion liquid lipase, makes the organo-functional group of functionalized ion liquid and the amino-acid residue of lipase pass through covalent attachment by following any one method:
Method I: carboxylic functionalized ion liquid, N-hydroxy-succinamide and 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride is dissolved in to morpholino b acid by the mol ratio of 1:1:1-3; under 25 ~ 35 ℃ of conditions, react 1-2h and activate, by activation after functionalized ion liquid and lipase in molar ratio 200-1000:1 at 0-4 ℃ of hybrid reaction 2-8h;
Method II: by containing amino functionalized ion liquid, N-hydroxy-succinamide, 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and lipase in molar ratio 100-300:1:1-3:1 be dissolved in morpholino b acid, at 0-4 ℃ of hybrid reaction 3-10h.
Wherein, the negatively charged ion of described functionalized ion liquid is to have different hydrophilic or electronegative polarity ion, and positively charged ion is the imidazole-like ionic containing different organo-functional groups, and organo-functional group is carboxyl or amino; Described functionalized ion liquid is preferably from 1-methyl-3-propyloic imidazoles villaumite, 1-ethyl-3-propyloic imidazoles villaumite, chlorination 1-methyl-3-(3-aminopropyl) imidazoles, 1-methyl-3-propyloic imidazoles hexafluorophosphate, bromination 1-methyl-3-(3-aminopropyl) imidazoles or 1-butyl-3-propyloic tetrafluoroborate.
The preferred porcine pancreatic lipase of described lipase, rizolipase, pseudomonas cepacia lipase, fold lipase from candida sp, antarctic candidia lipase or onion cloth gram Salmonella lipase.
Beneficial effect of the present invention:
(1) the present invention combines enzyme catalysis Study on Transformation in the chemically modified research of lipase and ionic liquid first, with functional ionic liquids, chemically modified is carried out in lipase surface, thereby the space structure that affects enzyme active center reaches the catalytic performance object of strengthening lipase.
(2) the present invention is with N-hydroxy-succinamide and 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride coupling mobilizing function ionic liquid, greatly improved activation efficiency, shortened the reaction times, the impact that agents useful for same is lived on enzyme is little, reduce the loss of enzyme in modification, reduce costs.
(3) the present invention by after functionalized ion liquid activation for modifying lipase, given full play to the advantage of functionalized ion liquid in catalyzed reaction, and the defect such as it is large to have overcome in prior art when functionalized ion liquid is as reaction medium consumption, and production cost is high.
(4) when intermediate ion liquid of the present invention and lipase reduce ionic liquid consumption by covalent attachment, make full use of that ionic liquid has compared with strong solubility energy, make enzymatic heterogeneous reaction there is the feature of accurate homogeneous reaction, alleviate the impact of diffusional limitation on enzyme catalysis efficiency, develop a kind of lipase modifying method of novelty, expanded the application of ionic liquid in enzyme engineering field.
(5) the lipase-catalyzed reaction of functionalized ion liquid covalent attachment provided by the invention, has chemical stability and the heatproof thermostabilitys such as good alkali resistance, organic solvent resistance.
(6) the present invention compares with immobilization way, simple to operate, mode is flexible, the cycle is short, instant effect, is a kind of biotechnology that has much practical value.
Accompanying drawing explanation
The mimic diagram of Fig. 1 modified by functionalized ion liquid lipase.
The affect figure of Fig. 2 PH on Ionic Liquid Modified porcine pancreatic lipase and free porcine pancreatic lipase.
The affect figure of Fig. 3 temperature on Ionic Liquid Modified porcine pancreatic lipase and free porcine pancreatic lipase.
The affect figure of Fig. 4 organic solvent on Ionic Liquid Modified porcine pancreatic lipase and free porcine pancreatic lipase.
Embodiment
Embodiment 1
1) 1-methyl-3-propyloic imidazoles villaumite is synthetic: according to people such as Jean Pierre Bazureau at (Rate accelerations of1,3-dipolar cycloaddition reactions in ionic liquids.Tetrahedron Letters, 2000,41:7351-7355.) in narration, prepare 1-methyl-3-propyloic imidazoles villaumite, yield 78.3%, purity 98.8%.
2) activation of functionalized ion liquid: 0.17g1-methyl-3-propyloic imidazoles villaumite and 0.192g EDCHCL and 0.115gNHS are dissolved in to 10ml MES, react 1h under 30 ℃ of conditions.
3) modification of enzyme: by 200: the 1 in molar ratio hybrid reactions of porcine pancreatic lipase after functionalized ion liquid and purifying after activation, temperature of reaction is controlled at 0-4 ℃, reaction times 4h, the modifier that the centrifugal removal of ultrafiltration is unnecessary, concentrated enzyme.
4) enzyme activity determination:
Lipase hydrolysis glycerine triacetate emulsion is probe reaction.At temperature 50 C, PH is 7.5, when substrate glycerine triacetate concentration is 6.8%, adopts the vigor of this enzyme of determination of acid-basetitration.The apparent activity of modifying enzyme is 863IU/g, and the apparent activity of free porcine pancreatic lipase is 760IU/g lipase.
The apparent activity calculation formula of enzyme is (following apparent enzyme is lived and all with this formula, calculated):
U=(V-V
0)×50/(10×0.02)=20(V-V
0)
Wherein: V---the volume of the NaOH solution that sample consumes, ml;
V
0---the volume of the NaOH solution that blank sample consumes, ml;
50---micromole's number of the NaOH that every milliliter of NaOH solution contains;
10---reaction times, min;
0.02---resolvase, g.
According to this area, investigate the ordinary method of enzyme PH stability and investigated respectively porcine pancreatic lipase and the PH stability of free porcine pancreatic lipase within the scope of PH6-9 that 1-methyl-3-propyloic imidazoles villaumite is modified, the results are shown in Figure 2.As seen from Figure 2, the optimum response PH of resolvase is 7.5, and the optimum response PH of the porcine pancreatic lipase of the prepared modified by functionalized ion liquid of the present invention is also 7.5, do not change but apparent enzyme activity significantly improves, and the enzyme activity under meta-alkalescence condition is still very high, the visible porcine pancreatic lipase by the prepared modified by functionalized ion liquid of the present invention, its PH stability obviously improves than free porcine pancreatic lipase, has widened the use range of enzyme under different PH.
According to this area, investigate the ordinary method of enzyme temperature stability and investigated respectively porcine pancreatic lipase and the temperature stability of free porcine pancreatic lipase within the scope of 30～65 ℃ that 1-methyl-3-propyloic imidazoles villaumite is modified, the results are shown in Figure 3.As seen from Figure 3, the optimal reaction temperature of resolvase is 45 ℃, and the optimal reaction temperature of the porcine pancreatic lipase of the prepared modified by functionalized ion liquid of the present invention is 55 ℃, and apparent enzyme activity significantly improves, the visible porcine pancreatic lipase by the prepared modified by functionalized ion liquid of the present invention, its temperature stability obviously improves than free porcine pancreatic lipase.
According to this area, investigated the ordinary method of the organic solvent resistance of enzyme and investigated free porcine pancreatic lipase and free porcine pancreatic lipase that 1-methyl-3-propyloic imidazoles villaumite is modified, result is as Fig. 4.Porcine pancreatic lipase through modified by functionalized ion liquid provided by the present invention has more superior organic solvent resistance than not modified porcine pancreatic lipase as shown in Figure 4, the former still preserves initial apparent activity under the DMF of 70% concentration exists still has initial apparent activity more than 50% more than 100% and in pure DMF solution, the enzyme of free-fat enzyme is lived and is being declined always, and in the situation that DMF concentration is high, few of enzyme is lived.
Embodiment 2
1) 1-ethyl-3-propyloic imidazoles villaumite is synthetic: according to people such as Jean Pierre Bazureau at (Rate accelerations of 1,3-dipolar cycloaddition reactions in ionic liquids.Tetrahedron Letters, 2000,41:7351-7355.) in narration, prepare 1-ethyl-3-propyloic imidazoles villaumite, yield 71.6%, purity 97.6%.
2) activation of functionalized ion liquid: 0.18g1-ethyl-3-propyloic imidazoles villaumite and 0.288g EDCHCL and 0.115gNHS are dissolved in to 10ml MES, react at ambient temperature 1.5h.
3) modification of enzyme: by 300: the 1 in molar ratio hybrid reactions of rizolipase after functionalized ion liquid and purifying after activation, temperature of reaction is controlled at 0-4 ℃, reaction times 6h, the modifier that the centrifugal removal of ultrafiltration is unnecessary, concentrated enzyme.
4) to prepare the operational condition of modifying enzyme identical with embodiment 1, and the apparent activity that records this modified by functionalized ion liquid rizolipase is 1120IU/g lipase, and the enzyme activity of free rizolipase is 990IU/g lipase.
Embodiment 3
1) chlorination 1-methyl-3-(3-aminopropyl) imidazoles is synthetic: according to people such as Wu Youting (a kind of containing amino imidazole like ion liquid compound and preparation method thereof, CN100999498) narration in, prepare chlorination 1-methyl-3-(3-aminopropyl) imidazoles, yield 82.7%, purity 98.1%.
2) modification of enzyme: by 0.18g chlorination 1-methyl-3-(3-aminopropyl) imidazoles, 1.44mg EDCHCL, 0.58mgNHS and 0.165 pseudomonas cepacia lipase are dissolved in 10ml MES, temperature of reaction is controlled at 0-4 ℃, reaction times 4h, the modifier that the centrifugal removal of ultrafiltration is unnecessary, concentrated enzyme.
3) to prepare the operational condition of modifying enzyme identical with embodiment 1, and the apparent activity that records this modified by functionalized ion liquid pseudomonas cepacia lipase is 2850IU/g lipase, and the enzyme of free pseudomonas cepacia lipase is lived as 2240IU/g lipase.
Embodiment 4
1) 1-methyl-3-propyloic imidazoles hexafluorophosphate is synthetic: according to people such as Jean Pierre Bazureau at (Rate accelerations of1,3-dipolar cycloaddition reactions in ionicliquids.Tetrahedron Letters, 2000,41:7351-7355.) in narration, prepare 1-methyl-3-propyloic imidazoles villaumite, yield 78.3%, purity 98.8%.By the 1-methyl-3-propyloic imidazoles villaumite and the KPF that obtain
6in acetone, stirring at room obtains ionic liquid for one day. and obtain product and filter, washing, separatory, underpressure distillation steams acetone and water, obtains 1-methyl-3-propyloic imidazoles hexafluorophosphate.Yield 58.8%, purity 95.8%
2) activation of functionalized ion liquid: 0.28g1-methyl-3-propyloic imidazoles hexafluorophosphate and 0.192g EDCHCL and 0.115gNHS are dissolved in to 10ml MES, react 2h under 30 ℃ of conditions.
3) modification of enzyme: by 700: the 1 in molar ratio hybrid reactions of pseudomonas cepacia lipase after functionalized ion liquid and purifying after activation, temperature of reaction is controlled at 0-4 ℃, reaction times 3h, the modifier that the centrifugal removal of ultrafiltration is unnecessary, concentrated enzyme.
4) to prepare the operational condition of modifying enzyme identical with embodiment 1, and the apparent activity that records this modified by functionalized ion liquid fold lipase from candida sp is 1600IU/g lipase, and the enzyme of free fold lipase from candida sp is lived as 1200IU/g lipase.
Embodiment 5
1) bromination 1-methyl-3-(3-aminopropyl) imidazoles is synthetic: according to people such as Wu Youting (a kind of containing amino imidazole like ion liquid compound and preparation method thereof, CN100999498) narration in, prepare bromination 1-methyl-3-(3-aminopropyl) imidazoles, yield 74.2%, purity 97.3%.
2) modification of enzyme: by 0.22g bromination 1-methyl-3-(3-aminopropyl) imidazoles, 1.44mg EDCHCL, 0.58mgNHS and 0.165 pseudomonas cepacia lipase are dissolved in 10ml MES, temperature of reaction is controlled at 0-4 ℃, reaction times 10h, the modifier that the centrifugal removal of ultrafiltration is unnecessary, concentrated enzyme.
3) to prepare the operational condition of modifying enzyme identical with embodiment 1, and the apparent activity that records this modified by functionalized ion liquid antarctic candidia lipase is 980IU/g lipase, and the enzyme of free antarctic candidia lipase is lived as 780IU/g lipase.
Embodiment 6
1) 1-butyl-3-propyloic tetrafluoroborate is synthetic: according to people such as Jean Pierre Bazureau at (Rate accelerations of1,3-dipolar cycloaddition reactions in ionicliquids.Tetrahedron Letters, 2000,41:7351-7355.) in narration, prepare 1-butyl-3-propyloic imidazoles villaumite, yield 65.1%, purity 97.2%.By the 1-butyl-3-propyloic imidazoles villaumite and the KBF that obtain
4in acetone, stirring at room obtains ionic liquid for one day. and obtain product and filter, washing, separatory, underpressure distillation steams acetone and water, obtains 1-butyl-3-propyloic tetrafluoroborate.Yield 62.7%, purity 96.3%
2) activation of functionalized ion liquid: 0.27g1-butyl-3-propyloic tetrafluoroborate and 0.576g EDCHCL and 0.115gNHS are dissolved in to 10ml MES, react 2h under 28 ℃ of conditions.
3) modification of enzyme: by 800: the 1 in molar ratio hybrid reactions of pseudomonas cepacia lipase after functionalized ion liquid and purifying after activation, temperature of reaction is controlled at 0-4 ℃, reaction times 5h, the modifier that the centrifugal removal of ultrafiltration is unnecessary, concentrated enzyme.
4) to prepare the operational condition of modifying enzyme identical with embodiment 1, and the apparent activity that records this modified by functionalized ion liquid onion cloth gram Salmonella lipase is 380IU/g lipase, and the enzyme of free onion cloth gram Salmonella lipase is lived as 220IU/g lipase.
Claims (4)
1. a lipase for modified by functionalized ion liquid, is characterized in that the lipase of described modified by functionalized ion liquid is to make by the following method the organo-functional group of functionalized ion liquid and the amino-acid residue of lipase obtain by covalent attachment:
By containing amino functionalized ion liquid, N-hydroxy-succinamide, 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and lipase in molar ratio 100-300:1:1-3:1 be dissolved in morpholino b acid, at 0-4 ℃ of hybrid reaction 3-10h; Wherein said functionalized ion liquid is selected from chlorination 1-methyl-3-(3-aminopropyl) imidazoles or bromination 1-methyl-3-(3-aminopropyl) imidazoles.
2. the lipase of modified by functionalized ion liquid according to claim 1, is characterized in that described lipase is porcine pancreatic lipase, rizolipase, pseudomonas cepacia lipase, fold lipase from candida sp, antarctic candidia lipase or onion cloth gram Salmonella lipase.
3. a method for modified by functionalized ion liquid lipase, is characterized in that making by the following method the organo-functional group of functionalized ion liquid and the amino-acid residue of lipase to pass through covalent attachment:
By containing amino functionalized ion liquid, N-hydroxy-succinamide, 1-(3-dimethylamino-propyl)-3-ethyl-carbodiimide hydrochloride and lipase in molar ratio 100-300:1:1-3:1 be dissolved in morpholino b acid, at 0-4 ℃ of hybrid reaction 3-10h; Wherein, described functionalized ion liquid is selected from chlorination 1-methyl-3-(3-aminopropyl) imidazoles or bromination 1-methyl-3-(3-aminopropyl) imidazoles.
4. the method for modified by functionalized ion liquid lipase according to claim 3, is characterized in that described lipase is porcine pancreatic lipase, rizolipase, pseudomonas cepacia lipase, fold lipase from candida sp, antarctic candidia lipase or onion cloth gram Salmonella lipase.
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| CN105907741A (en) * | 2016-06-06 | 2016-08-31 | 新疆大学 | Covalent binding method for preparing activated carbon immobilized lipase |
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