CN104357410A - Preparation method of freeze-dried powder for modifying ixtle SOD - Google Patents

Preparation method of freeze-dried powder for modifying ixtle SOD Download PDF

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CN104357410A
CN104357410A CN201410615294.9A CN201410615294A CN104357410A CN 104357410 A CN104357410 A CN 104357410A CN 201410615294 A CN201410615294 A CN 201410615294A CN 104357410 A CN104357410 A CN 104357410A
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陈冰
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Lingnan Normal University
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    • 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
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0089—Oxidoreductases (1.) acting on superoxide as acceptor (1.15)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y115/00—Oxidoreductases acting on superoxide as acceptor (1.15)
    • C12Y115/01—Oxidoreductases acting on superoxide as acceptor (1.15) with NAD or NADP as acceptor (1.15.1)
    • C12Y115/01001—Superoxide dismutase (1.15.1.1)

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Abstract

The invention relates to a preparation method of freeze-dried powder for modifying ixtle SOD and belongs to the technical field of organic synthesis and application. The preparation method is characterized in that microwave-assisted succinic anhydride (SA) and N-hydroxyl succinimide (NHS) activated monomethoxypolyethylene glycol (Mpeg) are used as a activation modifying agent and ixtle superoxide dismutase (SOD) is used as a modifying object to prepare a modifier for modifying ixtle SOD. According to the preparation method of the activation modifying agent, microwave-assisted heating is utilized, so that the activation time is shortened greatly, and the activation efficiency is improved; the ixtle SOD is modified by the activation modifying agent, and the modifier for modifying SOD is prepared; the obtained modifier has higher capability of heat resistance, acid-base resistance and proteolysis resistance compared with natural ixtle superoxide dismutase, and the immunogenicity is lowered or eliminated.

Description

A kind of preparation method modifying sisal SOD lyophilized powder
Technical field
The present invention relates to the preparation method of a kind of plant SOD lyophilized powder, be specifically related to a kind of preparation method modifying sisal SOD lyophilized powder, belong to organic synthesis and applications technical field.
Background technology
Superoxide-dismutase (superoxide dismutase, SOD) be the metalloenzyme that a class is extensively present in organism, have substantial connection with micro-(tace elements) metabolism such as copper (Cu), zinc (Zn), iron (Fe), manganese (Mn).SOD is a kind of important oxygen free radical scavenger in organism, the oxyradical of body can be balanced, thus avoid the untoward reaction that causes when Concentration of Superoxide Anion Radical is too high in body, unique function is demonstrated in radioprotective, anti-ageing, anti-inflammatory, Tumor suppression and cancer, autoimmune therapeutic etc., be subject to the extensive concern of Chinese scholars and investigator, research field has related to multiple subjects such as chemistry, biology, medical science, Food science and animal and veterinary.Therefore, further investigation SOD and the relation with the element metabolism such as copper, zinc, iron, manganese in body thereof, not only have important theory significance, and have important practical value.
Current SOD product used mostly is and extracts from human blood or animal blood, but to crow spreading of the communicable diseases such as epidemic disease along with mad cow disease in the world, mouth, and the use of the SOD extracted in animal blood has brought many Hazard Factor.Therefore, from plant, mushroom, extract SOD, edible safety be high, aboundresources, inexpensive, there is wide DEVELOPMENT PROSPECT.
In order to improve the stability of superoxide-dismutase, reply superoxide-dismutase molecule carries out necessary modification transformation.The approach at present carrying out molecular modification transformation to SOD molecule comprises SOD amino-acid residue is carried out to chemically modified, carries out covalent modification with water-soluble macromolecule, carry out enzyme cut modification to SOD SOD amino acid.Modifying enzyme not only remains the activity of natural enzyme completely, and comparatively natural enzyme is stablized, and is obviously better than natural enzyme in heat-resisting, acidproof, alkaline-resisting and antipepsin hydrolysis ability etc.CN201010137185.2 discloses a kind of SOD production process for modifying, using animal blood SOD enzyme as modification object, adopts high purity lauroyl chloride to make modifier; CN201410062990.1 discloses a kind of lauric acid and modifies the method for superoxide dismutase, and ox blood Cu/Zn-SOD is dissolved in weakly alkaline damping fluid by it, then adds the lauric acid after activation and modify.Although two patent small molecules is modified SOD molecule, the absorption rate of human body improves, and the stability of product is not high enough, limits its application to a certain extent, and there is the higher problem of preparation cost.(modern biomedical is in progress " preliminary study of Win-22118 " of Zhao Hua, 2008,8(02): 277-279) middle-molecular-weihydroxyethyl is the activated polyethylene glycol (PEG) of 6000Da is modifier, covalent modification is carried out to corn superoxide dismutase from corn (SOD), PEG-6000 utilize cyanuric chloride to activate, but this kind of method utilizes in other plant SOD thing, may not necessarily reach ideal effect.
At present, sisal is planted just in a large number in China's tropical and subtropical zone area, but it is developed, mainly be to extract fiber to make hawser, carpet, burlap etc., the biologically active substances such as SOD valuable in sisal then discard together with waste liquid, if extract the enzyme of high added value from sisal, will greatly improve the utilization ratio of sisal, and reduce SOD preparation cost.The present invention activates mono methoxy polyethylene glycol gained mono methoxy polyethylene glycol succinimidyl succinate for activation modifier with microwave-assisted succinyl oxide and N-hydroxy-succinamide, with sisal superoxide-dismutase for modifying object, prepare sisal superoxide-dismutase modifier, develop that a kind of cost-effective stability is high, the plant SOD of applied range.
Summary of the invention
The object of this invention is to provide a kind of preparation method modifying sisal SOD lyophilized powder, mono methoxy polyethylene glycol (mPEG) is activated for activation modifier with microwave-assisted succinyl oxide (SA) and N-hydroxy-succinamide (NHS), with sisal superoxide-dismutase (SOD) for modifying object, sisal SOD modifier is modified in preparation.This activation modifier preparation method, utilizes Microwave-assisted firing, greatly shortens soak time, improves activation efficiency.This sisal SOD utilizes above-mentioned activation modifier to modify, and prepares modification SOD modifier.
The Microwave-assisted synthesis of mono methoxy polyethylene glycol succinimidyl succinate and a purposes, comprise the following steps:
A. microwave-assisted activation mono methoxy polyethylene glycol (mPEG) synthesis activation modifier:
By 100 ~ 120 parts of quality mPEG, 3 ~ 6 parts of quality SA, 80 ~ 120 parts of quality DMF mix; Mixture is placed in microwave oven reflux, selects power 10 ~ 90%, microwave radiation 3 ~ 60 minutes, stop radiation; Thing cool to room temperature to be mixed, adds anhydrous diethyl ether or sherwood oil under stirring, make precipitation complete; Suction filtration, abandons filtrate and stays precipitation; In gained precipitation, add organic solvent under stirring, until precipitation is dissolved completely, suction filtration, abandons precipitation reserved filtrate; In gained filtrate, add anhydrous diethyl ether again or sherwood oil makes it precipitate completely, suction filtration, abandons filtrate and stays precipitation; Repeat aforesaid operations 3 ~ 5 times, obtain intermediate product I, i.e. mono methoxy polyethylene glycol succinate (mPEG-S); Described aforesaid operations adds organic solvent under referring to stirring in gained precipitation, until precipitation is dissolved completely, suction filtration, abandons precipitation reserved filtrate; In gained filtrate, add anhydrous diethyl ether again or sherwood oil makes it precipitate completely, suction filtration, abandons filtrate and stays precipitation;
Heating bath at 40 ~ 45 DEG C, adds 3 ~ 5 parts of quality NHS, 80 ~ 120 parts of quality DMF and 5 ~ 8 part of quality dicyclohexylcarbodiimide (DCC), mixes in intermediate product I, takes out, is placed in overnight at room temperature; Heating bath at reaction mixture is placed in 40 ~ 45 DEG C again, till the precipitation in mixture is no longer dissolved; Take out, suction filtration while hot, abandons filter residue reserved filtrate; It is made to precipitate completely to gained filtrate added drop-wise anhydrous diethyl ether or sherwood oil, suction filtration; After gained precipitation organic solvent dissolution, add anhydrous diethyl ether or sherwood oil makes it precipitate completely, suction filtration; Repeat aforesaid operations 3 ~ 5 times; Finally gained being precipitated vacuum-drying, is final product II, that is: activate modifier mono methoxy polyethylene glycol succinimidyl succinate (mPEG-SS);
B. mPEG-SS modifies sisal SOD
By sisal SOD powder and mPEG-SS, 350 ~ 550 parts of 0.1mol/L pH9.2 borate buffer solutions are dissolved in by 1:10 ~ 1:80 mol ratio, shaken at room temperature reaction 1 ~ 3 hour, then in reaction solution, add 1mol/L phosphate buffer soln (PBS) termination reaction of 100 ~ 150 parts of cold pH7.0 of volume, the gained mixing solutions PBS buffered soln of 3 ~ 50mmol/L pH7 ~ 8 is dialysed, concentrated, go up the good Sephadex sephadex chromatography post wash-out of pre-balance to be immediately separated, collect 280nm first peak, lyophilize must modify sisal SOD lyophilized powder.
Organic solvent described in steps A is DMF, methylene dichloride, trichloromethane or tetracol phenixin; The temperature of described cold phosphate buffer soln is 0 ~ 5 DEG C.
Sisal SOD powder described in step B is sisal SOD lyophilized powder.
the present invention relative to the beneficial effect of prior art is:
(1) activation modifier preparation method provided by the invention utilizes Microwave-assisted firing, greatly shortens soak time, improves activation efficiency; (2) sisal SOD utilizes above-mentioned activation modifier to modify, and gained modifier is heat-resisting, acid and alkali-resistance stability, and proteolysis resistant ability is all high than natural sisal superoxide-dismutase, and immunogenicity declines or eliminates; (3) modification sisal superoxide-dismutase provided by the invention derives from the modified rear gained of natural phant superoxide-dismutase, virus-free, without cross infection, can be used for medicine, food, healthcare products and makeup etc.
Accompanying drawing explanation
Fig. 1 is Technology Roadmap of the present invention;
Fig. 2 is the ultra-violet absorption spectrum of mPEG-SS and NHS of the present invention, and the ultraviolet absorption peak of final product II (mPEG-SS) and NHS, all at about 260nm, illustrates NHS and modifier raw material mPEG bonding;
Fig. 3,4,5 is modifier raw material (mPEG) of the present invention, activation modifier intermediate product I(mPEG-S) and final product II(mPEG-SS) infrared absorption spectrum, the infrared absorption spectrum (Fig. 4) of mPEG-S has more 1735 cm than the infrared absorption spectrum (Fig. 3) of mPEG -1place's absorption peak is the absorption peak of ester carbonyl group, and illustrate that the first step reaction is carried out and generates intermediate product I, the infrared absorption spectrum (Fig. 5) of mPEG-SS has more 1775cm than the infrared absorption spectrum (Fig. 3) of mPEG -1, 1736 cm -1, 1636 cm -1absorption peak, wherein 1775cm -1, 1736 cm -1the absorption of two kinds of ester carbonyl groups, 1636 cm -1be the stretching vibration peak of N-C=O, illustrate that second step reaction is carried out and generates final product II;
Fig. 6,7 is temperature of the present invention impacts on modification SOD, and wherein a is natural SOD, b is modification SOD;
Fig. 8 is the impact of pH of the present invention on modification SOD, and wherein a is natural SOD, b is modification SOD.
Embodiment
Be described in further details the present invention below by embodiment, these embodiments are only used for the present invention is described, do not limit the scope of the invention.
embodiment 1
A. microwave-assisted activation mono methoxy polyethylene glycol (mPEG) synthesis activation modifier:
By 100 parts of quality mPEG, 3 parts of quality SA, 80 parts of quality DMF mix; Mixture is placed in microwave oven reflux, selects power 10%, microwave radiation 3 minutes, stop radiation; Thing cool to room temperature to be mixed, adds anhydrous diethyl ether or sherwood oil under stirring, make precipitation complete; Suction filtration, abandons filtrate and stays precipitation; In gained precipitation, add DMF under stirring, until precipitation is dissolved completely, suction filtration, abandons precipitation reserved filtrate; In gained filtrate, add anhydrous diethyl ether again makes it precipitate completely, and suction filtration, abandons filtrate and stay precipitation; Repeat aforesaid operations 3 times, obtain intermediate product I, i.e. mono methoxy polyethylene glycol succinate (mPEG-S);
Heating bath at 40 DEG C, adds 3 parts of quality NHS, 80 parts of quality DMF and 5 part of quality dicyclohexylcarbodiimide (DCC), mixes in intermediate product I, takes out, is placed in overnight at room temperature; Heating bath at reaction mixture is placed in 40 DEG C again, till the precipitation in mixture is no longer dissolved; Take out, suction filtration while hot, abandons filter residue reserved filtrate; It is made to precipitate completely to gained filtrate added drop-wise anhydrous diethyl ether, suction filtration; Gained precipitation adds anhydrous diethyl ether and makes it precipitate completely, suction filtration after dissolving with DMF; Repeat aforesaid operations 3 times; Finally gained being precipitated vacuum-drying, is final product II, that is: activate modifier mono methoxy polyethylene glycol succinimidyl succinate (mPEG-SS);
B. mPEG-SS modifies sisal SOD
By sisal SOD powder and mPEG-SS, 350 parts of 0.1mol/L pH9.2 borate buffer solutions are dissolved in by 1:10 mol ratio, shaken at room temperature reacts 1 hour, then in reaction solution, add 1mol/L phosphate buffer soln (PBS) termination reaction of 100 parts of cold pH7.0 of volume, the gained mixing solutions PBS buffered soln of 3mmol/L pH7 is dialysed, concentrated, go up the good Sephadex sephadex chromatography post wash-out of pre-balance to be immediately separated, collect 280nm first peak, lyophilize must modify sisal SOD lyophilized powder.
embodiment 2
A. microwave-assisted activation mono methoxy polyethylene glycol (mPEG) synthesis activation modifier:
By 120 parts of quality mPEG, 6 parts of quality SA, 120 parts of quality DMF mix; Mixture is placed in microwave oven reflux, selects power 90%, microwave radiation 60 minutes, stop radiation; Thing cool to room temperature to be mixed, adds anhydrous diethyl ether or sherwood oil under stirring, make precipitation complete; Suction filtration, abandons filtrate and stays precipitation; In gained precipitation, add methylene dichloride under stirring, until precipitation is dissolved completely, suction filtration, abandons precipitation reserved filtrate; In gained filtrate, add sherwood oil again makes it precipitate completely, and suction filtration, abandons filtrate and stay precipitation; Repeat aforesaid operations 5 times, obtain intermediate product I, i.e. mono methoxy polyethylene glycol succinate (mPEG-S);
Heating bath at 45 DEG C, adds 5 parts of quality NHS, 120 parts of quality DMF and 8 part of quality dicyclohexylcarbodiimide (DCC), mixes in intermediate product I, takes out, is placed in overnight at room temperature; Heating bath at reaction mixture is placed in 45 DEG C again, till the precipitation in mixture is no longer dissolved; Take out, suction filtration while hot, abandons filter residue reserved filtrate; It is made to precipitate completely to gained filtrate added drop-wise sherwood oil, suction filtration; Gained precipitation adds sherwood oil and makes it precipitate completely, suction filtration after dissolving with methylene dichloride; Repeat aforesaid operations 5 times; Finally gained being precipitated vacuum-drying, is final product II, that is: activate modifier mono methoxy polyethylene glycol succinimidyl succinate (mPEG-SS);
B. mPEG-SS modifies sisal SOD
By sisal SOD powder and mPEG-SS, 550 parts of 0.1mol/L pH9.2 borate buffer solutions are dissolved in by 1:80 mol ratio, shaken at room temperature reacts 3 hours, then in reaction solution, add 1mol/L phosphate buffer soln (PBS) termination reaction of 150 parts of cold pH7.0 of volume, the gained mixing solutions PBS buffered soln of 50mmol/L pH8 is dialysed, concentrated, go up the good Sephadex sephadex chromatography post wash-out of pre-balance to be immediately separated, collect 280nm first peak, lyophilize must modify sisal SOD lyophilized powder.
embodiment 3
A. microwave-assisted activation mono methoxy polyethylene glycol (mPEG) synthesis activation modifier:
By 110 parts of quality mPEG, 5 parts of quality SA, 90 parts of quality DMF mix; Mixture is placed in microwave oven reflux, selects power 70%, microwave radiation 50 minutes, stop radiation; Thing cool to room temperature to be mixed, adds sherwood oil under stirring, makes precipitation complete; Suction filtration, abandons filtrate and stays precipitation; In gained precipitation, add trichloromethane under stirring, until precipitation is dissolved completely, suction filtration, abandons precipitation reserved filtrate; In gained filtrate, add anhydrous diethyl ether again makes it precipitate completely, and suction filtration, abandons filtrate and stay precipitation; Repeat aforesaid operations 4 times, obtain intermediate product I, i.e. mono methoxy polyethylene glycol succinate (mPEG-S);
Heating bath at 42 DEG C, adds 4 parts of quality NHS, 100 parts of quality DMF and 6 part of quality dicyclohexylcarbodiimide (DCC), mixes in intermediate product I, takes out, is placed in overnight at room temperature; Heating bath at reaction mixture is placed in 42 DEG C again, till the precipitation in mixture is no longer dissolved; Take out, suction filtration while hot, abandons filter residue reserved filtrate; It is made to precipitate completely to gained filtrate added drop-wise anhydrous diethyl ether, suction filtration; Gained precipitation adds anhydrous diethyl ether and makes it precipitate completely, suction filtration after dissolving with trichloromethane; Repeat aforesaid operations 4 times; Finally gained being precipitated vacuum-drying, is final product II, that is: activate modifier mono methoxy polyethylene glycol succinimidyl succinate (mPEG-SS);
B. mPEG-SS modifies sisal SOD
By sisal SOD powder and mPEG-SS, 450 parts of 0.1mol/L pH9.2 borate buffer solutions are dissolved in by 1:70 mol ratio, shaken at room temperature reacts 2 hours, then in reaction solution, add 1mol/L phosphate buffer soln (PBS) termination reaction of 145 parts of cold pH7.0 of volume, the gained mixing solutions PBS buffered soln of 40mmol/L pH7.8 is dialysed, concentrated, go up the good Sephadex sephadex chromatography post wash-out of pre-balance to be immediately separated, collect 280nm first peak, lyophilize must modify sisal SOD lyophilized powder.
embodiment 4
A. microwave-assisted activation mono methoxy polyethylene glycol (mPEG) synthesis activation modifier:
By 120 parts of quality mPEG, 3 parts of quality SA, 90 parts of quality DMF mix; Mixture is placed in microwave oven reflux, selects power 60%, microwave radiation 40 minutes, stop radiation; Thing cool to room temperature to be mixed, adds sherwood oil under stirring, makes precipitation complete; Suction filtration, abandons filtrate and stays precipitation; In gained precipitation, add tetracol phenixin under stirring, until precipitation is dissolved completely, suction filtration, abandons precipitation reserved filtrate; In gained filtrate, add anhydrous diethyl ether again makes it precipitate completely, and suction filtration, abandons filtrate and stay precipitation; Repeat aforesaid operations 4 times, obtain intermediate product I, i.e. mono methoxy polyethylene glycol succinate (mPEG-S);
Heating bath at 42 DEG C, adds 4 parts of quality NHS, 100 parts of quality DMF and 6 part of quality dicyclohexylcarbodiimide (DCC), mixes in intermediate product I, takes out, is placed in overnight at room temperature; Heating bath at reaction mixture is placed in 42 DEG C again, till the precipitation in mixture is no longer dissolved; Take out, suction filtration while hot, abandons filter residue reserved filtrate; It is made to precipitate completely to gained filtrate added drop-wise anhydrous diethyl ether, suction filtration; Gained precipitation adds anhydrous diethyl ether and makes it precipitate completely, suction filtration after dissolving with tetracol phenixin; Repeat aforesaid operations 4 times; Finally gained being precipitated vacuum-drying, is final product II, that is: activate modifier mono methoxy polyethylene glycol succinimidyl succinate (mPEG-SS);
B. mPEG-SS modifies sisal SOD
By sisal SOD powder and mPEG-SS, 450 parts of 0.1mol/L pH9.2 borate buffer solutions are dissolved in by 1:70 mol ratio, shaken at room temperature reacts 2 hours, then in reaction solution, add 1mol/L phosphate buffer soln (PBS) termination reaction of 145 parts of cold pH7.0 of volume, the gained mixing solutions PBS buffered soln of 40mmol/L pH7.8 is dialysed, concentrated, go up the good Sephadex sephadex chromatography post wash-out of pre-balance to be immediately separated, collect 280nm first peak, lyophilize must modify sisal SOD lyophilized powder.
embodiment 5
A. microwave-assisted activation mono methoxy polyethylene glycol (mPEG) synthesis activation modifier:
By 115 parts of quality mPEG, 4.5 parts of quality SA, 88 parts of quality DMF mix; Mixture is placed in microwave oven reflux, selects power 65%, microwave radiation 50 minutes, stop radiation; Thing cool to room temperature to be mixed, adds sherwood oil under stirring, makes precipitation complete; Suction filtration, abandons filtrate and stays precipitation; In gained precipitation, add trichloromethane under stirring, until precipitation is dissolved completely, suction filtration, abandons precipitation reserved filtrate; In gained filtrate, add anhydrous diethyl ether again makes it precipitate completely, and suction filtration, abandons filtrate and stay precipitation; Repeat aforesaid operations 4 times, obtain intermediate product I, i.e. mono methoxy polyethylene glycol succinate (mPEG-S);
Heating bath at 42 DEG C, adds 4 parts of quality NHS, 100 parts of quality DMF and 6 part of quality dicyclohexylcarbodiimide (DCC), mixes in intermediate product I, takes out, is placed in overnight at room temperature; Heating bath at reaction mixture is placed in 42 DEG C again, till the precipitation in mixture is no longer dissolved; Take out, suction filtration while hot, abandons filter residue reserved filtrate; It is made to precipitate completely to gained filtrate added drop-wise anhydrous diethyl ether, suction filtration; Gained precipitation adds anhydrous diethyl ether and makes it precipitate completely, suction filtration after dissolving with trichloromethane; Repeat aforesaid operations 4 times; Finally gained being precipitated vacuum-drying, is final product II, that is: activate modifier mono methoxy polyethylene glycol succinimidyl succinate (mPEG-SS);
B. mPEG-SS modifies sisal SOD
By sisal SOD powder and mPEG-SS, 450 parts of 0.1mol/L pH9.2 borate buffer solutions are dissolved in by 1:70 mol ratio, shaken at room temperature reacts 2 hours, then in reaction solution, add 1mol/L phosphate buffer soln (PBS) termination reaction of 145 parts of cold pH7.0 of volume, the gained mixing solutions PBS buffered soln of 40mmol/L pH7.8 is dialysed, concentrated, go up the good Sephadex sephadex chromatography post wash-out of pre-balance to be immediately separated, collect 280nm first peak, lyophilize must modify sisal SOD lyophilized powder.
Utilize ultra-violet absorption spectrum, infrared absorption spectrum to prepare sisal SOD lyophilized powder to embodiment to identify, the results are shown in Figure 2 ~ 5.
Prepare sisal SOD lyophilized powder to embodiment and carry out temperature capacity, acid and alkali-resistance ability, proteolysis resistant ability mensuration, result is as follows:
(1) temperature capacity of modification SOD measures
Natural SOD and modification SOD are incubated 2 hours respectively in 25 ~ 90 DEG C of water-baths, respectively its vigor of sampling and measuring.At 30 ~ 50 DEG C, temperature improves, and the vigor of modification SOD and natural SOD improves gradually.When temperature is greater than 50 DEG C, modification SOD vigor declines gradually, and the vigor of natural SOD declines rapidly.Illustrate that modification SOD thermotolerance strengthens.Experimental result as shown in Figure 6.
Natural SOD and modification SOD are incubated 3.5 hours in the water-bath of 70 DEG C, at regular intervals its vigor of sampling and measuring.Along with the prolongation of time, the two vigor all progressively reduces.Soaking time 2.5 hours, obviously comparatively modification SOD is fast for natural SOD vigor lowering speed.Illustrate that gained modification SOD thermotolerance improves.Experimental result as shown in Figure 7.
(2) the acid and alkali-resistance ability of modification SOD measures
Natural SOD and modification SOD are dissolved in the buffered soln of pH4 ~ 11 respectively, mix, and 25 DEG C are incubated 2 hours, measure its vigor.PH4 ~ 6, the two vigor raises gradually.PH is greater than 6, and the two vigor declines gradually.PH is greater than 9, and natural SOD vigor declines rapidly; And pH is greater than 10, modification SOD vigor lowering speed is just accelerated.Illustrate that the more natural SOD of acid and alkali-resistance ability of modification SOD is high, and the acid-fast ability of modification SOD is higher than resistance to alkali ability.Experimental result as shown in Figure 8.
(3) modification SOD proteolysis resistant ability measures
Add stomach en-or trypsinase respectively at natural SOD and modification SOD, measure its vigor after 2 hours, vigor declines all to some extent.Wherein, the residual vigor of natural SOD is recorded after adding stomach en-or trypsinase all lower than the residual vigor of modification SOD.Illustrate that the natural SOD of proteolysis resistant energy force rate of modification SOD is high.Therefore natural SOD is after modifying, and effectively improves proteolysis resistant ability.

Claims (7)

1. modify a preparation method for sisal SOD lyophilized powder, it is characterized in that: comprise the following steps:
A. microwave-assisted activation mono methoxy polyethylene glycol mPEG synthesis activation modifier:
By 100 ~ 120 parts of quality mPEG, 3 ~ 6 parts of quality SA, 80 ~ 120 parts of quality DMF mix to obtain mixture; Mixture is placed in microwave oven reflux microwave radiation, stops radiation thing cool to room temperature to be mixed, add anhydrous diethyl ether or sherwood oil under stirring, make precipitation complete; Suction filtration, abandons filtrate and stays precipitation; In gained precipitation, add organic solvent under stirring, until precipitation is dissolved completely, suction filtration, abandons precipitation reserved filtrate; In gained filtrate, add anhydrous diethyl ether again or sherwood oil makes it precipitate completely, suction filtration, abandons filtrate and stays precipitation; Repeat aforesaid operations 3 ~ 5 times, obtain intermediate product I: mono methoxy polyethylene glycol succinate mPEG-S; Described aforesaid operations adds organic solvent under referring to stirring in gained precipitation, until precipitation is dissolved completely, suction filtration, abandons precipitation reserved filtrate; In gained filtrate, add anhydrous diethyl ether again or sherwood oil makes it precipitate completely, suction filtration, abandons filtrate and stays precipitation;
Heating bath at 40 ~ 45 DEG C, adds 3 ~ 5 parts of quality NHS, 80 ~ 120 parts of quality DMF and 5 ~ 8 part of quality dicyclohexylcarbodiimide (DCC), mixes in intermediate product I, takes out, is placed in overnight at room temperature and obtains reaction mixture; Heating bath at reaction mixture is placed in 40 ~ 45 DEG C again, till the precipitation in mixture is no longer dissolved; Take out, suction filtration while hot, abandons filter residue reserved filtrate; It is made to precipitate completely to gained filtrate added drop-wise anhydrous diethyl ether or sherwood oil, suction filtration; After gained precipitation organic solvent dissolution, add anhydrous diethyl ether or sherwood oil makes it precipitate completely, suction filtration; Repetitive operation 3 ~ 5 times; Finally gained being precipitated vacuum-drying, is final product II: activation modifier mono methoxy polyethylene glycol succinimidyl succinate mPEG-SS;
B. mPEG-SS modifies sisal SOD
By sisal SOD powder and mPEG-SS, 350 ~ 550 parts of borate buffer solutions are dissolved in by 1:10 ~ 1:80 mol ratio, shaken at room temperature reaction 1 ~ 3 hour, then in reaction solution, add the cold phosphate buffer soln PBS of 100 ~ 150 parts of volumes, termination reaction, gained mixing solutions PBS buffered soln is dialysed, is concentrated, and goes up the good Sephadex sephadex chromatography post wash-out of pre-balance immediately and is separated, collect 280nm first peak, lyophilize must modify sisal SOD lyophilized powder.
2. a kind of preparation method modifying sisal SOD lyophilized powder according to claim 1, is characterized in that: the organic solvent described in steps A is DMF, methylene dichloride, trichloromethane or tetracol phenixin.
3. a kind of preparation method modifying sisal SOD lyophilized powder according to claim 1, is characterized in that: the radiation power of the microwave radiation described in steps A is 10 ~ 90%.
4. a kind of preparation method modifying sisal SOD lyophilized powder according to claim 1, is characterized in that: the radiated time of the microwave radiation described in steps A is 3 ~ 60 minutes.
5. a kind of preparation method modifying sisal SOD lyophilized powder according to claim 1, is characterized in that: the concentration of the borate buffer solution described in step B is 0.1mol/L, pH is 9.2.
6. a kind of preparation method modifying sisal SOD lyophilized powder according to claim 1, is characterized in that: the concentration of the cold phosphate buffer soln PBS described in step B is 1mol/L, and pH value is 7.0, and temperature is 0 ~ 5 DEG C.
7. a kind of preparation method modifying sisal SOD lyophilized powder according to claim 1, is characterized in that: the concentration of the PBS buffered soln of the dialysis described in step B is 3 ~ 50mmol/L, pH is 7 ~ 8.
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