CN104404027A - Method for treating multi-walled carbon nanotube immobilized enzyme by plasma - Google Patents
Method for treating multi-walled carbon nanotube immobilized enzyme by plasma Download PDFInfo
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- FFBHFFJDDLITSX-UHFFFAOYSA-N benzyl N-[2-hydroxy-4-(3-oxomorpholin-4-yl)phenyl]carbamate Chemical compound OC1=C(NC(=O)OCC2=CC=CC=C2)C=CC(=C1)N1CCOCC1=O FFBHFFJDDLITSX-UHFFFAOYSA-N 0.000 claims description 2
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- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Enzymes And Modification Thereof (AREA)
Abstract
The invention discloses a method for treating a multi-walled carbon nanotube immobilized enzyme by plasma, which comprises the steps of treating the multi-walled carbon nanotube by a low-temperature vacuum plasma technology, ultrasonically dispersing the multi-walled carbon nanotube in a phosphate buffer solution, carrying out physical adsorption reaction with free enzyme, filtering, washing for multiple times and drying after the reaction to obtain the immobilized enzyme taking the multi-walled carbon nanotube as a carrier. According to the invention, on one hand, impurities on the surface of the multi-walled carbon nanotube are removed by using plasma cleaning, on the other hand, the specific surface area of the multi-walled carbon nanotube is increased by corroding the surface of the multi-walled carbon nanotube with the plasma, the surface free energy of the wall of the multi-walled carbon nanotube is increased, and the enzyme immobilization efficiency is effectively improved. The method has the advantages of simple and easy operation, low immobilization cost, high enzyme protein immobilization capacity of the immobilized enzyme, high enzyme activity, high thermal stability and chemical stability, high mechanical strength of the immobilized enzyme, and potential application value in the fields of biochemical industry, fine chemical industry, food industry, pharmaceutical industry and the like.
Description
Technical field
The invention belongs to enzyme immobilization technology and plasma surface treatment technical field, be specifically related to a kind of method of Cement Composite Treated by Plasma multi-walled carbon nano-tubes immobilized enzyme.
Background technology
Immobilized enzyme be utilize the enzyme of physics or chemical process treatment of water-soluble to make it to become water insoluble or be fixed on solid phase carrier but still there is the enzyme derivative of enzymic activity.Enzyme immobilization technology has become a core key technology in modern biotechnology and industrialization link thereof.About 12 kinds of immobilized enzyme, comprise amylase, saccharase, proteolytic enzyme, nitrilase, L-Aminoacylase, multiple lipase, penicillin G acylase etc., being used in many industrial fields, is indispensable catalyzer.The basic skills of enzyme immobilization can be divided into roughly 5 kinds: absorption, covalent linkage and, embedding, microencapsulation and crosslinked, and these methods derive nearly hundreds of methods by combination.In order to ensure multiple biological fixnig and bioseparation, go out the carrier of different physical property and chemical property by a lot of patten's design.The immobilization scheme of these fixation techniques for enzyme and other carrier various types of and reasonable, ensure that the enzyme of wide material sources can obtain effective immobilization, make it be applied to the fields such as biochemical industry, fine chemicals industry, foodstuffs industry, medicine industry better.The immobilized enzyme one side of these application is solved and cannot be solved or masty problem by chemical means in the past, can realize the continuity of technique on the other hand, better control catalytic process and reduction running cost.
Carbon nanotube is the monodimension nanometer material that one has special construction (radial dimension belongs to nanometer scale, and axial dimension belongs to micron dimension, and the top of each mono-layer tube has pentagon or heptagon to participate in closing).It is lightweight, and hexagonal structure connects perfect, has shown the efficient mechanics of many exceptions, chemistry and electric property.It is the nano level hollow tube that the class graphite plane that is made up of carbon six-ring is curling.Carbon atom in carbon nanotube is with sp
2be hybridized to master, simultaneously to exist again to a certain degree bending for hexagonal mesh, forms Space expanding, define certain sp
3hybrid bond, the chemical bond namely formed can have sp concurrently simultaneously
2and sp
3mixed hybridization state, cause these P tracks to overlap each other and form highly delocalized large π key outward at carbon nanotube lamella, the large π key of carbon nanotube outside surface is the chemical fundamentals that carbon nanotube and other macromole with conjugation performance meet with non covalent bond.Therefore, when utilizing carbon nanotube to carry out enzyme immobilization, except the effect of Van der Waals force, the large π key of carbon nanotube outside surface can produce conjugation with the die aromatischen Aminosaeuren in zymoprotein, enhances enzyme stability on the carbon nanotubes.
Patent document CN102373192A(201110024154.0) disclose the method for the immobilized protease molecule of nano material, the carrier of the method using carbon nanotube as ankyrin enzyme molecule, it is first made to be dispersed into homogeneous solution, the nitration mixture re-using concentrated nitric acid and the vitriol oil carries out acidification to it, NHS and EDC is finally utilized to connect the connection amidine functional group of functionalization at carbon nanotube tube wall, amido that is last and enzyme reacts, by enzyme immobilization at tube wall.
Patent document CN1912200A(200610052954.2) disclose the nanofiber of carbon nanotube and the method for preparation and oxidation reduction fix thereof, the method by multi-walled carbon nano-tubes (or Single Walled Carbon Nanotube) first ultrasonic disperse in solvent, again vinyl cyanide/acrylic copolymer is dissolved in wherein, by the composite nano fiber of electrostatic spinning preparation containing carbon nanotube, responding property of surface band group carboxyl, by carbodiimide hydrochloride/succinimide activated, oxydo-reductase is fixed on fiber surface.
Above-mentioned patent utilization carbon nanotube or the composite nano materials containing carbon nanotube, all need to carry out certain modification and just can carry out enzyme immobilizatio, although recyclable recycling and improve utilization ratio and the storage stability of enzyme, but the surface-area of the multi-walled carbon nano-tubes used is relatively little, and the price of Single Walled Carbon Nanotube costly, the method that enzyme immobilization is taked is damaged to some extent the catalytic center of enzyme and its enzyme activity is decreased, and immobilization process is loaded down with trivial details and cost is higher.
Summary of the invention
In order to solve carbon nanotube immobilized enzyme prior art Problems existing, the invention provides a kind of method of Cement Composite Treated by Plasma multi-walled carbon nano-tubes immobilized enzyme, adopt low-cost multi-walled carbon nano-tubes to be carrier, reduce costs, and widen the Application Areas of immobilized enzyme.
Technical scheme of the present invention is as follows:
A method for Cement Composite Treated by Plasma multi-walled carbon nano-tubes immobilized enzyme, comprises the following steps:
Step 1, Cement Composite Treated by Plasma multi-walled carbon nano-tubes
The multi-wall carbon nano-tube pipe powder of 0.1 ~ 0.5 g is positioned on low temperature vacuum plasma body equipment Stage microscope, plasma electrically interpolar spacing is regulated to be 2 ~ 4 cm, regulate the pulse width of electric current to make power reach 22 ~ 84 W again, process 1 ~ 10 min under vacuum;
Step 2, multi-walled carbon nano-tubes and the physical adsorption of enzyme liquid are reacted
Be dissolved in phosphoric acid buffer by the multi-wall carbon nano-tube pipe powder after process, in ultrasonic cleaner, ultrasonic 10 ~ 30 min, then add resolvase, fix 30 ~ 240 min, obtain mixed solution in the water bath with thermostatic control of 20 ~ 40 DEG C;
Step 3, by mixed solution suction filtration and with phosphoric acid buffer washing, wash away the resolvase do not adsorbed, dried overnight under room temperature condition, obtain multi-walled carbon nano-tubes immobilized enzyme.
As preferably, step 1, the number of plies of described multi-walled carbon nano-tubes is 2 ~ 30 layers.
As preferably, step 2, described phosphoric acid buffer pH value is 6 ~ 8.
As preferably, step 2, described resolvase is any one in lipase, proteolytic enzyme, amylase or glucose oxidase.
As preferably, described lipase is any one in lipase from candida sp, porcine pancreatic lipase, Candida rugosa lipase, Lipozyme CALB L lipase, Palatase 20000 L lipase or Lipozyme TL100 L lipase.
As preferably, the rotating speed stirred during water-bath in step 2 is 50 ~ 400 rpm.
As preferably, in step 3, the pH value of phosphoric acid buffer is 6 ~ 8.
As preferably, step 1, vacuum tightness is-99.99 ~-98.90 KPa.
beneficial effect
1. method of the present invention use carrier multi-walled carbon nano-tubes low price, easily obtain.
2. method of the present invention uses Cement Composite Treated by Plasma carrier, removes the impurity of removing many walls nanotube surface, improves specific surface area and the surface free energy of carrier.
3. procedure of the present invention is simple and efficient, and running cost is lower, and does not produce any objectionable impurities, waste.
4. the immobilized enzyme particle diameter prepared of the present invention is less, and the length of gained immobilized lipase is 10 ~ 30 μm, and diameter is 20 ~ 40nm, can be further used for carrying out enzymic catalytic reaction in enzyme classes reactor.
Accompanying drawing explanation
Fig. 1 is that multi-walled carbon nano-tubes TEM schemes after Cement Composite Treated by Plasma;
Fig. 2 is that multi-walled carbon nano-tubes immobilized lipase TEM prepared by embodiment 2 schemes, and the length of immobilized lipase is 10 ~ 30 μm, and diameter is 20 ~ 40nm.
Embodiment
The following examples can make the present invention of those skilled in the art comprehend, but do not limit the present invention in any way.
embodiment 1
Cement Composite Treated by Plasma multi-walled carbon nano-tubes fixes porcine pancreatic lipase
Step 1, the multi-walled carbon nano-tubes being 20 by 0.1 g parietal layer is placed in low temperature vacuum plasma body equipment, regulates the pulse width of electric current by power, is set in 74.8 W, regulate plasma electrically interpolar spacing to be 4 cm, under vacuum tightness is for-99.99KPa, processes 10 min.
Step 2, transfers to the multi-walled carbon nano-tubes processed in 50 ml Erlenmeyer flasks, and to add 50 ml pH value be the phosphoric acid buffer of 7.0, ultrasonic 10 min.The porcine pancreatic lipase taking 0.15 g adds Erlenmeyer flask, puts into constant temperature magnetic force heating stirrer, and design temperature is 30 DEG C, and rotating speed 200 rpm, fixes 1 h, obtain mixed solution;
Step 3, washs mixed solution suction filtration with a large amount of phosphoric acid buffer, washes away the resolvase do not adsorbed, dried overnight under room temperature condition, obtain multi-walled carbon nano-tubes immobilized lipase.
Adopt volumetry to measure enzyme to live, the pH value adding the deionized water of 50 ml, 2 g nitrilotriacetic glyceride and 10 ml in the Erlenmeyer flask of 100 ml is the phosphoric acid buffer of 7.0, and magnetic stirring apparatus strong stirring 10 min becomes emulsion.By pH instrument, record is carried out to the pH value of emulsion, after the pH value of emulsion is stable, be recorded as initial pH value.Add the multi-walled carbon nano-tubes immobilized lipase of 0.1 g, envrionment temperature is 40 DEG C.The acetone termination reaction of 4.5 ml is added immediately after steady stirring reaction 30 min.Measure solution ph and be recorded as end of a period value, then slowly the titration of solution end of a period value is returned pH initial value, the amount of sodium hydroxide that record consumes with the sodium hydroxide solution of 0.02 mol/L.Enzyme activity unit 1U is defined as when envrionment temperature is 40 DEG C, and per minute catalysis nitrilotriacetic glyceride discharges the enzyme amount required for 1 μm of ol acetic acid.And utilize Bradford method to measure the protein concn of fixing front and back zymoprotein solution, calculate the charge capacity of zymoprotein by the difference of protein concn.
Multi-walled carbon nano-tubes is after Cement Composite Treated by Plasma, and material surface is corroded, and become coarse, specific surface area increases, for resolvase provides larger immobilization region.117 U/g with the enzyme work of the immobilized enzyme that is carrier of the multi-walled carbon nano-tubes after Cement Composite Treated by Plasma after measured, every gram of multi-walled carbon nano-tubes immobilized lipase zymoprotein charge capacity is 0.13g, than not improve 33% with the enzyme of the carrier immobilized enzyme of Cement Composite Treated by Plasma is alive, zymoprotein charge capacity improves 30%.
embodiment 2
Cement Composite Treated by Plasma multi-walled carbon nano-tubes fixed L ipozyme CALB L lipase
Step 1, the multi-walled carbon nano-tubes of 0.15 g is placed in low temperature vacuum plasma body equipment, regulates the pulse width of electric current by power setting at 74.8 W, the distance between adjustment plasma body two electrode, be 4 cm, process 4 min in vacuum tightness under-99.00KPa.
Step 2, puts into 50 ml Erlenmeyer flasks by the multi-walled carbon nano-tubes processed, adding 50 ml pH value is the phosphoric acid buffer of 7.0, ultrasonic 10 min.The Lipozyme CALB L lipase of 3 ml is added Erlenmeyer flask, and put into constant temperature magnetic force heating stirrer, design temperature is 30 DEG C, and rotating speed 250 rpm, after fixing 40 min, obtains mixed solution;
Step 3, washs mixed solution suction filtration with a large amount of phosphoric acid buffer, washes away the resolvase do not adsorbed, dried overnight under room temperature condition, obtain multi-walled carbon nano-tubes immobilized lipase.
Enzyme activity determination method and zymoprotein charge capacity measuring method are with embodiment 1.580 U/g with the enzyme work of the immobilized enzyme that is carrier of the multi-walled carbon nano-tubes after Cement Composite Treated by Plasma after measured, every gram of multi-walled carbon nano-tubes immobilized lipase zymoprotein charge capacity is 0.16g, than not improve 22% with the enzyme of the carrier immobilized enzyme of Cement Composite Treated by Plasma is alive, zymoprotein charge capacity improves 21%.
embodiment 3
Cement Composite Treated by Plasma multi-walled carbon nano-tubes fixed L ipozyme TL100 L lipase
As described in Example 2, difference adopts Lipozyme TL100 L lipase enzyme liquid as being fixed of resolvase.146 U/g with the enzyme work of the immobilized enzyme that is carrier of the multi-walled carbon nano-tubes after Cement Composite Treated by Plasma after measured, every gram of multi-walled carbon nano-tubes immobilized lipase zymoprotein charge capacity is 0.06g, than not improve 19% with the enzyme of the carrier immobilized enzyme of Cement Composite Treated by Plasma is alive, zymoprotein charge capacity improves 17%.
embodiment 4
Cement Composite Treated by Plasma multi-walled carbon nano-tubes fixes porcine pancreatic lipase
Step 1, the multi-walled carbon nano-tubes being 2 by 0.1 g parietal layer is placed in low temperature vacuum plasma body equipment, regulates the pulse width of electric current by power, is set in 74.8 W, regulate plasma electrically interpolar spacing to be 4 cm, under vacuum tightness is for-99.00KPa, processes 10 min.
Step 2, transfers to the multi-walled carbon nano-tubes processed in 50 ml Erlenmeyer flasks, and to add 50 ml pH value be the phosphoric acid buffer of 7.0, ultrasonic 10 min.The porcine pancreatic lipase taking 0.15 g adds Erlenmeyer flask, puts into constant temperature magnetic force heating stirrer, and design temperature is 30 DEG C, and rotating speed 50 rpm, fixes 1 h, obtain mixed solution;
Step 3, washs mixed solution suction filtration with a large amount of phosphoric acid buffer, washes away the resolvase do not adsorbed, dried overnight under room temperature condition, obtain multi-walled carbon nano-tubes immobilized lipase.
Enzyme activity determination method and zymoprotein charge capacity measuring method are with embodiment 1.320 U/g with the enzyme work of the immobilized enzyme that is carrier of the multi-walled carbon nano-tubes after Cement Composite Treated by Plasma after measured, every gram of multi-walled carbon nano-tubes immobilized lipase zymoprotein charge capacity is 0.28g, than not improve 15% with the enzyme of the carrier immobilized enzyme of Cement Composite Treated by Plasma is alive, zymoprotein charge capacity improves 18%.
embodiment 5
Cement Composite Treated by Plasma multi-walled carbon nano-tubes fixes porcine pancreatic lipase
Step 1, the multi-walled carbon nano-tubes being 30 by 0.1 g parietal layer is placed in low temperature vacuum plasma body equipment, regulates the pulse width of electric current by power, is set in 74.8 W, regulate plasma electrically interpolar spacing to be 4 cm, under vacuum tightness is for-98.90KPa, processes 10 min.
Step 2, transfers to the multi-walled carbon nano-tubes processed in 50 ml Erlenmeyer flasks, and to add 50 ml pH value be the phosphoric acid buffer of 7.0, ultrasonic 10 min.The porcine pancreatic lipase taking 0.15 g adds Erlenmeyer flask, puts into constant temperature magnetic force heating stirrer, and design temperature is 30 DEG C, and rotating speed 400 rpm, fixes 1 h, obtain mixed solution;
Step 3, washs mixed solution suction filtration with a large amount of phosphoric acid buffer, washes away the resolvase do not adsorbed, dried overnight under room temperature condition, obtain multi-walled carbon nano-tubes immobilized lipase.
Enzyme activity determination method and zymoprotein charge capacity measuring method are with embodiment 1.260 U/g with the enzyme work of the immobilized enzyme that is carrier of the multi-walled carbon nano-tubes after Cement Composite Treated by Plasma after measured, every gram of multi-walled carbon nano-tubes immobilized lipase zymoprotein charge capacity is 0.23g, than not improve 20% with the enzyme of the carrier immobilized enzyme of Cement Composite Treated by Plasma is alive, zymoprotein charge capacity improves 19%.
Can find out that the enzyme of immobilized enzyme prepared by the present invention is lived high by embodiment 1-5, zymoprotein charge capacity is large, vacuum tightness during Cement Composite Treated by Plasma and the performance of the stirring velocity of heating in water bath to immobilized enzyme all have a certain impact, and optimum vacuum tightness is-99.99KPa, and rotating speed is 200rpm.
Claims (8)
1. a method for Cement Composite Treated by Plasma multi-walled carbon nano-tubes immobilized enzyme, is characterized in that, comprises the following steps:
Step 1, Cement Composite Treated by Plasma multi-walled carbon nano-tubes
0.1 ~ 0.5 g multi-wall carbon nano-tube pipe powder is positioned on low temperature vacuum plasma body equipment Stage microscope, plasma electrically interpolar spacing is regulated to be 2 ~ 4 cm, regulate the pulse width of electric current to make power reach 22 ~ 84 W again, process 1 ~ 10 min under vacuum;
Step 2, multi-walled carbon nano-tubes and the physical adsorption of enzyme liquid are reacted
Be dissolved in phosphoric acid buffer by the multi-wall carbon nano-tube pipe powder after process, in ultrasonic cleaner, ultrasonic 10 ~ 30 min, then add resolvase, fix 30 ~ 240 min, obtain mixed solution in the water bath with thermostatic control of 20 ~ 40 DEG C;
Step 3, by mixed solution suction filtration and with phosphoric acid buffer washing, wash away the resolvase do not adsorbed, dried overnight under room temperature condition, obtain multi-walled carbon nano-tubes immobilized enzyme.
2. the method for Cement Composite Treated by Plasma multi-walled carbon nano-tubes immobilized enzyme according to claim 1, is characterized in that: step 1, and the number of plies of described multi-walled carbon nano-tubes is 2 ~ 30 layers.
3. the method for Cement Composite Treated by Plasma multi-walled carbon nano-tubes immobilized enzyme according to claim 1, is characterized in that: step 2, and described phosphoric acid buffer pH value is 6 ~ 8.
4. the method for Cement Composite Treated by Plasma multi-walled carbon nano-tubes immobilized enzyme according to claim 1, is characterized in that: step 2, and described resolvase is any one in lipase, proteolytic enzyme, amylase or glucose oxidase.
5. the method for Cement Composite Treated by Plasma multi-walled carbon nano-tubes immobilized enzyme according to claim 4, is characterized in that: described lipase is any one in lipase from candida sp, porcine pancreatic lipase, Candida rugosa lipase, Lipozyme CALB L lipase, Palatase 20000 L lipase or Lipozyme TL100 L lipase.
6. the method for Cement Composite Treated by Plasma multi-walled carbon nano-tubes immobilized enzyme according to claim 1, is characterized in that: the rotating speed stirred during water-bath in step 2 is 50 ~ 400 rpm.
7. the method for Cement Composite Treated by Plasma multi-walled carbon nano-tubes immobilized enzyme according to claim 1, is characterized in that: in step 3, the pH value of phosphoric acid buffer is 6 ~ 8.
8. the method for Cement Composite Treated by Plasma multi-walled carbon nano-tubes immobilized enzyme according to claim 1, is characterized in that: step 1, and vacuum tightness is-99.99 ~-98.90KPa.
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| CN109468185A (en) * | 2018-12-29 | 2019-03-15 | 盐城工学院 | A kind of preparation method of mulberries alcohol-free beer |
| CN112169757A (en) * | 2020-09-29 | 2021-01-05 | 上海交通大学 | Low-temperature plasma modified carbon nanotube and application thereof in water treatment |
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| CN102373192A (en) * | 2011-01-21 | 2012-03-14 | 华东理工大学 | Immobilization method of proteinase molecule by using nano-material and application thereof |
| CN104109662A (en) * | 2014-06-23 | 2014-10-22 | 华中科技大学 | Immobilized Burkholderia cepacia lipase and preparation method thereof |
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| CN109468185A (en) * | 2018-12-29 | 2019-03-15 | 盐城工学院 | A kind of preparation method of mulberries alcohol-free beer |
| CN112169757A (en) * | 2020-09-29 | 2021-01-05 | 上海交通大学 | Low-temperature plasma modified carbon nanotube and application thereof in water treatment |
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