CN105602930A - Reverse micelle emulsion used for biological enzyme immobilization and biological enzyme immobilization method thereof - Google Patents
Reverse micelle emulsion used for biological enzyme immobilization and biological enzyme immobilization method thereof Download PDFInfo
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- CN105602930A CN105602930A CN201610063882.5A CN201610063882A CN105602930A CN 105602930 A CN105602930 A CN 105602930A CN 201610063882 A CN201610063882 A CN 201610063882A CN 105602930 A CN105602930 A CN 105602930A
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- reverse micelle
- parts
- enzyme
- biology enzyme
- biological enzyme
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
- C12N11/02—Enzymes or microbial cells immobilised on or in an organic carrier
- C12N11/08—Enzymes or microbial cells immobilised on or in an organic carrier the carrier being a synthetic polymer
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
- C12N9/18—Carboxylic ester hydrolases (3.1.1)
- C12N9/20—Triglyceride splitting, e.g. by means of lipase
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y301/00—Hydrolases acting on ester bonds (3.1)
- C12Y301/01—Carboxylic ester hydrolases (3.1.1)
- C12Y301/01003—Triacylglycerol lipase (3.1.1.3)
Abstract
The invention discloses reverse micelle emulsion used for biological enzyme immobilization. The reverse micelle emulsion used for the biological enzyme immobilization comprises the following components in parts by weight: 5-20 parts of OP-10, 10-35 parts of n-octyl alcohol, 30-60 parts of isooctane, 0.1-1.0 part of Tris-HCl buffer solution and 0.1-8.0 parts of distilled water. The reverse micelle emulsion used for the biological enzyme immobilization has the advantages that a surfactant is dispersed into a continuous non-polar solvent, and a nanoscale stable aggregate is spontaneously formed; when the reverse micelle system is taken as a medium for carrying out biological enzyme immobilization, a biological enzyme orientationally exists at an oil-water interface of a reverse micelle, active groups of the biological enzyme are orientationally and regularly toward an organic solvent phase, and groups reacting with nano magnetic particles are regularly toward a water pool, so that loss of the active groups of the biological enzyme is greatly avoided, and the immobilized biological enzyme has efficient catalytic performance; meanwhile, after reaction is finished, the reverse micelle system can be recycled and repeatedly used, so that the reverse micelle emulsion used for the biological enzyme immobilization has a broad development prospect.
Description
Technical field
The present invention relates to the fixing technical field of biology enzyme, be specially a kind of biology enzyme fixing with reverse micelle emulsion and fixed biologically thereofThe method of enzyme.
Background technology
The characteristics such as catalytic activity is high although biology enzyme has, selectivity strong and selectively get well, it exists expensive, difficultReclaim and easily the drawbacks limit such as reunion its application. In order to overcome this series of shortcomings, biology enzyme is fixed on to carrier and receivesThe research of rice magnetic-particle is subject to scientific research personnel and more and more payes attention to, the method has that preparation technology is simple, low price andBe convenient to the advantages such as recovery, wherein carrier representative has nanometer Fe3O4Magnetic-particle etc. But conventional fixed form is normally in waterCarry out, this method makes biology enzyme and nano magnetic particle that the combination in any site occur, and causes the activity decreased of biology enzyme.
Summary of the invention
The technical problem to be solved in the present invention is to overcome in prior art, to use nanometer Fe3O4After magnetic-particle fixed biologically enzyme, causeThe technological deficiency that bioenzyme activity reduces; And then provide a kind of biology enzyme the fixing method with reverse micelle emulsion and fixed biologically enzyme thereof.
In order to solve the problems of the technologies described above, the invention provides following technical scheme:
A kind of biology enzyme is fixing uses reverse micelle emulsion, comprises each component of following parts by weight:
OP-10:5～20 part
N-octyl alcohol: 10～35 parts
Isooctane: 30～60 parts
Tris-HCl buffer solution: 0.1～1.0 part
Distilled water: 0.1～8.0 part.
Further, a kind of biology enzyme is fixing uses reverse micelle emulsion, comprises each component of following parts by weight:
OP-10:15 part
N-octyl alcohol: 21 parts
Isooctane: 48 parts
Tris-HCl buffer solution: 0.5 part
Distilled water: 3.5 parts.
Further, a kind of biology enzyme is fixing uses reverse micelle emulsion, comprises each component of following parts by weight:
OP-10:14 part
N-octyl alcohol: 22 parts
Isooctane: 45 parts
Tris-HCl buffer solution: 0.8 part
Distilled water: 3.2 parts.
Further, a kind of biology enzyme is fixing uses reverse micelle emulsion, comprises each component of following parts by weight:
OP-10:12 part
N-octyl alcohol: 20 parts
Isooctane: 46 parts
Tris-HCl buffer solution: 0.8 part
Distilled water: 3.2 parts.
By the fixing method with reverse micelle emulsion fixed biologically enzyme of biology enzyme, comprise following step:
1), by OP-10:5～20 weight portion, n-octyl alcohol: 10～35 weight portions, isooctane: 30～60 weight portions, Tris-HClBuffer solution: 0.1～1.0 weight portion, distilled water: 0.1～8.0 weight portion, mixing and stirring, obtains mixed emulsion;
2), to 1) mixed emulsion of gained carries out ultrasonic wave processing, forms reverse micelle system;
3), to 2) add in the reverse micelle system of gained and treat fixing biology enzyme 1～10 weight portion, nanometer Fe3O4Magnetic-particle5～20 weight portions, fully stir, and make nanometer Fe3O4Magnetic-particle is fixed biology enzyme;
4), with distilled water cleaning step 3) product of gained, obtain nanometer Fe3O4The biology enzyme that magnetic-particle is fixing.
Further, described step 3) in biology enzyme be lipase from candida sp.
Further, described step 2) in ultrasonic wave process time hyperacoustic intensity be 0.5～1.2KW/m3, supersonic frequencyBe 18～40KHz.
Further, described step 2) in time of ultrasonic wave processing be 0.5～2 hour.
Further, described step 3) in nanometer Fe3O4The particle diameter of magnetic-particle is 40～60nm.
Reverse micelle of the present invention is assembled stable a kind of nanoscale of surfactant-dispersed spontaneous formation in continuous non-polar solvenBody, the spherical polarity core wherein being formed by surfactant hydrophilic group is as " pond ", water that not only can solubilising someOr the aqueous solution, and can effectively limit existence and the interaction of molecule wherein or ion. Taking this reverse micelle system as JieMatter is carried out biology enzyme and is fixed, and makes biology enzyme directionally be present in the oil-water interfaces place of reverse micelle, the court of its active group rule orientedTo organic solvent mutually in, and with the reactive group rule of nano magnetic particle in pond, greatly avoided biology enzyme to liveProperty base loss, the biology enzyme after fixing has efficient catalytic performance. On the other hand, adopt reverse micelle system fixed biologically enzymeThe advantage such as simple, the environmental friendliness of method technique and security height, what is more important this method has been avoided bioenzyme activity siteMinimizing, make its catalytic activity more efficient, after simultaneous reactions completes, reverse micelle system can reclaim and reuse, therefore toolThere is very vast potential for future development.
Brief description of the drawings
Accompanying drawing is used to provide a further understanding of the present invention, and forms a part for description, with embodiments of the invention oneBe used from explanation the present invention, be not construed as limiting the invention. In the accompanying drawings:
Fig. 1 is the catalyze and synthesize rates of different immobilized enzymes to amyl valerate;
Fig. 2 is the repeat performance of reverse micelle immobilized enzyme in embodiment 3;
Fig. 3 is the magnetic effect before and after Fe3O4 magnetic-particle fixed biologically enzyme.
Detailed description of the invention
Below in conjunction with accompanying drawing, the preferred embodiments of the present invention are described, only should be appreciated that preferred embodiment described hereinFor description and interpretation the present invention, be not intended to limit the present invention.
Embodiment 1
A kind of biology enzyme is fixing uses reverse micelle emulsion, comprises each component of following parts by weight:
OP-10:15 part
N-octyl alcohol: 21 parts
Isooctane: 48 parts
Tris-HCl buffer solution: 0.5 part
Distilled water: 3.5 parts.
The method of the raw fixed biologically enzyme of this embodiment, comprises following step:
1), by OP-10:15 weight portion, n-octyl alcohol: 21 weight portions, isooctane: 48 weight portions, Tris-HCl buffer solution:0.5 weight portion, distilled water: 3.5 weight portions, mixing and stirring, obtains mixed emulsion;
2), to 1) mixed emulsion of gained carries out ultrasonic wave processing, forms reverse micelle system; Hyperacoustic intensity is 0.5KW/m3，Supersonic frequency is 18KHz, and the time of processing is 2 hours;
3), to 2) add lipase from candida sp 2 weight portions, nanometer Fe in the reverse micelle system of gained3O4Magnetic-particle 10Weight portion, fully stirs, and makes nanometer Fe3O4Magnetic-particle is fixed biology enzyme; Nanometer Fe3O4The particle diameter of magnetic-particle is 40nm;
4), with distilled water cleaning step 3) product of gained, obtain nanometer Fe3O4The biology enzyme that magnetic-particle is fixing.
Embodiment 2
A kind of biology enzyme is fixing uses reverse micelle emulsion, comprises each component of following parts by weight:
OP-10:14 part
N-octyl alcohol: 22 parts
Isooctane: 45 parts
Tris-HCl buffer solution: 0.8 part
Distilled water: 3.2 parts.
The method of the raw fixed biologically enzyme of this embodiment, comprises following step:
1), by OP-10:14 weight portion, n-octyl alcohol: 22 weight portions, isooctane: 45 weight portions, Tris-HCl buffer solution:0.8 weight portion, distilled water: 3.2 weight portions, mixing and stirring, obtains mixed emulsion;
2), to 1) mixed emulsion of gained carries out ultrasonic wave processing, forms reverse micelle system; Hyperacoustic intensity is 1.2KW/m3，Supersonic frequency is 40KHz, and the time of processing is 0.5 hour;
3), to 2) add lipase from candida sp 5 weight portions, nanometer Fe in the reverse micelle system of gained3O4Magnetic-particle 10Weight portion, fully stirs, and makes nanometer Fe3O4Magnetic-particle is fixed biology enzyme; Nanometer Fe3O4The particle diameter of magnetic-particle is 60nm;
4), with distilled water cleaning step 3) product of gained, obtain nanometer Fe3O4The biology enzyme that magnetic-particle is fixing.
Embodiment 3
A kind of biology enzyme is fixing uses reverse micelle emulsion, comprises each component of following parts by weight:
OP-10:12 part
N-octyl alcohol: 20 parts
Isooctane: 46 parts
Tris-HCl buffer solution: 0.8 part
Distilled water: 3.2 parts.
The method of the raw fixed biologically enzyme of this embodiment, comprises following step:
1), by OP-10:12 weight portion, n-octyl alcohol: 20 weight portions, isooctane: 46 weight portions, Tris-HCl buffer solution:0.8 weight portion, distilled water: 3.2 weight portions, mixing and stirring, obtains mixed emulsion;
2), to 1) mixed emulsion of gained carries out ultrasonic wave processing, forms reverse micelle system; Hyperacoustic intensity is 0.8KW/m3，Supersonic frequency is 30KHz, and the time of processing is 1 hour;
3), to 2) add lipase from candida sp 8 weight portions, nanometer Fe in the reverse micelle system of gained3O4Magnetic-particle 10Weight portion, fully stirs, and makes nanometer Fe3O4Magnetic-particle is fixed biology enzyme; Nanometer Fe3O4The particle diameter of magnetic-particle is 50nm;
4), with distilled water cleaning step 3) product of gained, obtain nanometer Fe3O4The biology enzyme that magnetic-particle is fixing.
Embodiment 4
A kind of biology enzyme is fixing uses reverse micelle emulsion, comprises each component of following parts by weight:
OP-10:5 part
N-octyl alcohol: 10 parts
Isooctane: 30 parts
Tris-HCl buffer solution: 0.1 part
Distilled water: 0.1 part.
The method of the raw fixed biologically enzyme of this embodiment, comprises following step:
1), by OP-10:5 weight portion, n-octyl alcohol: 10 weight portions, isooctane: 30 weight portions, Tris-HCl buffer solution:0.1 weight portion, distilled water: 0.1 weight portion, mixing and stirring, obtains mixed emulsion;
2), to 1) mixed emulsion of gained carries out ultrasonic wave processing, forms reverse micelle system; Hyperacoustic intensity is 0.8KW/m3，Supersonic frequency is 30KHz, and the time of processing is 1.2 hours;
3), to 2) add lipase from candida sp 1 weight portion, nanometer Fe in the reverse micelle system of gained3O4Magnetic-particle 5Weight portion, fully stirs, and makes nanometer Fe3O4Magnetic-particle is fixed biology enzyme; Nanometer Fe3O4The particle diameter of magnetic-particle is 50nm;
4), with distilled water cleaning step 3) product of gained, obtain nanometer Fe3O4The biology enzyme that magnetic-particle is fixing.
Embodiment 5
A kind of biology enzyme is fixing uses reverse micelle emulsion, comprises each component of following parts by weight:
OP-10:20 part
N-octyl alcohol: 35 parts
Isooctane: 60 parts
Tris-HCl buffer solution: 1.0 parts
Distilled water: 8.0 parts.
The method of the raw fixed biologically enzyme of this embodiment, comprises following step:
1), by OP-10:20 weight portion, n-octyl alcohol: 35 weight portions, isooctane: 60 weight portions, Tris-HCl buffer solution:1.0 weight portions, distilled water: 8.0 weight portions, mixing and stirring, obtains mixed emulsion;
2), to 1) mixed emulsion of gained carries out ultrasonic wave processing, forms reverse micelle system; Hyperacoustic intensity is 1KW/m3，Supersonic frequency is 35KHz, and the time of processing is 1.4 hours;
3), to 2) add lipase from candida sp 10 weight portions, nanometer Fe in the reverse micelle system of gained3O4Magnetic-particle 20Weight portion, fully stirs, and makes nanometer Fe3O4Magnetic-particle is fixed biology enzyme; Nanometer Fe3O4The particle diameter of magnetic-particle is 50nm;
4), with distilled water cleaning step 3) product of gained, obtain nanometer Fe3O4The biology enzyme that magnetic-particle is fixing.
The biology enzyme of embodiment of the present invention 1-3 is fixed by reverse micelle emulsion applications to catalyzing and synthesizing in amyl valerate. By immobilized enzymeBe placed in n-amyl alcohol, positive valeric acid and cyclohexane mixed solution, make it under the stirring condition of 37 DEG C, carry out synthetic reaction, measureThe synthetic ratio of amyl valerate under different time, and be the immobilized enzyme of reaction medium and free enzyme with making under the same conditions waterActivity contrasts, and as shown in Figure 1, free enzyme < of time embodiment 3 < embodiment 2 < that reach 100% synthetic ratio are real for resultExecute the immobilised enzymes that the conventional water of example 1 < is medium, and the time that the conventional water immobilised enzymes that is medium reaches 100% synthetic ratio is 35hLeft and right, and the application's embodiment 3 only needs 16h left and right, is in close proximity to free enzyme, the immobilization of visible gained of the present inventionThe catalytic activity of enzyme is more efficient.
Fig. 2 has shown the repeat performance of reverse micelle immobilized enzyme in embodiment 3, and visible immobilised enzymes of the present invention can repeatUse, the stability of enzyme is high, reuses after 5 times, and it catalyzes and synthesizes rate still more than 85%.
Fig. 3 can find out, Fe3O4Heat treatment before and after magnetic-particle fixed biologically enzyme is little, and this illustrates Fe3O4Magnetic-particle warpAfter crossing reverse micelle fixed biologically enzyme, still can keep higher magnetic, be conducive to recovery and reuse.
Finally it should be noted that: the foregoing is only the preferred embodiments of the present invention, be not limited to the present invention, althoughWith reference to previous embodiment, the present invention is had been described in detail, for a person skilled in the art, it still can be to frontState the technical scheme that each embodiment records and modify, or part technical characterictic is wherein equal to replacement. All at thisWithin bright spirit and principle, any amendment of doing, be equal to replacement, improvement etc., all should be included in protection scope of the present inventionWithin.
Claims (9)
1. biology enzyme is fixing uses a reverse micelle emulsion, it is characterized in that, comprises each component of following parts by weight:
OP-10:5～20 part
N-octyl alcohol: 10～35 parts
Isooctane: 30～60 parts
Tris-HCl buffer solution: 0.1～1.0 part
Distilled water: 0.1～8.0 part.
2. biology enzyme as claimed in claim 1 is fixing uses reverse micelle emulsion, it is characterized in that, comprises the each of following parts by weightComponent:
OP-10:15 part
N-octyl alcohol: 21 parts
Isooctane: 48 parts
Tris-HCl buffer solution: 0.5 part
Distilled water: 3.5 parts.
3. biology enzyme as claimed in claim 1 is fixing uses reverse micelle emulsion, it is characterized in that, comprises the each of following parts by weightComponent:
OP-10:14 part
N-octyl alcohol: 22 parts
Isooctane: 45 parts
Tris-HCl buffer solution: 0.8 part
Distilled water: 3.2 parts.
4. biology enzyme as claimed in claim 1 is fixing uses reverse micelle emulsion, it is characterized in that, comprises the each of following parts by weightComponent:
OP-10:12 part
N-octyl alcohol: 20 parts
Isooctane: 46 parts
Tris-HCl buffer solution: 0.8 part
Distilled water: 3.2 parts.
5. by the fixing method with reverse micelle emulsion fixed biologically enzyme of biology enzyme as claimed in claim 1, it is characterized in that,Comprise following step:
1), by OP-10:5～20 weight portion, n-octyl alcohol: 10～35 weight portions, isooctane: 30～60 weight portions, Tris-HClBuffer solution: 0.1～1.0 weight portion, distilled water: 0.1～8.0 weight portion, mixing and stirring, obtains mixed emulsion;
2), to 1) mixed emulsion of gained carries out ultrasonic wave processing, forms reverse micelle system;
3), to 2) add in the reverse micelle system of gained and treat fixing biology enzyme 1～10 weight portion, nanometer Fe3O4Magnetic-particle5～20 weight portions, fully stir, and make nanometer Fe3O4Magnetic-particle is fixed biology enzyme;
4), with distilled water cleaning step 3) product of gained, obtain nanometer Fe3O4The biology enzyme that magnetic-particle is fixing.
6. as claimed in claim 5 by the fixing method with reverse micelle emulsion fixed biologically enzyme of biology enzyme, it is characterized in that instituteThe step 3 of stating) in biology enzyme be lipase from candida sp.
7. as claimed in claim 5 by the fixing method with reverse micelle emulsion fixed biologically enzyme of biology enzyme, it is characterized in that instituteThe step 2 of stating) in ultrasonic wave process time hyperacoustic intensity be 0.5～1.2KW/m3, supersonic frequency is 18～40KHz.
8. as claimed in claim 7 by the fixing method with reverse micelle emulsion fixed biologically enzyme of biology enzyme, it is characterized in that instituteThe step 2 of stating) in time of ultrasonic wave processing be 0.5～2 hour.
9. as claimed in claim 5 by the fixing method with reverse micelle emulsion fixed biologically enzyme of biology enzyme, it is characterized in that instituteThe step 3 of stating) in nanometer Fe3O4The particle diameter of magnetic-particle is 40～60nm.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108686575A (en) * | 2018-05-15 | 2018-10-23 | 江南大学 | A kind of compound emulsifying agent with magnetic responsiveness energy |
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| CN103203192A (en) * | 2013-02-05 | 2013-07-17 | 东华大学 | Nonionic reverse micelle system and method for modifying polar material by using same |
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| CN108686575A (en) * | 2018-05-15 | 2018-10-23 | 江南大学 | A kind of compound emulsifying agent with magnetic responsiveness energy |
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Application publication date: 20160525 |