CN109251924A - A kind of preparation method of dynamic key polymer and its application in terms of improving enzyme heat stability - Google Patents
A kind of preparation method of dynamic key polymer and its application in terms of improving enzyme heat stability Download PDFInfo
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- CN109251924A CN109251924A CN201811157297.7A CN201811157297A CN109251924A CN 109251924 A CN109251924 A CN 109251924A CN 201811157297 A CN201811157297 A CN 201811157297A CN 109251924 A CN109251924 A CN 109251924A
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- C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
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- C08G65/00—Macromolecular compounds obtained by reactions forming an ether link in the main chain of the macromolecule
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Abstract
The preparation method and its application in terms of improving enzyme heat stability that the present invention provides a kind of dynamic key polymer, belong to polymeric material field.The dynamic key polymer is generated reactive polymeric through imines and is formed by the amine of aromatic aldehyde, amino-terminated polymer, and the group containing different function.The dynamic key polymer improves there are two types of the modes of enzyme heat stability: 1) dynamic key polymer being directly added into the catalyst system of enzyme, when reaction condition rises to the temperature that enzyme can originally inactivated, due to the presence of dynamic key polymer, the activity of enzyme is maintained；2) dynamic key polymer is added in the catalyst system for the enzyme that activity significantly lowers due to high temperature, dynamic key polymer can restore and improve the activity of enzyme after a period of time.
Description
Technical field
The invention belongs to polymeric material fields, are related to Enzyme catalyzed synthesis, are related specifically to a kind of dynamic key polymer
Preparation method and its application in terms of improving enzyme heat stability.
Background technique
Mild, processing efficient, selectivity are high because of catalytic reaction condition for biological enzyme, in the side such as organic synthesis, pharmacy, food
Face is widely used.However the catalysis of enzyme reaction often has its most suitable temperature and acid-base condition, in the high temperature for exceeding this range
In environment, the activity of enzyme is substantially reduced, or even causes the irreversible inactivation of zymoprotein, and the industry that high degree affects enzyme is answered
With.Therefore, how to improve the thermal stability, raising catalytic efficiency and enzyme utilization rate of enzyme is the major issue in biocatalysis.
In recent years, a variety of methods be used to improve the thermal stability of enzyme, specifically include that 1) protein engineering, such as orient into
The genes means such as change.However, the program generally requires longer screening period and a large amount of human and material resources；2) enzyme immobilization, packet
Including will be wrapped up or is connected on carrier using the methods of absorption, crosslinking, but the activity of immobilised enzymes usually decreases；3) it adds
Stabilizer, such as the conformational stability of enzyme is influenced by the way that surfactant, polyalcohol etc. is added, but it is imitated under severe conditions
Fruit is relatively limited.
Dynamic key polymer is the polymer by supermolecular mechanism or reversible covalent reaction forming, and being that one kind is novel is suitable for
The high molecular material of property.Its maximum feature is that polymer architecture composition can be regulated and controled by environmental stimuli, such as with the presence of biological enzyme
When, which can form and the optimal structure of enzyme surface action around enzyme.It has been reported that will with functional group,
The dynamic key polymer of imines connection is directly added into enzymic catalytic reaction system, and the activity of enzyme is been significantly enhanced
(Chem.Commun.2016,52,4053,Chem.Commun.2016,52,13768).The quasi polymer is improving enzyme thermostabilization
Property aspect should also have potential application, but effective exploitation is not yet received.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of dynamic key polymer and its in terms of improving enzyme heat stability
Application, the dynamic key polymer by aromatic aldehyde, amino-terminated polymer, and the group containing different function amine, through imines
Reactive polymeric is generated to form.The dynamic key polymer improves there are two types of the modes of enzyme heat stability: 1) dynamic key polymer is straight
It connects and is added in the catalyst system of enzyme, when reaction condition rises to the temperature that enzyme can originally inactivated, due to dynamic key polymer
In the presence of the activity of enzyme is maintained；2) dynamic key polymer is added to the catalyst system for the enzyme that activity significantly lowers due to high temperature
In, dynamic key polymer can restore and improve the activity of enzyme after a period of time.
Technical solution of the present invention:
A kind of preparation method of dynamic key polymer, steps are as follows:
1) aromatic aldehyde, amino-terminated polymer and amine containing functional group are mixed in organic solvent, 60~
80 DEG C are flowed back 3~4 days, are carried out imines and are generated reaction；Wherein, the ratio between aromatic aldehyde and the amino-terminated mole of polymer are 1:
2~2:1；Mass ratio 2:1~10:1 of aromatic aldehyde and the amine containing functional group；
2) solvent after reacting in system is generated with vacuum distillation removal imines, and distilled water is added, and concentration is prepared
For the dynamic key polymer solution of 10~20mM.
The aromatic aldehyde is equal benzene trioxin, o-phthalaldehyde, terephthalaldehyde or m-terephthal aldehyde；
The amino-terminated polymer be amino-terminated polyethylene glycol, polyetheramine, polytetrahydrofuran, chitosan or
Amido modified hyaluronic acid.
The amino-terminated polymer is bis- (3- aminopropyl) polyethylene glycol, polyetheramine D series, both-end (3- ammonia
Propyl) polytetrahydrofuran, the long chain alkane of double-end amino sealing end, four arms-polyethylene glycol-amino, polyetheramine T series, polyetheramine
Hyaluronic acid after ED series, O-CMC or amide hydrolysis, concrete structure formula are respectively (A)~(I):
The amine containing functional group be PAMAM dendritic, or comprising 1~3 primary amine, be embedded with 0-5 phase
With or different alkoxies, amide groups, hydroxy methylene or secondary amine functional group hydrocarbon chain, the carbon number of hydrocarbon chain is 2-12；Knot
Structure formula is followed successively by (a), (b), (c) and (d):
Wherein, X be carbon number 2-12, embedded with 0-5 identical or different alkoxies, amide groups, hydroxy methylene or secondary
The hydrocarbon chain of functional group including amido.It is a kind of using the above method preparation dynamic key polymer improve enzyme heat stability side
The application in face, there are two types of application modes:
Mode one, according to different target enzyme viability, it is anti-that target enzymatic is added in dynamic key polymer solution obtained
It answers in system, steps up reaction temperature.By adjusting the additive amount of different temperatures and Different Dynamic key polymer solution, and with
Plus in dynamic key polymeric system the activity of target enzyme does not compare, and considers dynamic key polymer to the most suitable of target enzyme activation
The amount of sharing and most suitable hyperthermia temperature range.
Mode two, the temperature for gradually rising target enzyme catalyst system, until the activity of target enzyme significantly reduces.In room temperature condition
Under, dynamic key polymer solution obtained is added in target enzyme catalystic converter system, re-measures mesh after standing 10~60min
The activity for marking enzyme obtains the most suitable dosage of dynamic key polymer by comparing the additive amount of Different Dynamic key polymer solution.
Beneficial effects of the present invention: the preparation process of dynamic key polymer is simple, bright to the thermal stability reinforcing effect of enzyme
It is aobvious.The means such as opposite other genetic modifications, enzyme immobilization, this method is simple and easy, the period that takes effect is short, and has centainly more
The general applicability of kind enzyme, and it is not limited to the thermal stability and activity of a certain enzyme.
Detailed description of the invention
Fig. 1 is lipase at different conditions to the hydrolysis effect figure of p-nitrophenol.
Fig. 2 is carbonic anhydrase at different conditions to the hydrolysis effect figure of p-nitrophenol.
Specific embodiment
Technical solution of the present invention is further detailed below in conjunction with specific embodiments and the drawings.
Embodiment 1: a method of fatty enzyme heat stability is enhanced based on hydrophobicity dynamic key polymer, including as follows
Step:
1) 16.2mg (0.1mmol) benzene trioxin, 112.5mg (0.075mmol) polypropylene glycol (mw are weighed
=1500) and 15.0mg (0.075mmol) 1,12- diamino dodecane, the round bottom for sequentially adding the methanol solution containing 20mL are burnt
It in bottle, is placed in 60 DEG C and flows back 3 days, carry out imines and generate reaction.
2) reaction terminates, and removes all solvents with distillation under vacuum, and 5mL distilled water is added, and obtaining concentration is 20mM's
Dynamic key polymer solution is stand-by.
3) 10mg lipase (deriving from pig pancreas, EC 3.1.1.3) is dissolved in 1mL distilled water, parallel three parts of preparation.One
Part is stored at room temperature 10min, and portion places 10min at 80 DEG C, and dynamic key polymer obtained by 50 μ L steps 2) is added in another, and
10min is placed at 80 DEG C.
4) ultraviolet spectrophotometer method is utilized, the lipase that measurement high-temperature process is crossed is in room temperature to substrate p-nitrophenol
Hydrolysis rate.Substrate is dissolved in obtained by 4mL acetonitrile by 2.5mg p-nitrophenol.It is successively that 20 μ L lipase are molten when specific detection
Liquid, 1.78mL (first and second part of lipase solution) or 1.73mL (third part lipase solution) phosphate buffered saline solution (pH
7.0) and cuvette is added in 200 μ L p-nitrophenyl phenol solutions, and preceding 10min mixed solution is continuously detected at interval of 0.5min
In the UV absorption of 400nm, influence of the dynamic key polymer to enzymatic activity is observed.
After Fig. 1 is lipase itself, 80 DEG C of high temperatures, and hydrophobicity dynamic key polymer is added and 80 DEG C of high temperature are broken
Active situation after bad.By 10min before ultraviolet spectrophotometry real-time monitoring to the hydrolysis situation of p-nitrophenol, as a result table
Bright, the lipase active after hydrophobicity dynamic key polymer and 80 DEG C of high temperatures are added is not compared with by the original fat of high temperature
The activity of enzyme is taller, illustrates that the dynamic key polymer of this method preparation is able to maintain and improves the activity of enzyme.
Embodiment 2: a method of the dynamic key polymer based on multiamide key restores carbonic anhydrase thermal stability, including
Following steps:
1) 16.2mg (0.1mmol) benzene trioxin, 112.5mg (0.075mmol) polypropylene glycol (mw are weighed
=1500) it and 109.3mg (0.015mmol) PAMAM dendritic (in 1.0 generations, 20wt.% is in methanol), sequentially adds
It in the round-bottomed flask of the methanol solution containing 20mL, is placed in 60 DEG C and flows back 3 days, carry out imines and generate reaction.
2) reaction terminates, and removes all solvents with distillation under vacuum, and 5mL distilled water is added, and obtaining concentration is 20mM's
Dynamic key polymer solution is stand-by.
3) 3.0mg carbonic anhydrase (deriving from bovine red blood cells, EC 4.2.1.1) is dissolved in 1mL distilled water, utilizes ultraviolet point
Light photometer measuring method measures activity index of the carbonic anhydrase in room temperature to the hydrolysis rate of p-nitrophenol, as enzyme.Hereafter, will
The carbonic anhydrase aqueous solution places 10min at 80 DEG C, and with same method measurement enzyme to the hydrolysis rate of p-nitrophenol, hair
Existing activity is substantially reduced.
4) dynamic key polymer obtained by 50 μ L steps 2) is added in the carbonic anhydrase aqueous solution that activity is substantially reduced, room temperature
Carbonic anhydrase is measured respectively to the hydrolysis rate of p-nitrophenol after standing 0min, 20min, 40min, 60min, finds 20min
Afterwards before its activation recovering to high temperature the case where, and with the extension of time, activity further increases.
Attached drawing 2 is the carbonic anhydrase that inactivates by high temperature after the dynamic key polymer that the key containing multiamide is added, activity
The case where being restored at any time records its hydrolysis situation to p-nitrophenol by ultraviolet spectrophotometry, the results show that
After standing 20min, the activation recovering of carbonic anhydrase is to the activity level of original carbonic anhydrase, and activity continues to improve later, until
When 60min, the hydrolysis rate to p-nitrophenol is active 3 times of original carbonic anhydrase；Illustrate the dynamic of this method preparation
Key polymer can restore and increase substantially the activity of enzyme.
Claims (9)
1. a kind of application of dynamic key polymer in terms of improving enzyme heat stability, which is characterized in that application method is as follows:
Firstly, dynamic key polymer solution is added in target enzyme catalystic converter system, gradually according to different target enzyme viability
Improve reaction temperature；Then, by adjusting the additive amount of different temperatures and Different Dynamic key polymer solution, and with not plus dynamic
The activity of target enzyme compares in key polymeric system, consider dynamic key polymer to the most suitable dosage of target enzyme activation and
Most suitable hyperthermia temperature range.
2. a kind of application of dynamic key polymer in terms of improving enzyme heat stability, which is characterized in that application method is as follows:
Firstly, the temperature of target enzyme catalyst system is gradually risen, until the activity of target enzyme significantly reduces；Then in room temperature condition
Under, dynamic key polymer solution is added in target enzyme catalystic converter system, re-measures target enzyme after standing 10~60min
Activity obtains the most suitable dosage of dynamic key polymer by comparing the additive amount of Different Dynamic key polymer solution.
3. application according to claim 1 or 2, which is characterized in that the dynamic key polymer the preparation method is as follows:
1) aromatic aldehyde, amino-terminated polymer and amine containing functional group are mixed in organic solvent, at 60~80 DEG C
Reflux 3~4 days carries out imines and generates reaction；Wherein, the ratio between aromatic aldehyde and the amino-terminated mole of polymer be 1:2~
2:1；Mass ratio 2:1~10:1 of aromatic aldehyde and the amine containing functional group；
2) solvent after reacting in system is generated with vacuum distillation removal imines, and distilled water is added, and it is 10 that concentration, which is prepared,
The dynamic key polymer solution of~20mM.
4. application according to claim 3, which is characterized in that in the preparation method of dynamic key polymer, the fragrance
Aldehyde is equal benzene trioxin, o-phthalaldehyde, terephthalaldehyde or m-terephthal aldehyde.
5. application according to claim 3, which is characterized in that in the preparation method of dynamic key polymer, the amino
The polymer of sealing end is amino-terminated polyethylene glycol, polyetheramine, polytetrahydrofuran, chitosan or amido modified hyalomitome
Acid.
6. application according to claim 4, which is characterized in that in the preparation method of dynamic key polymer, the amino
The polymer of sealing end is amino-terminated polyethylene glycol, polyetheramine, polytetrahydrofuran, chitosan or amido modified hyalomitome
Acid.
7. application according to claim 3, which is characterized in that in the preparation method of dynamic key polymer, described contains function
Can the amine of group be PAMAM dendritic, or comprising 1~3 primary amine, embedded with 0-5 identical or different alkoxy, acyls
The hydrocarbon chain of amido, hydroxy methylene or secondary amine functional group, the carbon number of hydrocarbon chain are 2-12；Structural formula be followed successively by (a), (b),
(c) and (d):
Wherein, X be carbon number 2-12, embedded with 0-5 identical or different alkoxy, amide groups, hydroxy methylene or secondary amines
The hydrocarbon chain of functional group inside.
8. according to application described in claim 4,5 or 6, which is characterized in that described in the preparation method of dynamic key polymer
Amine containing functional group is PAMAM dendritic, or comprising 1~3 primary amine, embedded with the identical or different alcoxyls of 0-5
Base, amide groups, hydroxy methylene or secondary amine functional group hydrocarbon chain, the carbon number of hydrocarbon chain is 2-12；Structural formula be followed successively by (a),
(b), (c) and (d):
Wherein, X be carbon number 2-12, embedded with 0-5 identical or different alkoxy, amide groups, hydroxy methylene or secondary amines
The hydrocarbon chain of functional group inside.
9. application according to claim 5 or 6, which is characterized in that in the preparation method of dynamic key polymer, the ammonia
Base sealing end polymer be bis- (3- aminopropyl) polyethylene glycol, polyetheramine D series, both-end (3- aminopropyl) polytetrahydrofuran,
The long chain alkane of double-end amino sealing end, four arms-polyethylene glycol-amino, polyetheramine T series, polyetheramine ED series, O- carboxymethyl shell
Hyaluronic acid after glycan or amide hydrolysis, concrete structure formula are respectively (A)-(I):
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111574756A (en) * | 2020-05-26 | 2020-08-25 | 中国科学院长春应用化学研究所 | Chitosan-based/functionalized chitosan-based hydrogel and preparation and application thereof |
| CN114085373A (en) * | 2021-12-09 | 2022-02-25 | 万华化学集团股份有限公司 | Hyaluronic acid polyether polyol and preparation method and application thereof |
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| WO2004085640A1 (en) * | 2003-03-28 | 2004-10-07 | Council Of Scientific And Industrial Research | A process for preparation of thermostable enzyme |
| WO2005073370A1 (en) * | 2004-01-31 | 2005-08-11 | Transparent Inc. | Enzyme composite |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111574756A (en) * | 2020-05-26 | 2020-08-25 | 中国科学院长春应用化学研究所 | Chitosan-based/functionalized chitosan-based hydrogel and preparation and application thereof |
| CN114085373A (en) * | 2021-12-09 | 2022-02-25 | 万华化学集团股份有限公司 | Hyaluronic acid polyether polyol and preparation method and application thereof |
| CN114085373B (en) * | 2021-12-09 | 2023-07-14 | 万华化学集团股份有限公司 | Hyaluronic acid polyether polyol and preparation method and application thereof |
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