CN104357410B - A kind of preparation method of modification ixtle SOD lyophilized powder - Google Patents
A kind of preparation method of modification ixtle SOD lyophilized powder Download PDFInfo
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Abstract
The present invention relates to a kind of preparation method of modification ixtle SOD lyophilized powder, belongs to organic synthesis and applications technical field, which is with microwave-assisted succinic anhydrides（SA）With N N-Hydroxysuccinimide（NHS）Activation mono methoxy polyethylene glycol（mPEG）To activate dressing agent, with ixtle superoxide dismutase（SOD）To modify object, modification ixtle SOD trims are prepared.The activation dressing agent preparation method, using Microwave-assisted firing, greatly shortens soak time, improves activation efficiency；Ixtle SOD is modified using above-mentioned activation dressing agent, prepares modification SOD trim；Gained trim is heat-resisting, acid and alkali-resistance, and proteolysis resistant ability is high than natural ixtle superoxide dismutase, and immunogenicity declines or eliminates.
Description
Technical field
The present invention relates to a kind of preparation method of plant SOD lyophilized powder, and in particular to a kind of modification ixtle SOD lyophilizing
The preparation method of powder, belongs to organic synthesis and applications technical field.
Background technology
Superoxide dismutase (superoxide dismutase, SOD) is that a class is widely present in biological internal gold
Category enzyme, has substantial connection with trace element (tace elements) metabolism such as copper (Cu), zinc (Zn), ferrum (Fe), manganese (Mn).SOD
It is a kind of biological important oxygen free radical scavenger in vivo, the oxygen-derived free radicals of body can be balanced, so as to avoids when super in body
The untoward reaction caused during oxygen anion free radical excessive concentration, in radioprotective, defying age, antiinflammatory, suppression tumor and cancer, certainly
The aspects such as body immunization therapy show the function of uniqueness, and by Chinese scholars and the extensive concern of researcher, research field is
It is related to multiple subjects such as chemistry, biology, medical science, Food Science and animal and veterinary.Therefore, further investigate SOD and its with
The relation of the element metabolism such as copper, zinc, ferrum, manganese in body, not only has important theory significance, and with important practical valency
Value.
Presently used SOD products mostly are and extract from human blood or animal blood, but as bovine spongiform encephalopathy, mouth in the world are crowed epidemic disease etc.
Infectious disease spreads, and the use of the SOD extracted in animal blood brought many risk factors.Therefore, from
Extract SOD in plant, mushroom, it is edible safety height, aboundresources, inexpensive, with wide DEVELOPMENT PROSPECT.
In order to improve the stability of superoxide dismutase, necessary modification should be carried out to superoxide dismutase molecule and is changed
Make.At present the approach that SOD molecules carry out molecular modification transformation is included being chemically modified SOD amino acid residues, with water-soluble
Property macromole carries out covalent modification, carries out enzyme action modification to SOD to SOD aminoacid.Modification enzyme has not only been fully retained native enzyme
Activity, it is stable compared with native enzyme, and be substantially better than day at aspects such as heat-resisting, acidproof, alkaline-resisting and antipepsin hydrolysis abilities
Right enzyme.CN201010137185.2 discloses a kind of SOD production process for modifying, using animal blood SOD enzymes as modification object, adopts
High-purity lauroyl chloride makees dressing agent；CN201410062990.1 discloses the side that a kind of lauric acid modifies superoxide dismutase
Method, which is dissolved in Sanguis Bovis seu Bubali Cu/Zn-SOD in alkalescence buffer, adds the lauric acid after activation and is modified.Although which two
Individual patent is modified to SOD molecules with small molecule, and the absorption rate of human body is improved, but the stability of product is not high enough,
Its application is limited to a certain extent, and there is a problem of that preparation cost is higher.Zhao Hua's《Polyethyleneglycol modified superoxides discrimination
Change the preliminary study of enzyme》（Modern biomedical is in progress, and 2008,8（02）：277-279）Middle-molecular-weihydroxyethyl is gathered for the activation of 6000Da
Ethylene glycol (PEG) is dressing agent, and covalent modification is carried out to corn superoxide dismutase from corn (SOD), and PEG -6000 utilize cyanogen urea acyl
Chlorine is activated, but this kind of method may not necessarily reach ideal effect using in other plant SOD things.
At present, ixtle is just being planted in China's tropical and subtropical zone area in a large number, but which is developed, and essentially consists in extraction
Fiber is to make hawser, carpet, burlap etc., and the bioactive substance such as SOD valuable in ixtle is then abandoned together with waste liquid
Go, if the enzyme of high added value is extracted from ixtle, the utilization rate of ixtle will be greatly improved, and reduce SOD preparations
Cost.The present invention single methoxy with microwave-assisted succinic anhydrides and obtained by N-hydroxy-succinamide activation mono methoxy polyethylene glycol
Base Polyethylene Glycol succinimidyl succinate is activation dressing agent, with ixtle superoxide dismutase to modify object,
Ixtle superoxide dismutase trim is prepared, a kind of cost-effective stability height, the plant of applied range is developed
SOD。
The content of the invention
It is an object of the invention to provide a kind of preparation method of modification ixtle SOD lyophilized powder, with microwave-assisted succinum
Anhydride（SA）And N-hydroxy-succinamide（NHS）Activation mono methoxy polyethylene glycol（mPEG）To activate dressing agent, with imperial tongue
Blue fiber crops superoxide dismutase（SOD）To modify object, modification ixtle SOD trims are prepared.It is prepared by the activation dressing agent
Method, using Microwave-assisted firing, greatly shortens soak time, improves activation efficiency.Ixtle SOD utilizes above-mentioned activation
Dressing agent is modified, and prepares modification SOD trim.
A kind of Microwave-assisted synthesis and purposes of mono methoxy polyethylene glycol succinimidyl succinate, including following step
Suddenly：
A. microwave-assisted activates mono methoxy polyethylene glycol（mPEG）Synthesis activation dressing agent：
By 100 ~ 120 parts of quality mPEG, 3 ~ 6 parts of quality SA, 80 ~ 120 parts of quality DMF mix homogeneously；Mixture is placed in
In microwave oven reflux, power 10 ~ 90%, microwave radiation 3 ~ 60 minutes is selected to stop radiation；Thing to be mixed is cooled to room temperature,
Stirring is lower to add absolute ether or petroleum ether, makes precipitation complete；Sucking filtration, abandons filtrate and stays precipitation；Add in gained precipitation under stirring
Enter organic solvent, until precipitation is completely dissolved, sucking filtration abandons precipitation reserved filtrate；Absolute ether or stone are added in gained filtrate
It is complete that oily ether precipitates which, and sucking filtration is abandoned filtrate and stays precipitation；Repeat aforesaid operations 3 ~ 5 times, obtain intermediate product I, i.e. mono methoxy
Polyethanediol succinate（mPEG-S）；The aforesaid operations refer to stirring under to gained precipitation in add organic solvent, until
Precipitation is completely dissolved, sucking filtration, abandons precipitation reserved filtrate；Absolute ether or petroleum ether are added in gained filtrate has precipitated which
Entirely, sucking filtration, abandons filtrate and stays precipitation；
The heating bath at 40 ~ 45 DEG C, adds 3 ~ 5 parts of quality NHS, 80 ~ 120 parts of quality DMF and 5 ~ 8 parts in intermediate product I
Quality dicyclohexylcarbodiimide（DCC）, mix homogeneously, taking-up are put at room temperature overnight；Reactant mixture is again placed in 40 ~
Heating bath at 45 DEG C, till the precipitation in mixture no longer dissolves；Take out, sucking filtration, abandons filtering residue reserved filtrate while hot；Filter to gained
Drop adds absolute ether or petroleum ether which is precipitated completely, sucking filtration；After gained precipitation is with organic solvent dissolving, absolute ether is added
Or petroleum ether makes which precipitate completely, sucking filtration；Repeat aforesaid operations 3 ~ 5 times；Finally gained precipitation is vacuum dried, is final product
II, i.e.,：Activation dressing agent mono methoxy polyethylene glycol succinimidyl succinate（mPEG-SS）；
B. mPEG-SS modifies ixtle SOD
By ixtle SOD powder and mPEG-SS, by 1:10～1:80 mol ratios are dissolved in 350 ~ 550 parts of 0.1mol/L
PH9.2 borate buffer solutions, shaken at room temperature react 1～3 hour, then in reactant liquor add 100 ~ 150 parts of volumes cold
The 1mol/L phosphate buffered solution of pH7.0（PBS）Terminating reaction, gained mixed solution 3～50mmol/L pH7～8
The dialysis of PBS buffer solution, concentration, go up the good Sephadex sephadex chromatography posts eluting of pre-balance immediately and separate, collect
280nm first peaks, lyophilization must modify ixtle SOD lyophilized powder.
Organic solvent described in step A is DMF, dichloromethane, chloroform or carbon tetrachloride；Described cold phosphate delays
The temperature for rushing solution is 0 ~ 5 DEG C.
Ixtle SOD powder described in step B is ixtle SOD lyophilized powder.
The present invention relative to the beneficial effect of prior art is：
（1）The activation dressing agent preparation method that the present invention is provided utilizes Microwave-assisted firing, greatly shortens soak time, carries
Overactivity efficiency；（2）Ixtle SOD is modified using above-mentioned activation dressing agent, and gained trim is heat-resisting, acid and alkali-resistance is stable
Property, and proteolysis resistant ability is high than natural ixtle superoxide dismutase, immunogenicity declines or eliminates；（3）
The modification ixtle superoxide dismutase that the present invention is provided derives from the modified rear institute of natural plants superoxide dismutase
, it is virus-free, without cross infection, can be used for medicine, food, health product and cosmetics etc..
Description of the drawings
Fig. 1 is the Technology Roadmap of the present invention；
Fig. 2 is the ultra-violet absorption spectrum of mPEG-SS of the present invention and NHS, final product II（mPEG-SS）It is ultraviolet with NHS
In 260nm or so, absworption peak illustrates that NHS is bonded with dressing agent raw material mPEG；
Fig. 3,4,5 are dressing agent raw materials of the present invention（mPEG）, activation dressing agent intermediate product I（mPEG-S）And final product
II（mPEG-SS）Infrared absorption spectroscopy, the infrared absorption spectroscopy of mPEG-S（Fig. 4）Than the infrared absorption spectroscopy of mPEG（Fig. 3）
Have more 1735 cm-1Place's absworption peak, is the absworption peak of ester carbonyl group, illustrates that first step reaction is carried out and generates intermediate product I, mPEG-
The infrared absorption spectroscopy of SS（Fig. 5）Than the infrared absorption spectroscopy of mPEG（Fig. 3）Have more 1775cm-1、1736 cm-1、1636 cm-1
Absworption peak, wherein 1775cm-1、1736 cm-1It is the absorption of two kinds of ester carbonyl groups, 1636 cm-1It is the stretching vibration peak of N-C=O, says
Bright second step reaction is carried out and generates final product II；
Fig. 6,7 are impact of the temperature of the present invention to modification SOD, and wherein a is natural SOD, and b is modification SOD；
Fig. 8 is impacts of the pH of the present invention to modification SOD, and wherein a is natural SOD, and b is modification SOD.
Specific embodiment
The present invention is described in further details below by embodiment, these embodiments are only used for illustrating the present invention, and
Do not limit the scope of the invention.
Embodiment 1
A. microwave-assisted activates mono methoxy polyethylene glycol（mPEG）Synthesis activation dressing agent：
By 100 parts of quality mPEG, 3 parts of quality SA, 80 parts of quality DMF mix homogeneously；Mixture is placed in into microwave oven backflow
In device, power 10%, microwave radiation 3 minutes is selected to stop radiation；Thing to be mixed is cooled to room temperature, and stirring is lower to add anhydrous second
Ether or petroleum ether, make precipitation complete；Sucking filtration, abandons filtrate and stays precipitation；DMF is added in gained precipitation under stirring, until having precipitated
CL, sucking filtration abandon precipitation reserved filtrate；Absolute ether is added in gained filtrate makes which precipitate completely, and sucking filtration is abandoned filtrate and stayed
Precipitation；Repeat aforesaid operations 3 times, obtain intermediate product I, i.e. mono methoxy polyethylene glycol succinate（mPEG-S）；
The heating bath at 40 DEG C, adds 3 parts of quality NHS, 80 parts of quality DMF and 5 parts of quality dicyclohexyls in intermediate product I
Carbodiimide（DCC）, mix homogeneously, taking-up are put at room temperature overnight；Reactant mixture is again placed in heating bath at 40 DEG C, until
Till precipitation in mixture no longer dissolves；Take out, sucking filtration, abandons filtering residue reserved filtrate while hot；To gained filtrate added drop-wise absolute ether
Precipitate which complete, sucking filtration；After gained precipitation is with DMF dissolvings, absolute ether is added to make its precipitation complete, sucking filtration；Repetition is above-mentioned
Operation 3 times；Finally gained precipitation is vacuum dried, is final product II, i.e.,：Activation dressing agent mono methoxy polyethylene glycol succinum
Sour succinimide ester（mPEG-SS）；
B. mPEG-SS modifies ixtle SOD
By ixtle SOD powder and mPEG-SS, by 1:10 mol ratios are dissolved in 350 parts of 0.1mol/L pH9.2 borates
Buffer solution, shaken at room temperature react 1 hour, and then in reactant liquor, the 1mol/L phosphate of the cold pH7.0 of 100 parts of volumes of addition delays
Rush solution（PBS）Terminating reaction, gained mixed solution is dialysed with the PBS buffer solution of 3mmol/L pH7, concentration, goes up immediately pre-
The Sephadex sephadex chromatography posts eluting for first balancing is separated, and collects 280nm first peaks, and lyophilization must modify imperial tongue
Blue fiber crops SOD lyophilized powder.
Embodiment 2
A. microwave-assisted activates mono methoxy polyethylene glycol（mPEG）Synthesis activation dressing agent：
By 120 parts of quality mPEG, 6 parts of quality SA, 120 parts of quality DMF mix homogeneously；Mixture is placed in microwave oven to return
In stream device, power 90%, microwave radiation 60 minutes is selected to stop radiation；Thing to be mixed is cooled to room temperature, and stirring is lower to add nothing
Water ether or petroleum ether, make precipitation complete；Sucking filtration, abandons filtrate and stays precipitation；Dichloromethane is added in gained precipitation under stirring, directly
It is completely dissolved to precipitation, sucking filtration, abandons precipitation reserved filtrate；Petroleum ether is added in gained filtrate makes which precipitate completely, and sucking filtration is abandoned
Filtrate stays precipitation；Repeat aforesaid operations 5 times, obtain intermediate product I, i.e. mono methoxy polyethylene glycol succinate（mPEG-S）；
The heating bath at 45 DEG C, adds 5 parts of quality NHS, 120 parts of quality DMF and 8 parts of two hexamethylenes of quality in intermediate product I
Base carbodiimide（DCC）, mix homogeneously, taking-up are put at room temperature overnight；Reactant mixture is again placed in heating bath at 45 DEG C, directly
No longer dissolving to the precipitation in mixture；Take out, sucking filtration, abandons filtering residue reserved filtrate while hot；To gained filtrate added drop-wise petroleum ether
Precipitate which complete, sucking filtration；After gained precipitation is with dichloromethane dissolving, petroleum ether is added which is precipitated completely, sucking filtration；In repetition
State operation 5 times；Finally gained precipitation is vacuum dried, is final product II, i.e.,：Activation dressing agent mono methoxy polyethylene glycol amber
Amber acid succinimide ester（mPEG-SS）；
B. mPEG-SS modifies ixtle SOD
By ixtle SOD powder and mPEG-SS, by 1:80 mol ratios are dissolved in 550 parts of 0.1mol/L pH9.2 borates
Buffer solution, shaken at room temperature react 3 hours, and then in reactant liquor, the 1mol/L phosphate of the cold pH7.0 of 150 parts of volumes of addition delays
Rush solution（PBS）Terminating reaction, gained mixed solution is dialysed with the PBS buffer solution of 50mmol/L pH8, concentration, goes up immediately pre-
The Sephadex sephadex chromatography posts eluting for first balancing is separated, and collects 280nm first peaks, and lyophilization must modify imperial tongue
Blue fiber crops SOD lyophilized powder.
Embodiment 3
A. microwave-assisted activates mono methoxy polyethylene glycol（mPEG）Synthesis activation dressing agent：
By 110 parts of quality mPEG, 5 parts of quality SA, 90 parts of quality DMF mix homogeneously；Mixture is placed in into microwave oven backflow
In device, power 70%, microwave radiation 50 minutes is selected to stop radiation；Thing to be mixed is cooled to room temperature, and stirring is lower to add oil
Ether, makes precipitation complete；Sucking filtration, abandons filtrate and stays precipitation；Chloroform is added in gained precipitation under stirring, it is completely molten until precipitating
Solution, sucking filtration abandon precipitation reserved filtrate；Absolute ether is added in gained filtrate makes which precipitate completely, and sucking filtration is abandoned filtrate and stays heavy
Form sediment；Repeat aforesaid operations 4 times, obtain intermediate product I, i.e. mono methoxy polyethylene glycol succinate（mPEG-S）；
The heating bath at 42 DEG C, adds 4 parts of quality NHS, 100 parts of quality DMF and 6 parts of two hexamethylenes of quality in intermediate product I
Base carbodiimide（DCC）, mix homogeneously, taking-up are put at room temperature overnight；Reactant mixture is again placed in heating bath at 42 DEG C, directly
No longer dissolving to the precipitation in mixture；Take out, sucking filtration, abandons filtering residue reserved filtrate while hot；To the anhydrous second of gained filtrate added drop-wise
It is complete that ether precipitates which, sucking filtration；After gained precipitation is with chloroform dissolving, absolute ether is added which is precipitated completely, sucking filtration；Weight
Multiple aforesaid operations 4 times；Finally gained precipitation is vacuum dried, is final product II, i.e.,：The poly- second of activation dressing agent mono methoxy two
Alcohol succinimidyl succinate（mPEG-SS）；
B. mPEG-SS modifies ixtle SOD
By ixtle SOD powder and mPEG-SS, by 1:70 mol ratios are dissolved in 450 parts of 0.1mol/L pH9.2 borates
Buffer solution, shaken at room temperature react 2 hours, and then in reactant liquor, the 1mol/L phosphate of the cold pH7.0 of 145 parts of volumes of addition delays
Rush solution（PBS）Terminating reaction, gained mixed solution is dialysed with the PBS buffer solution of 40mmol/L pH7.8, concentration, goes up immediately
The good Sephadex sephadex chromatography posts eluting of pre-balance is separated, and collects 280nm first peaks, and lyophilization must modify dragon
The blue fiber crops SOD lyophilized powder of tongue.
Embodiment 4
A. microwave-assisted activates mono methoxy polyethylene glycol（mPEG）Synthesis activation dressing agent：
By 120 parts of quality mPEG, 3 parts of quality SA, 90 parts of quality DMF mix homogeneously；Mixture is placed in into microwave oven backflow
In device, power 60%, microwave radiation 40 minutes is selected to stop radiation；Thing to be mixed is cooled to room temperature, and stirring is lower to add oil
Ether, makes precipitation complete；Sucking filtration, abandons filtrate and stays precipitation；Carbon tetrachloride is added in gained precipitation under stirring, it is completely molten until precipitating
Solution, sucking filtration abandon precipitation reserved filtrate；Absolute ether is added in gained filtrate makes which precipitate completely, and sucking filtration is abandoned filtrate and stays heavy
Form sediment；Repeat aforesaid operations 4 times, obtain intermediate product I, i.e. mono methoxy polyethylene glycol succinate（mPEG-S）；
The heating bath at 42 DEG C, adds 4 parts of quality NHS, 100 parts of quality DMF and 6 parts of two hexamethylenes of quality in intermediate product I
Base carbodiimide（DCC）, mix homogeneously, taking-up are put at room temperature overnight；Reactant mixture is again placed in heating bath at 42 DEG C, directly
No longer dissolving to the precipitation in mixture；Take out, sucking filtration, abandons filtering residue reserved filtrate while hot；To the anhydrous second of gained filtrate added drop-wise
It is complete that ether precipitates which, sucking filtration；After gained precipitation is with carbon tetrachloride dissolving, absolute ether is added which is precipitated completely, sucking filtration；Weight
Multiple aforesaid operations 4 times；Finally gained precipitation is vacuum dried, is final product II, i.e.,：The poly- second of activation dressing agent mono methoxy two
Alcohol succinimidyl succinate（mPEG-SS）；
B. mPEG-SS modifies ixtle SOD
By ixtle SOD powder and mPEG-SS, by 1:70 mol ratios are dissolved in 450 parts of 0.1mol/L pH9.2 borates
Buffer solution, shaken at room temperature react 2 hours, and then in reactant liquor, the 1mol/L phosphate of the cold pH7.0 of 145 parts of volumes of addition delays
Rush solution（PBS）Terminating reaction, gained mixed solution is dialysed with the PBS buffer solution of 40mmol/L pH7.8, concentration, goes up immediately
The good Sephadex sephadex chromatography posts eluting of pre-balance is separated, and collects 280nm first peaks, and lyophilization must modify dragon
The blue fiber crops SOD lyophilized powder of tongue.
Embodiment 5
A. microwave-assisted activates mono methoxy polyethylene glycol（mPEG）Synthesis activation dressing agent：
By 115 parts of quality mPEG, 4.5 parts of quality SA, 88 parts of quality DMF mix homogeneously；Mixture is placed in microwave oven to return
In stream device, power 65%, microwave radiation 50 minutes is selected to stop radiation；Thing to be mixed is cooled to room temperature, and stirring is lower to add stone
Oily ether, makes precipitation complete；Sucking filtration, abandons filtrate and stays precipitation；Chloroform is added in gained precipitation under stirring, it is complete until precipitating
Precipitation reserved filtrate is abandoned in dissolving, sucking filtration；Absolute ether is added in gained filtrate makes which precipitate completely, and sucking filtration is abandoned filtrate and stays heavy
Form sediment；Repeat aforesaid operations 4 times, obtain intermediate product I, i.e. mono methoxy polyethylene glycol succinate（mPEG-S）；
The heating bath at 42 DEG C, adds 4 parts of quality NHS, 100 parts of quality DMF and 6 parts of two hexamethylenes of quality in intermediate product I
Base carbodiimide（DCC）, mix homogeneously, taking-up are put at room temperature overnight；Reactant mixture is again placed in heating bath at 42 DEG C, directly
No longer dissolving to the precipitation in mixture；Take out, sucking filtration, abandons filtering residue reserved filtrate while hot；To the anhydrous second of gained filtrate added drop-wise
It is complete that ether precipitates which, sucking filtration；After gained precipitation is with chloroform dissolving, absolute ether is added which is precipitated completely, sucking filtration；Weight
Multiple aforesaid operations 4 times；Finally gained precipitation is vacuum dried, is final product II, i.e.,：The poly- second of activation dressing agent mono methoxy two
Alcohol succinimidyl succinate（mPEG-SS）；
B. mPEG-SS modifies ixtle SOD
By ixtle SOD powder and mPEG-SS, by 1:70 mol ratios are dissolved in 450 parts of 0.1mol/L pH9.2 borates
Buffer solution, shaken at room temperature react 2 hours, and then in reactant liquor, the 1mol/L phosphate of the cold pH7.0 of 145 parts of volumes of addition delays
Rush solution（PBS）Terminating reaction, gained mixed solution is dialysed with the PBS buffer solution of 40mmol/L pH7.8, concentration, goes up immediately
The good Sephadex sephadex chromatography posts eluting of pre-balance is separated, and collects 280nm first peaks, and lyophilization must modify dragon
The blue fiber crops SOD lyophilized powder of tongue.
Ixtle SOD lyophilized powder is prepared to embodiment and is identified using ultra-violet absorption spectrum, infrared absorption spectroscopy,
As a result Fig. 2～5 are seen.
Ixtle SOD lyophilized powder is prepared to embodiment carries out temperature capacity, acidproof alkali ability, proteolysis resistant ability
Determine, it is as a result as follows：
（1）The temperature capacity of modification SOD is determined
Natural SOD and modification SOD are incubated 2 hours respectively in 25～90 DEG C of water-baths, separately sampled its vigor of measure.30
～50 DEG C, temperature is improved, and the vigor of modification SOD and natural SOD is gradually stepped up.When temperature is more than 50 DEG C, modification SOD vigor is gradually
Decline, and the vigor of natural SOD declines rapidly.Illustrate that modification SOD thermostability strengthens.Experimental result is as shown in Figure 6.
Natural SOD and modification SOD are incubated 3.5 hours in 70 DEG C of water-bath, and its vigor is measured by sampling at regular intervals.
Prolongation over time, the two vigor are gradually reduced.Temperature retention time 2.5 hours, natural SOD vigor decrease speed are substantially relatively repaiied
SOD is fast for decorations.Illustrate that gained modification SOD thermostability is improved.Experimental result is as shown in Figure 7.
（2）The acidproof alkali ability of modification SOD is determined
Natural SOD and modification SOD are dissolved in the buffer solution of pH4～11 respectively, mix homogeneously, and 25 DEG C are incubated 2 hours, determine
Its vigor.PH4～6, the two vigor gradually rise.PH is more than 6, and the two vigor is gradually reduced.PH is more than 9, and natural SOD vigor is fast
Speed declines；And pH is more than 10, modification SOD vigor decrease speed is just accelerated.Illustrate the more natural SOD of acidproof alkali ability of modification SOD
Height, and the acid-fast ability of modification SOD is higher than resistance to alkali ability.Experimental result is as shown in Figure 8.
（3）Modification SOD proteolysis resistant ability is determined
Pepsin or trypsin are separately added in natural SOD and modification SOD, its vigor after 2 hours, is determined, vigor is equal
Decline.Wherein, the residual vigor that natural SOD is measured after adding pepsin or trypsin is below the residual of modification SOD
Stay vigor.Illustrate that the proteolysis resistant ability of modification SOD is higher than natural SOD.Therefore natural SOD is effectively carried after modification
High proteolysis resistant ability.
Claims (5)
1. it is a kind of modification ixtle SOD lyophilized powder preparation method, it is characterised in that：Comprise the following steps：
A. microwave-assisted activation mono methoxy polyethylene glycol mPEG synthesis activates dressing agent：
100 ~ 120 parts of quality mPEG, 3 ~ 6 parts of quality SA, 80 ~ 120 parts of quality DMF mix homogeneously are obtained into mixture；By mixture
Be placed in microwave radiation in microwave oven reflux, stop radiation thing to be mixed and be cooled to room temperature, stirring is lower add absolute ether or
Petroleum ether, makes precipitation complete；Sucking filtration, abandons filtrate and stays precipitation；Organic solvent is added in gained precipitation under stirring, until having precipitated
CL, sucking filtration abandon precipitation reserved filtrate；Absolute ether or petroleum ether are added in gained filtrate makes which precipitate completely, sucking filtration,
Abandon filtrate and stay precipitation；Repeat aforesaid operations 3 ~ 5 times, obtain intermediate product I：Mono methoxy polyethylene glycol succinate mPEG-S；
The aforesaid operations to be referred to and add organic solvent in gained precipitation under stirring, and until precipitation is completely dissolved, sucking filtration abandons heavy
Shallow lake reserved filtrate；Absolute ether or petroleum ether are added in gained filtrate makes which precipitate completely, and sucking filtration is abandoned filtrate and stays precipitation；
The heating bath at 40 ~ 45 DEG C, adds 3 ~ 5 parts of quality NHS, 80 ~ 120 parts of quality DMF and 5 ~ 8 parts of quality in intermediate product I
Dicyclohexylcarbodiimide（DCC）, mix homogeneously, taking-up are put；Reactant mixture is again
It is secondary to be placed in heating bath at 40 ~ 45 DEG C, till the precipitation in mixture no longer dissolves；Take out, sucking filtration, abandons filtering residue and stay filter while hot
Liquid；Which is made to precipitate completely to gained filtrate added drop-wise absolute ether or petroleum ether, sucking filtration；After gained precipitation is with organic solvent dissolving,
Absolute ether or petroleum ether is added which is precipitated completely, sucking filtration；Repetitive operation 3 ~ 5 times；Finally gained precipitation is vacuum dried, is
Final product II：Activation dressing agent mono methoxy polyethylene glycol succinimidyl succinate mPEG-SS；
B.mPEG-SS modifies ixtle SOD：
By ixtle SOD powder and mPEG-SS, by 1:10～1:80 mol ratios are dissolved in 350 ~ 550 parts of borate buffer solutions,
Shaken at room temperature reacts 1～3 hour, cold phosphate buffered solution PBS of 100 ~ 150 parts of volumes is then added in reactant liquor, eventually
Only react, gained mixed solution is dialysed with PBS buffer solution, concentration, goes up the good Sephadex glucosans of pre-balance immediately and coagulates
Glue chromatographic column eluting is separated, and collects 280nm first peaks, and lyophilization must modify ixtle SOD lyophilized powder；
Organic solvent described in step A is DMF, dichloromethane, chloroform or carbon tetrachloride；
The concentration of cold phosphate buffered solution PBS described in step B is 1mol/L, and pH value is 7.0, and temperature is 0 ~ 5 DEG C.
2. it is according to claim 1 it is a kind of modification ixtle SOD lyophilized powder preparation method, it is characterised in that：Step A
The radiant power of described microwave radiation is 10 ~ 90%.
3. it is according to claim 1 it is a kind of modification ixtle SOD lyophilized powder preparation method, it is characterised in that：Step A
The radiated time of described microwave radiation is 3 ~ 60 minutes.
4. it is according to claim 1 it is a kind of modification ixtle SOD lyophilized powder preparation method, it is characterised in that：Step B
The concentration of described borate buffer solution is 0.1mol/L, and pH is 9.2.
5. it is according to claim 1 it is a kind of modification ixtle SOD lyophilized powder preparation method, it is characterised in that：Step B
The concentration of the PBS buffer solution of described dialysis is 3～50mmol/L, and pH is 7～8.
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| CN1323345A (en) * | 1998-10-13 | 2001-11-21 | 诺沃奇梅兹有限公司 | A modified polypeptide with reduced immune response |
| CN103215247A (en) * | 2013-04-11 | 2013-07-24 | 莫宏春 | Method of modifying natural superoxide dismutase(SOD) enzyme solution, posttreatment modified SOD enzyme solution and myristic acid-SOD lyophilized powder |
| CN103805588A (en) * | 2013-02-28 | 2014-05-21 | 无锡华奇生物科技有限公司 | Method for modifying superoxide dismutase by using lauric acid |
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| CN103805588A (en) * | 2013-02-28 | 2014-05-21 | 无锡华奇生物科技有限公司 | Method for modifying superoxide dismutase by using lauric acid |
| CN103215247A (en) * | 2013-04-11 | 2013-07-24 | 莫宏春 | Method of modifying natural superoxide dismutase(SOD) enzyme solution, posttreatment modified SOD enzyme solution and myristic acid-SOD lyophilized powder |
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