CN106753943A - A kind of enzyme immobilization method and its application - Google Patents
A kind of enzyme immobilization method and its application Download PDFInfo
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- CN106753943A CN106753943A CN201611031203.2A CN201611031203A CN106753943A CN 106753943 A CN106753943 A CN 106753943A CN 201611031203 A CN201611031203 A CN 201611031203A CN 106753943 A CN106753943 A CN 106753943A
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- enzyme
- ice pellets
- polymeric membrane
- solvent
- ice
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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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12C—BEER; PREPARATION OF BEER BY FERMENTATION; PREPARATION OF MALT FOR MAKING BEER; PREPARATION OF HOPS FOR MAKING BEER
- C12C7/00—Preparation of wort
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- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Biochemistry (AREA)
- Food Science & Technology (AREA)
- Biomedical Technology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
The present invention proposes a kind of enzyme immobilization method and its application, belongs to biological chemical field, can not influence the activity of enzyme while the excellent carrier with organic solvent as solvent and enzyme immobilizatio are combined together.The enzyme immobilization method comprises the following steps：Buffer solution containing enzyme is made ice pellets；Ice pellets is immersed in the macromolecule casting solution using organic solvent as solvent, macromolecule casting solution is immersed in ice pellets surface；Take out ice pellets, in placing it in Cryo Equipment, with the evaporation of solvent in macromolecule casting solution, by way of directly being reacted in ice pellets surface formation polymeric membrane or with the ice surface progressively dissolved and to form polymeric membrane, the enzyme product coated by polymeric membrane is obtained.The present invention can be applied in beer production.
Description
Technical field
The invention belongs to biological chemical field, more particularly to a kind of enzyme immobilization method and its application.
Background technology
Enzyme preparation has obtained widely available as one of the outstanding achievement of bio-engineering research in every profession and trade.Such as Beer Brewage
In industry, the deficiency of enzyme activity in malt can be made up by using enzyme preparation, improve wort components, improve beer fermentation degree, contracting
Short fermentation period, increases yield etc..Enzyme preparation is usually used in liquid form, but is difficult to point once adding under this form
From and recycle.Because enzyme preparation price is typically higher, it is unfavorable to cost that single is utilized, and these limiting factors result in it should
With being under some influence.
It is to improve one of above-mentioned enzyme preparation problem effective means by enzyme immobilization.Immobilised enzymes keep its efficiently it is single-minded and
While gentle enzymic catalytic reaction characteristic, the above-mentioned weak point of resolvase is overcome, be presented that bin stability is high, separate back
House easily, can repeatedly use, operate the series of advantages such as continuously controllable, technique is easy, therefore enzyme immobilizatio to be ground
The person's of studying carefully pay attention to day by day, the problem that people exist according to traditional enzyme immobilization technology is constantly explored and improves, improves and develop new enzyme
Process for fixation.
Due to enzyme characteristic in itself, influence of the external environment to its activity is very big, and every kind of enzyme all has oneself in itself
Distinctive optimal pH, temperature etc., therefore under the conditions of discomfort, the activity of enzyme will reduce even irreversible inactivation, for example
Substantial amounts of organic solvent, such as common alcohols, aldehydes can inactivate enzyme denaturation.With the development of macromolecular material, more
The material for carrying out more excellent performances continuously emerges, but most materials are required for participation or the hot conditions of organic solvent
It is lower to dissolve one-step shaping of going forward side by side.The extremely strong polytetrafluoroethylene (PTFE) of such as resistance to acids and bases and hydrophobicity can only be by high-temperature fusion
Mode process, and the dissolving of Kynoar is accomplished by highly polar organic solvent, such as DMF, N- methyl
Pyrrolidones and triethyl phosphate etc..These conditions limit polymer shaping process enzyme and cannot directly be immobilized onto it
In.In a word, although current people can prepare the excellent carrier of Various Functions, often related in these carrier preparation process
The use of organic solvent or hot conditions, cause it to be difficult to be used as enzyme immobilizatio carrier.
The content of the invention
The invention provides a kind of enzyme immobilization method and its application, can be by the excellent load with organic solvent as solvent
While body is combined together with enzyme immobilizatio, the activity of enzyme is not influenceed.
An aspect of of the present present invention provides a kind of enzyme immobilization method, comprises the following steps：
Buffer solution containing enzyme is made ice pellets；
Ice pellets is immersed in the macromolecule casting solution using organic solvent as solvent, macromolecule casting solution is immersed in ice
Grain surface；
Ice pellets is taken out, in placing it in Cryo Equipment, with the evaporation of solvent in macromolecule casting solution, by directly existing
Ice pellets surface forms polymeric membrane or the mode to form polymeric membrane is reacted with the ice surface progressively dissolved, and obtains by macromolecule
The enzyme product of film cladding.
As optional technical scheme, the buffer solution containing enzyme is added in calcium chloride solution and reacts formation with preliminary solid
Surely change the solution of the nanometer flower structure of enzyme, the solution is made ice pellets.
It is also residual on polymeric membrane including washing away directly after ice pellets surface forms polymeric membrane as optional technical scheme
The solvent for staying, and the step of rise high-temperature and dissolve ice pellets.
As optional technical scheme, reacted with the ice surface progressively dissolved after forming polymeric membrane, also including washing away high score
The step of solvent remained on sub- film.
Used as optimal technical scheme, the macromolecule in described macromolecule casting solution can react in zero degree and subzero
Shaping.
Used as optimal technical scheme, described enzyme irreversible will not be inactivated in zero degree or subzero, be completed in immobilization
After, enzyme activity answers reversible recovery.
Another aspect provides a kind of enzyme immobilization method as described in above-mentioned any one technical scheme in beer
Application in wine production process.
The invention provides a kind of enzyme immobilization method, the excellent carrier with organic solvent as solvent is utilized in the method
To being fixed of enzyme, due to the use of organic solvent so that the material of excellent performance can participate in the fixation of enzyme as carrier
During change, so as to realize the excellent carrier of Various Functions to enzyme immobilizatio while enzymatic activity is not influenceed.
Specific embodiment
The technical scheme in the embodiment of the present invention will be clearly and completely described below, it is clear that described implementation
Example is only a part of embodiment of the invention, rather than whole embodiments.Based on the embodiment in the present invention, this area is common
The every other embodiment that technical staff is obtained under the premise of creative work is not made, belongs to the model of present invention protection
Enclose.
The embodiment of one aspect of the present invention provides a kind of enzyme immobilization method, comprises the following steps：
S1：Buffer solution containing enzyme is made ice pellets.
In this step, the buffer solution containing enzyme can be directly prepared into ice pellets.Wherein, it is used for when buffer solution can be purchase
Preserve enzyme and the buffer solution of balanced action is played to enzyme.For in specific this step of species of buffer solution and being not specifically limited.
In addition, for the buffer solution containing enzyme to be made the concrete mode of ice pellets also and is not specifically limited, as long as the mesh can be realized
.Wherein, made ice pellets can be in ice pellets shape, it is also possible in other arbitrary shapes, but should have certain surface
Product, is beneficial to follow-up in its surface impregnation macromolecule casting solution.
S2：Ice pellets is immersed in the macromolecule casting solution using organic solvent as solvent, macromolecule casting solution is impregnated
On ice pellets surface.
In this step, primarily to the macromolecule casting solution using organic solvent as solvent is immersed in into ice pellets surface
On, to realize cladding of the polymeric membrane for ice pellets.It should be noted that, due to be on ice pellets surface to be coated on, therefore, now
The temperature of system is relatively low, and the state of ice should be kept not change.It is understood that in the realization of this method, this step
In macromolecule casting solution in also may not include organic solvent.
S3：Ice pellets is taken out, in placing it in Cryo Equipment, with the evaporation of middle solvent, by directly on ice pellets surface
Form polymeric membrane or the mode to form polymeric membrane is reacted with the ice surface progressively dissolved, obtain what is coated by polymeric membrane
Enzyme product.
In this step, will be impregnated with after the ice pellets of macromolecule casting solution takes out, in order to continue to keep the state of ice, can be by
It is placed in Cryo Equipment such as refrigerator.It is molten in macromolecule casting solution with the change of its interior air pressure in Cryo Equipment
Agent slowly start evaporation, so in one case, polymeric membrane can progressively forming covered on ice pellets surface, in another situation
Under, it is dissolved as under the critical condition of water in ice, ice can progressively dissolve, and with the slow mechanism dissolved of ice, macromolecule casting solution can
Ice with dissolving reacts, so as to form polymeric membrane.Based on above-mentioned two situations, finally can obtain by polymeric membrane bag
The enzyme product for covering.
In an alternative embodiment, the buffer solution containing enzyme is added in calcium chloride solution and reacts formation with preliminary solid
Surely change the solution of the nanometer flower structure of enzyme, the solution is made ice pellets.In the embodiment, the buffer solution containing enzyme can also be entered
The treatment of one step, makes it be made ice pellets based on the solution of the nanometer flower structure with preliminary immobilised enzymes.
It is also residual on polymeric membrane including washing away directly after ice pellets surface forms polymeric membrane in an alternative embodiment
The solvent for staying, and the step of rise high-temperature and dissolve ice pellets.In another alternative embodiment, reacted with the ice surface progressively dissolved
Formed polymeric membrane after, also including wash away on polymeric membrane remain solvent the step of.In the above-described embodiments, due to ice pellets table
Also remaining has fraction of solvent in the polymeric membrane in face, can produce a little influence to enzyme immobilizatio, therefore need to be gone out, with
Ensure the premium properties of final immobilization product.
In a preferred embodiment, the macromolecule in described macromolecule casting solution can react in zero degree and subzero
Shaping.In another preferred embodiment, described enzyme irreversible will not be inactivated in zero degree or subzero, immobilization complete with
Afterwards, enzyme activity answers reversible recovery.In the above-described embodiments, the enzyme used in it and carrier macromolecule are defined,
Specifically, the carrier being applicable need to can react in zero degree and subzero, and itself react at low temperature it is extremely slow or
Nonreactive carrier is not particularly suited in the various embodiments described above, in addition, above-described embodiment is that enzyme is processed at low temperature, with
Ensure its non-inactivation, and enzyme activity can be recovered after the completion of immobilization.Therefore, do not applied to without the specific enzyme of the property above-mentioned
Method.It is understood that the carrier and enzyme that meet above-mentioned condition are alternative extensively, those skilled in the art can be according to reality
Situation carries out reasonability must be selected.
The embodiment of another aspect of the present invention provides a kind of enzyme immobilization method as described in above-mentioned any one embodiment
Application in beer production.The method that the present embodiment is provided has universality, is particularly suited for beer, beverage/food
Deng production process in.
In order to become apparent from introducing enzyme immobilization method and its application that the embodiment of the present invention is provided in detail, below will knot
Specific embodiment is closed to be described.
Embodiment 1
Buffer solution containing certain enzyme is directly prepared into ice pellets shape or other arbitrary shapes, ice pellets is immersed into certain macromolecule
In casting solution, make its floor height molecule casting solution of surface impregnation one, ice pellets is taken out, be put into refrigerator or other Cryo Equipments, with
The evaporation of solvent in macromolecule casting solution, polymeric membrane is formed on ice pellets surface.After end to be formed, wash away on polymeric membrane
The solvent of residual, then rising high-temperature dissolves ice pellets, obtains by the enzyme product of polymeric PTC materials.
Embodiment 2
Reaction in calcium chloride solution will be added in cushioning liquid containing alpha -acetolactate decarboxylase obtains being formed and carry
The nanometer flower structure of preliminary immobilised enzymes, ice pellets or other shapes are made by the solution, and ice pellets then is immersed into Kynoar
In macromolecule casting solution, temperature is lifted with given pace, treat that ice surface is progressively dissolved and reacted with macromolecule casting solution, in ice
Grain surface forms polymeric membrane.After membrane forming polymer terminates, the solvent remained on polymeric membrane is washed away, obtained by macromolecule
The enzyme product of cladding.
Claims (7)
1. a kind of enzyme immobilization method, it is characterised in that comprise the following steps：
Buffer solution containing enzyme is made ice pellets；
Ice pellets is immersed in the macromolecule casting solution using organic solvent as solvent, macromolecule casting solution is immersed in ice pellets table
Face；
Ice pellets is taken out, in placing it in Cryo Equipment, with the evaporation of solvent in macromolecule casting solution, by directly in ice pellets
Surface forms polymeric membrane or the mode to form polymeric membrane is reacted with the ice surface progressively dissolved, and obtains by polymeric membrane bag
The enzyme product for covering.
2. enzyme immobilization method according to claim 1, it is characterised in that the buffer solution containing enzyme is added to calcium chloride
The solution for forming the nanometer flower structure with preliminary immobilised enzymes is reacted in solution, the solution is made ice pellets.
3. enzyme immobilization method according to claim 1, it is characterised in that directly form polymeric membrane on ice pellets surface
Afterwards, also including washing away the solvent remained on polymeric membrane, and rising high-temperature the step of dissolving ice pellets.
4. enzyme immobilization method according to claim 1, it is characterised in that react to form height with the ice surface progressively dissolved
After molecular film, also including wash away on polymeric membrane remain solvent the step of.
5. the enzyme immobilization method according to claim any one of 1-4, it is characterised in that in described macromolecule casting solution
Macromolecule can react shaping in zero degree and subzero.
6. the enzyme immobilization method according to claim any one of 1-4, it is characterised in that described enzyme is in zero degree or zero degree
Irreversible will not below inactivate, after immobilization completion, enzyme activity answers reversible recovery.
7. application of a kind of enzyme immobilization method as described in claim any one of 1-6 in beer production.
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| Application Number | Priority Date | Filing Date | Title |
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| CN201611031203.2A CN106753943A (en) | 2016-11-18 | 2016-11-18 | A kind of enzyme immobilization method and its application |
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| Application Number | Priority Date | Filing Date | Title |
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| CN201611031203.2A CN106753943A (en) | 2016-11-18 | 2016-11-18 | A kind of enzyme immobilization method and its application |
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4276381A (en) * | 1979-04-09 | 1981-06-30 | Mitsubishi Rayon Company, Ltd. | Preparation of immobilized enzymes of microorganisms |
| CN102517277A (en) * | 2012-01-12 | 2012-06-27 | 南京林业大学 | Enzyme immobilization technology by film forming through carrier surface phase transformation method |
| CN104877980A (en) * | 2015-05-14 | 2015-09-02 | 江南大学 | Method for immobilizing protease capable of degrading soyabean protein allergen |
| CN105950604A (en) * | 2016-06-03 | 2016-09-21 | 中国科学院青岛生物能源与过程研究所 | Enzyme immobilization method |
-
2016
- 2016-11-18 CN CN201611031203.2A patent/CN106753943A/en active Pending
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4276381A (en) * | 1979-04-09 | 1981-06-30 | Mitsubishi Rayon Company, Ltd. | Preparation of immobilized enzymes of microorganisms |
| CN102517277A (en) * | 2012-01-12 | 2012-06-27 | 南京林业大学 | Enzyme immobilization technology by film forming through carrier surface phase transformation method |
| CN104877980A (en) * | 2015-05-14 | 2015-09-02 | 江南大学 | Method for immobilizing protease capable of degrading soyabean protein allergen |
| CN105950604A (en) * | 2016-06-03 | 2016-09-21 | 中国科学院青岛生物能源与过程研究所 | Enzyme immobilization method |
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Application publication date: 20170531 |