CN102229923A - Lipase nano-sized polymer biocatalyst particle and preparation method thereof - Google Patents
Lipase nano-sized polymer biocatalyst particle and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a lipase nano-sized polymer biocatalyst particle and a preparation method thereof. The method comprises the following core steps: chemically modifying the surface of a lipase with glycidyl methacrylate which is used as a hydrophobic reagent, introducing a C-C double-bond group, and then, without needing to separate intermediates, directly adding a vinyl monomer containing C-C double bonds as a raw material to initiate the free radical polymerization to obtain the target product. The method is carried out under mild production conditions, has a simple preparation process, can effectively prevent enzyme inactivation in the middle modification procedure to ensure high enzyme activity, and can also greatly improve the heat stability of the lipase nano-sized polymer biocatalyst particle. Besides, the novel method simplifies the procedure of separating the intermediates to reduce the production cost, and is favorable for the large-scale industrial production.
Description
Technical field
The present invention relates to a kind of lipase nano-polymer biocatalyst particle and preparation method thereof.
Background technology
Lipase (Lipase) is as one of industrial enzyme of being most widely used, can catalytic hydrolysis, kinds of reaction such as esterification, and have higher chirality/regioselectivity.Its common Application Areas comprises organic synthesis, foodstuffs industry, detergent industry, energy industry, bio-transformation, biological medicine, biosensor or the like.Chemical modification method is the common methods in the enzyme molecular modification process, and along with the development of nanosecond science and technology in recent years, nano-polymer biocatalyst particle becomes novel problem of biochemical catalytic field just gradually.The lipase nano-polymer biocatalyst particle not only can effectively be realized homogeneous catalysis, solve existing immobilized lipase and cause the lower problem of enzymatic activity because of high resistance to mass transfer, but also can effectively improve thermostability, strengthen the organic solvent tolerance.Conventional two-step prepares the lipase nano-polymer biocatalyst particle and adopts the modifier of vinylformic acid succinimide ester as the two key groups of the first step introducing protein surface, enters the water original position radical polymerization of second step after separating through intermediate product then and obtains target product.The modifier cost height of this method, middle separating step length consuming time has seriously restricted its application in actual industrial production.Therefore, develop that a kind of raw materials cost is low, technology is simple, the preparation method of the lipase nano-polymer biocatalyst particle of less energy consumption is significant.
Summary of the invention
The purpose of this invention is to provide a kind of lipase nano-polymer biocatalyst particle and preparation method thereof.
Lipase nano-polymer biocatalyst particle provided by the present invention is that raw material is made with following materials in parts by mass: 10 parts in lipase, glycidyl methacrylate (enzyme modification agent) 2-200 part, vinyl monomer 75-375 part and initiator system 20-30 part; Concrete preparation method comprises the steps: 1) described lipase and described glycidyl methacrylate are added the pH value in the buffered soln of 3-8, reacted 0.5～3 hour down at 0-50 ℃; 2) in the reaction solution of step 1), add described vinyl monomer and described initiator system, continue reaction 5-12 hour, obtain the lipase nano-polymer biocatalyst particle at 0-50 ℃.
Wherein, described enzyme modification agent glycidyl methacrylate is to add with the dimethyl formamide solution form of glycidyl methacrylate, because the mutual solubility of dimethyl formamide and modifier and water is better, so, can promote it to react in the aqueous phase dissolving and with lipase as the solvent of modifier.
Lipase described in the present invention derives from commercial enzyme, animal extracts or microorganism extracts.
Among the present invention, described vinyl monomer is meant the vinyl monomer that can form polymkeric substance by Raolical polymerizable; Described vinyl monomer specifically can be selected from following at least a: acrylamide, N-N-isopropylacrylamide, vinylformic acid, acryloyl polyethylene glycol, methacrylic acid, methyl methacrylate, hydroxyethyl methylacrylate, Hydroxyethyl acrylate, Propylene glycol monoacrylate, Rocryl 410, N, N '-methylene diacrylamide, suitable divinyl and trihydroxy methyl propane trimethyl acrylic ester.
Among the present invention, described initiator system is meant under 0-50 ℃ of condition can cause the generation free radical, causes the thermal initiator system of vinyl monomer generation radical polymerization or redox to draw the agent system.Described initiator system specifically can be made up of the material among following A and the B, described A is selected from following at least a: Potassium Persulphate, ammonium persulphate, hydrogen peroxide, tertbutyl peroxide and peroxidation phenyl-diformyl, described B is selected from following at least a: ferrous salt, sulphite, N, N '-xylidine, ammoniacal liquor, N, N '-dimethyl-para-totuidine, N, N, N ', N '-Tetramethyl Ethylene Diamine, piperidines and N-methylmorpholine.Described initiator system is preferably by ammonium persulphate and N, N, and N ', N '-Tetramethyl Ethylene Diamine is formed, and its mass ratio can be 1.5: 2.
Described buffered soln can be the aqueous solution of following at least a material: acetic acid, sodium-acetate, sodium phosphate, Sodium phosphate dibasic, SODIUM PHOSPHATE, MONOBASIC, potassiumphosphate, potassium hydrogen phosphate, SODIUM PHOSPHATE, MONOBASIC, boric acid, Sodium Tetraborate, potassium borate, yellow soda ash, sodium bicarbonate, salt of wormwood and saleratus.
Described method also comprises step 2) reaction solution of the fatty enzyme nano-polymer biocatalyst particle that obtains is dialysed, and the freeze dried step of the liquid after will dialysing.
In preparation method provided by the invention, vinyl monomer also can add in step 1, and concrete grammar is as follows:
1) described lipase, described glycidyl methacrylate and described vinyl monomer being added the pH value is in the buffered soln of 3-8, reacts 0.5～3 hour down at 0-50 ℃; 2) in the reaction solution of step 1), add described initiator system, continue reaction 5-12 hour, obtain the lipase nano-polymer biocatalyst particle at 0-50 ℃.
The above-mentioned method that adds earlier vinyl monomer back adds the method for vinyl monomer, and the polyreaction yield is lower, considers from final polymerisate yield aspect, and preferred back adds the method for vinyl monomer.
The present invention adopts the hydrophobic agents glycidyl methacrylate as the enzyme modification agent, introduce the carbon-carbon double bond group on the lipase surface, need not intermediate product separates, directly add the vinyl monomer that contains carbon-carbon double bond and cause radical polymerization, obtain target lipase nano-polymer biocatalyst particle.The particle diameter of this lipase nano-polymer biocatalyst particle is 20-50nm, and its core is a lipase, and shell is a macromolecular material, has chemical bond to connect between the nucleocapsid.
The inventive method core is the introducing of novel hydrophobically modified dose (glycidyl methacrylate), can effectively realize exciting of lipase activity, obtains high enzyme yield alive, avoids the modification step loss that work causes to the lipase enzyme in the traditional preparation process method.Simultaneously, the preparation process of one kettle way can significantly improve production efficiency, reduces production costs, and prepares and have the more lipase nano-polymer biocatalyst particle of the following yardstick of 100 nanometers of high thermal stability, the industrial production of being more convenient for and amplification.
Description of drawings
Fig. 1 modifies for utilizing conventional two-step vinylformic acid succinimide ester (NAS), new two-step approach glycidyl methacrylate (GMA) is modified, new one kettle way glycidyl methacrylate (One-Pot) is modified, three kinds of two key modification degree (Acryloylation%) of method, and respectively go on foot product remaining activity relatively (Intermediate ProductRA%/CRL-pAM nanogel RA%).
Fig. 2 modifies method gained target product for glycidyl methacrylate of the present invention (GMA), and conventional acrylic succinimide ester (NAS) modification method obtains product, and each enzyme kinetics parameter of native lipase (Km, Vmax, Kcat) comparison diagram; NAS:CRL-pNIPAAM and GMA:CRL-pNIPAAM are meant with the N-N-isopropylacrylamide to be the catalysed particulate of vinyl monomer preparation among the figure.
Fig. 3 is preparation method of the present invention (One-pot), and conventional two-step (Two-step) and native lipase are at 50 ℃, 60 ℃ following thermostability comparison diagrams.
Fig. 4 compares for the schematic flow sheet of preparation method of the present invention (One-pot) technological process and conventional two-step (Two-step) technological process.
Fig. 5 is lipase nanogel product polymerisate yield and the remaining activity yield comparison diagram of embodiment 3 under different modifying agent charging capacity scope.
Fig. 6 is lipase nanogel product polymerisate yield and the remaining activity yield comparison diagram of embodiment 4 under different monomers charging capacity scope.
Fig. 7 is lipase nanogel product polymerisate yield and the remaining activity yield comparison diagram of embodiment 5 under different pH scopes.
Embodiment
Below in conjunction with embodiment the inventive method is described further, but the present invention is not limited thereto.
Experimental technique described in the embodiment if no special instructions, is ordinary method; Described reagent and material if no special instructions, all can obtain from commercial channels.Use lipase to be Lipase from Canadida rugosa among the embodiment, lipase is from sigma company, and cat. no is L1754, Type VII, 〉=700unit/mg.
Above-mentioned lipase need carry out purifying before use, concrete grammar is as follows: above-mentioned 100 parts of lipase are dissolved in the damping fluid of pH value 3～8, centrifugal 5～20min under 0～30 ℃, getting supernatant liquor, to place molecular weight cut-off be the dialysis tubing of 10KDa, be 3～8 at pH, dialysis 24 hours in the 50mM damping fluid, measure protein concentration and determine the lipase protein content.The amount of used lipase is in the lipase protein mass among the following embodiment.
Embodiment 1, One-pot legal system are equipped with the lipase nano-polymer biocatalyst particle
Raw material is the weight part meter: 10 parts in lipase, and 200 parts of enzyme modification agent (glycidyl methacrylate), 250 parts of vinyl monomers (acrylamide), initiator system are 15 parts of ammonium persulphates and 12 parts of N, N, N ', the mixture of N '-Tetramethyl Ethylene Diamine.
Above-mentioned lipase is dissolved in that 5000 mass parts pH are 4, in the 50mM acetate buffer, adds enzyme modification agent (50%v/v dimethyl formamide solution), and reaction is 2 hours under 30 ℃ of conditions.After adding 250 parts of acrylamide dissolving backs and adding above-mentioned initiator system, under agitation condition, continue reaction 12 hours in 30 ℃, then reaction solution being placed molecular weight cut-off is the dialysis tubing of 10KDa, be 7 at pH, dialysis 24 hours in the 50mM phosphoric acid buffer, collect the liquid freeze-drying of dialysis back and can obtain target product lipase nano-polymer biocatalyst particle.
The biology catalytic activity total recovery (RA%=lipase enzyme activity to be measured/natural fat enzyme activity * 100%) of measuring the lipase nano-polymer biocatalyst particle as substrate with p-nitrophenol cetylate class is 108%, and the polyreaction yield is 50%.
Concrete activity determination method is as follows:
When adopting the p-nitrophenol cetylate to live as substrate mensuration enzyme, at first the p-nitrophenol cetylate is dissolved in the acetone, under agitation slowly join Sodium phosphate dibasic-phosphate sodium dihydrogen buffer solution (50mM of the Triton X-100 that contains 1.25% (w/v) then, pH 7.0) in be mixed with the substrate solution of 0.5mM, acetone concentration is controlled at 5% (v/v) in the solution.When the survey enzyme was lived, a certain amount of enzyme liquid joined in the substrate solution, mixed the absorption value of measuring the 348nm place after 10 seconds on ultraviolet spectrophotometer and changed, and obtained the hydrolysis relative reactivity of lipase by slope calculations.The hydrolysis of catalysis p-nitrophenol cetylate produces the required enzyme amount of 1 μ mol palmitinic acid and is defined as enzyme unit alive in 1 minute.
The polymer reaction yield is as the criterion with the Size Exclusion Chromatograph SEC figure under the 280nm ultraviolet detection.Wherein, enzyme nanogel product polymerization yield %=polymkeric substance goes out peak area/total peak area * 100%.
Comparative Examples 1, conventional two-step
Raw material is the weight part meter: 10 parts in lipase, and 15 parts of enzyme modification agent (vinylformic acid succinimide ester), 250 parts of vinyl monomers (acrylamide), initiator system are 15 parts of ammonium persulphates and 12 parts of N, N, N ', the mixture of N '-Tetramethyl Ethylene Diamine.
Above-mentioned lipase is dissolved in that 5000 mass parts pH are 4, in the 50mM acetate buffer, adds enzyme modification agent (2% dimethyl sulfoxide (DMSO)), and reaction is 6 hours under 30 ℃ of conditions, is 7 at pH, dialysis 24h removes excessive modifier in the 50mM phosphoric acid buffer.Add 250 parts of acrylamides, temperature keeps 30 ℃, magnetic agitation, add above-mentioned initiator system, temperature keeps 30 ℃, continues reaction 12 hours, and then reaction solution being placed molecular weight cut-off is the dialysis tubing of 10KDa, be 7 at pH, dialysis 24 hours in the 50mM phosphoric acid buffer, collect the liquid freeze-drying of dialysis back and promptly get the conventional acrylic succinimide ester and modify the lipase nano-polymer biocatalyst particle that two-step approach down prepares.
Two-step approach under Comparative Examples 2, the new modifier
Raw material is the weight part meter: 10 parts in lipase, and 200 parts of enzyme modification agent (glycidyl methacrylate), 250 parts of vinyl monomers (acrylamide), initiator system are 15 parts of ammonium persulphates and 12 parts of N, N, N ', the mixture of N '-Tetramethyl Ethylene Diamine.
It is that reaction is 2 hours under 30 ℃ of conditions in 4 the 50mM acetate buffer that above-mentioned lipase and enzyme modification agent (50%v/v dimethyl formamide solution) are added pH, and dialysis 24h removes excessive modifier in damping fluid.After adding 250 parts of acrylamide dissolving backs and adding above-mentioned initiator system, under agitation condition, continue reaction 12 hours, then reaction solution being placed molecular weight cut-off is the dialysis tubing of 10KDa, be 7 at pH, dialysis 24 hours in the 50mM phosphoric acid buffer, collect the liquid freeze-drying of dialysis back and promptly obtain target product lipase nano-polymer biocatalyst particle.
Embodiment 1, Comparative Examples 1, Comparative Examples 2 are utilized the TNBS method to measure two key modification degree respectively and respectively gone on foot the biology catalytic activity yield of product with p-nitrophenol cetylate class as the mensuration of substrate, and its comparing result as shown in Figure 1.As shown in Figure 1, embodiment 1 method can effectively realize alive the exciting of lipase enzyme, obtains high enzyme yield alive.
Further detect of the influence of new modifier for the zymetology fundamental property, adopt above-mentioned enzyme activity determination method, in 0.1～0.9mM concentration of substrate solution, measure lipase enzyme digestion reaction initial reaction rate respectively, is X-coordinate according to Michaelis-Menton equation with the substrate inverse, the initial reaction rate inverse calculates Km (slope for ordinate zou carries out double-reciprocal plot
-1), Vmax (intercept
-1), Kcat=Vmax/[E] total, as shown in Figure 2, find the Km of former modifier modification after product (CRL-NAS) and polymerisate (NANOGEL (N)), Vmax, the Kcat value all descends to some extent, and Km and Vmax value that new modifier is modified after product (CRL-GMA) and polymerisate (NANOGEL (G)) slightly rise, and the Kcat value significantly improves simultaneously.Illustrate that new hydrophobically modified dose can influence enzyme-to-substrate bonded tightness degree to a certain extent, but the hydrophobic pathway of its formation can effectively improve the maximum speed of reaction of enzyme, significantly improve catalytic efficiency, reduce the disadvantageous effect of the introducing of polymkeric substance shell enzyme catalysis efficient.
Measure native lipase down at 50 ℃ and 60 ℃ respectively, the lipase nano-polymer biocatalyst particle thermostability that conventional two-step vinylformic acid succinimide ester modification target product and One-pot method of the present invention prepare compares, as shown in Figure 3.According to Fig. 3 and transformation period calculation formula: t
1/2=-ln2/k (wherein k is ln (RA%)～t slope) can obtain following result:
This shows that the present invention compares natural enzyme and traditional method, can effectively improve enzyme and live the transformation period that prepared lipase nanogel has better thermostability.
Fig. 4 has shown the contrast of comparative example 1 and embodiment 1 preparation flow synoptic diagram, illustrate that novel method can carry out in gentle steady temperature fixed reactor, and effectively save the intermediate product separating step, reduce production costs, enhance productivity (shortening about 24 hours/batch of production time).
Raw material is the weight part meter: 10 parts in lipase, and 200 parts of enzyme modification agent (glycidyl methacrylate), vinyl monomer (acrylamide) is 250 parts, initiator system is 15 parts of ammonium persulphates and 12 parts of N, N, N ', the mixture of N '-Tetramethyl Ethylene Diamine.
It is that reaction is 2 hours under 30 ℃ of conditions in 4 the 50mM acetate buffer that above-mentioned lipase, enzyme modification agent (50%v/v dimethyl formamide solution) and 250 parts of acrylamides are added pH.The back adds above-mentioned initiator system and causes radical polymerization, under agitation condition, continue reaction 12 hours, then reaction solution being placed molecular weight cut-off is the dialysis tubing of 10KDa, be 7 at pH, dialysis 24 hours in the 50mM phosphoric acid buffer, collect the liquid freeze-drying of dialysis back and can obtain target product lipase nano-polymer biocatalyst particle.
The biology catalytic activity total recovery of measuring nano-polymer biocatalyst particle as substrate with p-nitrophenol cetylate class is 94%, and the polyreaction yield is about 45%.
Embodiment 3, One-pot method change the modifier charging capacity
Change the modifier charging capacity among the embodiment 1 into 2-200 part glycidyl methacrylate respectively, all the other prescriptions are identical with embodiment 1 with step.At this moment, the product that obtains is measured the biology catalytic activity total recovery of nano-polymer biocatalyst particle and polyreaction yield as shown in Figure 5 with p-nitrophenol cetylate class as substrate.
Wherein, bulk polymerization reaction yield (Nanogel yield%) is suitable, and the biology catalytic activity total recovery is embodied on the relative enzyme of residue (CRL-pAM nanogel RA%) alive of product.Feed intake down at low modifier, the activation phenomenon is not remarkable because enzyme is lived, therefore the yield that obtains the lipase nanogel is lower, after the modifier charging capacity surpasses 50 parts, the enzyme phenomenon that activates alive begins to manifest, enzyme yield alive begins to improve, and when the modifier charging capacity increases to 200 parts, can obtain being equivalent to the enzyme nanogel enzyme yield alive of natural enzyme enzyme alive 114%.
Change the monomeric charge amount among the embodiment 1 into 75,150,225,300,375 parts of acrylamides respectively, all the other prescriptions are identical with embodiment 1 with step.At this moment, the product that obtains is measured the biology catalytic activity total recovery of nano-polymer biocatalyst particle and polyreaction yield as shown in Figure 6 with p-nitrophenol cetylate class as substrate.
The polyreaction yield of lipase nanogel (Nanogel yield%) improves with the increase of monomeric charge amount, reaches capacity when charging capacity surpasses 300 parts.And the biology catalytic activity total recovery of product (CRL-pAM nanogelRA%) also can reach the highest yield during at 225～300 parts in the monomeric charge amount.
Embodiment 5, One-pot method change system pH
Changing the reaction system damping fluid among the embodiment 1 into the pH value respectively is 3.0,4.0,6.0,8.0, and all the other prescriptions are identical with embodiment 1 with step.At this moment, the product that obtains is measured the biology catalytic activity total recovery of nano-polymer biocatalyst particle and polyreaction yield as shown in Figure 7 with p-nitrophenol cetylate class as substrate.
When pH＞3, the polyreaction yield (yield%) of lipase nanogel is significantly improved, and it is certain that the back keeps.Biology catalytic activity total recovery (RA%) then reaches optimal yield near pH6.
The present invention can summarize with other the specific form without prejudice to spirit of the present invention or principal character.Therefore, above-mentioned embodiment of the present invention all can only be thought can not limit the present invention to explanation of the present invention, claims have been pointed out scope of the present invention, therefore, suitable with claims of the present invention contain with scope in any change, all will be understood that it is to be included in the scope of claims.
Claims (10)
1. method for preparing the lipase nano-polymer biocatalyst particle, it is characterized in that: described lipase nano-polymer biocatalyst particle is that raw material is made with following materials in parts by mass: 10 parts in lipase, glycidyl methacrylate 2-200 part, vinyl monomer 75-375 part and initiator system 20-30 part;
Comprise the steps: 1) described lipase and described glycidyl methacrylate are added the pH value in the buffered soln of 3-8, reacted 0.5～3 hour down at 0-50 ℃;
2) in the reaction solution of step 1), add described vinyl monomer and described initiator system, continue reaction 5-12 hour, promptly obtain the lipase nano-polymer biocatalyst particle at 0-50 ℃.
2. method according to claim 1 is characterized in that: described vinyl monomer is the vinyl monomer that can form polymkeric substance by Raolical polymerizable; Described vinyl monomer is preferably from following at least a: acrylamide, N-N-isopropylacrylamide, vinylformic acid, acryloyl polyethylene glycol, methacrylic acid, methyl methacrylate, hydroxyethyl methylacrylate, Hydroxyethyl acrylate, Propylene glycol monoacrylate, Rocryl 410, N, N '-methylene diacrylamide, suitable divinyl and trihydroxy methyl propane trimethyl acrylic ester;
Described initiator system is can cause the generation free radical under 0-50 ℃ of condition, causes the thermal initiator system of vinyl monomer generation radical polymerization or redox to draw the agent system; Described initiator system preferably is made up of the material among following A and the B, described A is selected from following at least a: Potassium Persulphate, ammonium persulphate, hydrogen peroxide, tertbutyl peroxide and peroxidation phenyl-diformyl, described B is selected from following at least a: ferrous salt, sulphite, N, N '-xylidine, ammoniacal liquor, N, N '-dimethyl-para-totuidine, N, N, N ', N '-Tetramethyl Ethylene Diamine, piperidines and N-methylmorpholine; Described initiator system is most preferably by ammonium persulphate and N, N, and it is 1.5: 2 that N ', N '-Tetramethyl Ethylene Diamine form its mass ratio;
Described buffered soln is the aqueous solution of following at least a material: acetate, sodium acetate, sodium phosphate, Sodium phosphate dibasic, SODIUM PHOSPHATE, MONOBASIC, potassiumphosphate, potassium hydrogen phosphate, SODIUM PHOSPHATE, MONOBASIC, boric acid, Sodium Tetraborate, potassium borate, yellow soda ash, sodium bicarbonate, salt of wormwood and saleratus.
3. method according to claim 1 is characterized in that: described glycidyl methacrylate is that to add the pH value with the dimethyl formamide solution form of glycidyl methacrylate be in the buffered soln of 3-8.
4. according to each described method among the claim 1-3, it is characterized in that: described method also comprises step 2) reaction solution of the fatty enzyme nano-polymer biocatalyst particle that obtains carries out the dialysis that molecular weight cut-off is 10KDa, and the freeze dried step of the liquid after will dialysing.
5. method for preparing the lipase nano-polymer biocatalyst particle, it is characterized in that: described lipase nano-polymer biocatalyst particle is that raw material is made with following materials in parts by mass: 10 parts in lipase, glycidyl methacrylate 2-200 part, vinyl monomer 75-375 part and initiator system 20-30 part;
Comprise the steps: 1) described lipase, described glycidyl methacrylate and described vinyl monomer are added the pH value in the buffered soln of 3-8, reacted 0.5～3 hour down at 0-50 ℃;
2) in the reaction solution of step 1), add described initiator system, continue reaction 5-12 hour, promptly obtain the lipase nano-polymer biocatalyst particle at 0-50 ℃.
6. method according to claim 5 is characterized in that: described vinyl monomer is the vinyl monomer that can form polymkeric substance by Raolical polymerizable; Described vinyl monomer is preferably from following at least a: acrylamide, N-N-isopropylacrylamide, vinylformic acid, acryloyl polyethylene glycol, methacrylic acid, methyl methacrylate, hydroxyethyl methylacrylate, Hydroxyethyl acrylate, Propylene glycol monoacrylate, Rocryl 410, N, N '-methylene diacrylamide, suitable divinyl and trihydroxy methyl propane trimethyl acrylic ester;
Described initiator system is can cause the generation free radical under 0-50 ℃ of condition, causes the thermal initiator system of vinyl monomer generation radical polymerization or redox to draw the agent system; Described initiator system preferably is made up of the material among following A and the B, described A is selected from following at least a: Potassium Persulphate, ammonium persulphate, hydrogen peroxide, tertbutyl peroxide and peroxidation phenyl-diformyl, described B is selected from following at least a: ferrous salt, sulphite, N, N '-xylidine, ammoniacal liquor, N, N '-dimethyl-para-totuidine, N, N, N ', N '-Tetramethyl Ethylene Diamine, piperidines and N-methylmorpholine; Described initiator system is most preferably by ammonium persulphate and N, N, and it is 1.5: 2 that N ', N '-Tetramethyl Ethylene Diamine form its mass ratio;
Described buffered soln is the aqueous solution of following at least a material: acetate, sodium acetate, sodium phosphate, Sodium phosphate dibasic, SODIUM PHOSPHATE, MONOBASIC, potassiumphosphate, potassium hydrogen phosphate, SODIUM PHOSPHATE, MONOBASIC, boric acid, Sodium Tetraborate, potassium borate, yellow soda ash, sodium bicarbonate, salt of wormwood and saleratus.
7. method according to claim 5 is characterized in that: described glycidyl methacrylate is that to add the pH value with the dimethyl formamide solution form of glycidyl methacrylate be in the buffered soln of 3-8.
8. according to each described method among the claim 5-7, it is characterized in that: described method also comprises step 2) reaction solution of the fatty enzyme nano-polymer biocatalyst particle that obtains carries out the dialysis that molecular weight cut-off is 10KDa, and the freeze dried step of the liquid after will dialysing.
9. the lipase nano-polymer biocatalyst particle that each described method prepares among the claim 1-8.
10. lipase nano-polymer biocatalyst particle according to claim 9 is characterized in that: the particle diameter of described lipase nano-polymer biocatalyst particle is 20-50nm.
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| CN103451174A (en) * | 2013-09-22 | 2013-12-18 | 清华大学 | Enzyme-macromolecule conjugate and preparing method and application thereof |
| CN107312767A (en) * | 2017-07-17 | 2017-11-03 | 安徽工程大学 | A kind of combination immobilization β glucoside enzyme granulates and preparation method thereof |
| CN107312767B (en) * | 2017-07-17 | 2020-11-06 | 安徽工程大学 | Combined immobilized beta-glucosidase particle and preparation method thereof |
| CN115873841A (en) * | 2022-12-06 | 2023-03-31 | 中南林业科技大学 | Enzyme-metal composite catalyst for biological catalysis and preparation method thereof |
| CN115873841B (en) * | 2022-12-06 | 2024-04-09 | 中南林业科技大学 | Enzyme-metal composite catalyst for biocatalysis and preparation method thereof |
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