CN102618614B - Method for preparing urechis unicinctus glycosaminoglycan and antibacterial peptide simultaneously - Google Patents
Method for preparing urechis unicinctus glycosaminoglycan and antibacterial peptide simultaneously Download PDFInfo
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Abstract
The invention discloses a method for preparing urechis unicinctus glycosaminoglycan and antibacterial peptide simultaneously. The method includes: raw material process, producing raw material of urechis unicinctus into a homogenate; enzymolysis; ultrafitration; preparation of glycosaminoglycan, eliminating protein, ethanol precipitation and washing, anion exchange column chromatography, gel filtration chromatography to obtain glycosaminoglycan with high purity; and preparation of the antibacterial peptide. According to the method, the reaction condition is mild, the preparation process is environment-friendly and energy-saving, and the added value of products is high; the purity of the prepared glycosaminoglycan and the antibacterial peptide is high; a large amount of protein can be recycled, the comprehensive utilization of the urechis unicinctus is facilitated, the prepared glycosaminoglycan has obvious effects of anticoagulant and free radical scavenging, and the prepared antibacterial peptide has obvious bacteriostatic activity to staphylococcus aureus, Escherichia coli, Aeromonas hydrophila and the like; and the obtained glycosaminoglycan and the urechis unicinctus can provide fine immunopotentiators and novel antibacterial agents for marine rare life aquaculture.
Description
Technical field
The invention belongs to bioengineering field, relate to the preparation of glycosaminoglycan and antibacterial peptide, especially relate to a kind of method for preparing Urechis uniconctus glycosaminoglycan and antibacterial peptide simultaneously.
Background technology
Urechis uniconctus popular name sea intestines, extra large intestines belong to Echiuroidea, Yi guiding principle, do not have pipe Yi order, sting Yi section.Body is thick, is about 100-300mm, and wide about 25-27mm, body surface are abound with the granular projection that differs in size, and kiss is conical; 1 pair of neuroseta, thick; One circle 9-13 bar brown caudal seta is arranged around the anus, is the benthic common kind in the coastal silt bank of northern China tideland inferior segment and subtidal zone shoaling water.Research concentrates on morphological structure, fetal development, the life history, artificial breeding technique, Analysis of Nutritional and thrombus dissolving enzyme etc. to Urechis uniconctus at present, does not appear in the newspapers as yet for the Application and Development of Urechis uniconctus glycosaminoglycan and antibacterial peptide.
Glycosaminoglycan is the more rich material of content in the marine organisms, is the long-chain polysaccharide that is made of the multiple disaccharide unit, has the effect of tangible anticoagulation, antitumor, antiviral and enhance immunity power; Antibacterial peptide is the defensive peptide class active substance that a class that the animal immune system of defense produces under inductive condition is resisted exogenous pathogenic agent pathogenic effects, extensively be present in the plant and animal body, molecular mass is generally about 4ku, antibacterial peptide is biological intravital endogenous microbiotic, is the important component part of organism innate immunity system.
Summary of the invention
The purpose of this invention is to provide a kind of is starting material with the Urechis uniconctus, prepares the method for Urechis uniconctus glycosaminoglycan and antibacterial peptide simultaneously.
Technical scheme of the present invention is summarized as follows:
A kind of method for preparing Urechis uniconctus glycosaminoglycan and antibacterial peptide simultaneously comprises the steps:
(1) raw material is handled: after the raw material Urechis uniconctus was cleaned, homogenate was made in homogenate;
(2) enzymolysis: in described homogenate, add bacillus subtilis neutral proteinase, enzymolysis 4-6h under 43 ℃ of-47 ℃ of water bath condition, after enzymolysis solution boiled the enzyme that goes out, the centrifugal 10min-15min of 5000r-8000r/min got supernatant liquor;
(3) ultrafiltration: with the hollow fiber filter membrane ultra-filtration and separation of described supernatant liquor by relative molecular mass 10,000, obtain relative molecular mass 10,000 below with the liquid of the two kind components of relative molecular mass more than 10,000;
(4) preparation of glycosaminoglycan
1. isolating protein: with the liquid concentration of the component of described relative molecular mass more than 10,000 1/3-1/4 to this liquid volume, transfer pH to 2.0 with the HCl aqueous solution earlier, the centrifugal precipitation of removing, supernatant liquor is transferred pH to 7.0 with the NaOH aqueous solution, the centrifugal precipitation of removing, it is 3%-5% that supernatant liquor adding trichoroacetic acid(TCA) makes the final quality concentration of trichoroacetic acid(TCA), the centrifugal protein precipitation of removing;
2. alcohol precipitation, washing: the supernatant liquor that 1. step is obtained under agitation adds ethanol, making the alcoholic acid volumetric concentration is 70%-80%, under 4 ℃ of conditions, left standstill 20-28 hour, the centrifugal 10min-15min of 5000r-8000r/min, remove supernatant liquor, precipitation is used acetone and absolute ethanol washing 2-4 time successively, must slightly put forward glycosaminoglycan;
3. anion exchange chromatography: will slightly put forward glycosaminoglycan and be dissolved in the distilled water, making its mass concentration is 1%-3%, be added in the anion-exchange column, earlier with pH=5.0-5.5 acetate-sodium acetate buffer solution wash-out, use the NaCl aqueous solution wash-out of 0-2mol/L gradient again, elutriant is concentrated into the 1/2-1/4, dialysis, reconcentration of effluent volume to the long-pending 1/3-1/4 of dialysis back liquid;
4. gel permeation chromatography: the concentrated solution that 3. step is obtained is splined on the SephadexG-100 gel-filtration column, further separation and purification, and the effluent liquid of collection is concentrated into the 1/4-1/6 of effluent volume, and the concentrated solution lyophilize gets high-purity glycosaminoglycan;
(5) antibacterial peptide preparation: with the liquid concentration of described relative molecular weight 10,000 following components 1/2-1/4 to this liquid volume, be splined on the SephadexG-100 gel-filtration column, with 0.2mol/L pH=7.0 aqueous sodium acetate solution wash-out, elutriant is concentrated into the 1/2-1/3 of this effluent volume, concentrated solution is splined on the SephadexG-25 gel-filtration column, 0.2mol/L pH=7.0 aqueous sodium acetate solution wash-out, elutriant is concentrated into the 1/4-1/6 of this effluent volume, and the concentrated solution lyophilize gets the pure product of antibacterial peptide.
Best is that the addition of bacillus subtilis neutral proteinase is the 0.8%-1% of homogenate quality in the step (2).
Preferred DEAE-52 Mierocrystalline cellulose of the filler of anion-exchange column or Q-Sepharose Fast Flow.
Advantage of the present invention and positively effect are:
1. method reaction conditions gentleness of the present invention, preparation process environment protecting and power-saving, a kind of raw material prepares two kinds of active substances, added value of product height simultaneously;
2. it is raw material that method of the present invention adopts Urechis uniconctus, the glycosaminoglycan of preparation and antibacterial peptide purity height;
3. present method also can reclaim protein in a large number in preparation glycosaminoglycan and antibacterial peptide, help the comprehensive utilization of Urechis uniconctus, prepared glycosaminoglycan has tangible anticoagulation (three indexs of AT, PT and APTT all are significantly higher than the blank group), removes the effect of free radical (the removing ability for hydroxy radical qiao and ultra-oxygen anion free radical is significantly higher than the blank group), and the antibacterial peptide of preparation has tangible bacteriostatic activity for streptococcus aureus, intestinal bacteria, Aeromonas hydrophila etc.; The glycosaminoglycan of gained and antibacterial peptide also can be extra large precious animal farming industry good immunostimulant and novel antibacterial medicine are provided.
Description of drawings
Fig. 1 is the influence of Urechis uniconctus glycosaminoglycan to white rabbit TT experiment.
Fig. 2 is the influence of Urechis uniconctus glycosaminoglycan to white rabbit PT experiment.
Fig. 3 is the influence of Urechis uniconctus glycosaminoglycan to white rabbit APTT experiment.
Fig. 4 is an antibacterial peptide inhibition zone measurement result.
Embodiment
The invention will be further described below in conjunction with embodiment, and following embodiment is illustrative, is not determinate, can not limit protection scope of the present invention with following embodiment.
The extraction object that present embodiment adopted is to be selected from echiuran-Urechis uniconctus that the Bohai Sea extensively distributes.
Embodiment 1
A kind of method for preparing Urechis uniconctus glycosaminoglycan and antibacterial peptide simultaneously may further comprise the steps:
(1) raw material is handled: after Urechis uniconctus was cleaned, homogenate was made in homogenate;
(2) enzymolysis: add bacillus subtilis neutral proteinase in homogenate, the addition of bacillus subtilis neutral proteinase is 1% of a homogenate quality, enzymolysis 5h under 45 ℃ of water bath condition, and after enzymolysis solution boiled the enzyme that goes out, the centrifugal 10min of 6000r/min got supernatant liquor;
(3) ultrafiltration: with the hollow fiber filter membrane ultra-filtration and separation of supernatant liquor by relative molecular mass 10,000, obtain relative molecular mass 10,000 below with the liquid of the two kind components of relative molecular mass more than 10,000;
(4) preparation of glycosaminoglycan
1. isolating protein: with the liquid concentration of the component of relative molecular mass more than 10,000 to 1/3 of this liquid volume, transfer pH to 2.0 with the HCl aqueous solution earlier, the centrifugal precipitation of removing, supernatant liquor is transferred pH to 7.0 with the NaOH aqueous solution, the centrifugal precipitation of removing, it is 4% that supernatant liquor adding trichoroacetic acid(TCA) makes the final quality concentration of trichoroacetic acid(TCA), the centrifugal protein precipitation of removing;
2. alcohol precipitation, washing: the supernatant liquor that 1. step is obtained under agitation adds ethanol, and making the alcoholic acid volumetric concentration is 80%, leaves standstill 24 hours under 4 ℃ of conditions, the centrifugal 10min of 6000r/min, remove supernatant liquor, precipitation is used acetone and absolute ethanol washing 3 times successively, slightly put forward glycosaminoglycan;
3. anion exchange chromatography: will slightly put forward glycosaminoglycan and be dissolved in the distilled water, making its mass concentration is 2%, being added to filler is in the cellulosic anion-exchange column of DEAE-52, earlier with pH=5.3 acetate-sodium acetate buffer solution wash-out, use the NaCl aqueous solution wash-out of 0-2mol/L gradient again, elutriant (anticoagulant active is stronger) be concentrated into effluent volume 1/3, dialysis, reconcentration to dialysis back liquid long-pending 1/3;
4. gel permeation chromatography: the concentrated solution that 3. step is obtained is splined on the SephadexG-100 gel-filtration column, further separation and purification, and the effluent liquid of collection is concentrated into 1/5 of effluent volume, and the concentrated solution lyophilize gets high-purity glycosaminoglycan;
(5) antibacterial peptide preparation: with the liquid concentration of described relative molecular weight 10,000 following components to 1/3 of this liquid volume, be splined on the SephadexG-100 gel-filtration column, with 0.2mol/L pH=7.0 aqueous sodium acetate solution wash-out, elutriant is concentrated into 1/3 of this effluent volume, concentrated solution is splined on the SephadexG-25 gel-filtration column, 0.2mol/L pH=7.0 aqueous sodium acetate solution wash-out, elutriant is concentrated into 1/4 of this effluent volume, and the concentrated solution lyophilize gets the pure product of antibacterial peptide.
By the check, the glycosaminoglycan purity of preparation reaches 94.6%, yield be raw material heavy 0.32%; Antibacterial peptide purity reaches 92.1%, yield be raw material heavy 0.08%.
A kind of method for preparing Urechis uniconctus glycosaminoglycan and antibacterial peptide simultaneously comprises the steps:
(1) raw material is handled: after the raw material Urechis uniconctus was cleaned, homogenate was made in homogenate;
(2) enzymolysis: in described homogenate, add bacillus subtilis neutral proteinase, the addition of bacillus subtilis neutral proteinase is 0.8% of a homogenate quality, and enzymolysis 6h under 43 ℃ of water bath condition is after enzymolysis solution boils the enzyme that goes out, the centrifugal 10min of 8000r/min gets supernatant liquor;
(3) ultrafiltration: with the hollow fiber filter membrane ultra-filtration and separation of described supernatant liquor by relative molecular mass 10,000, obtain relative molecular mass 10,000 below with the liquid of the two kind components of relative molecular mass more than 10,000;
(4) preparation of glycosaminoglycan
1. isolating protein: with the liquid concentration of the component of described relative molecular mass more than 10,000 to 1/3 of this liquid volume, transfer pH to 2.0 with the HCl aqueous solution earlier, the centrifugal precipitation of removing, supernatant liquor is transferred pH to 7.0 with the NaOH aqueous solution, the centrifugal precipitation of removing, it is 3% that supernatant liquor adding trichoroacetic acid(TCA) makes the final quality concentration of trichoroacetic acid(TCA), the centrifugal protein precipitation of removing;
2. alcohol precipitation, washing: the supernatant liquor that 1. step is obtained under agitation adds ethanol, and making the alcoholic acid volumetric concentration is 75%, leaves standstill 24 hours under 4 ℃ of conditions, the centrifugal 13min of 7000r/min, remove supernatant liquor, precipitation is used acetone and absolute ethanol washing 3 times successively, slightly put forward glycosaminoglycan;
3. anion exchange chromatography: will slightly put forward glycosaminoglycan and be dissolved in the distilled water, making its mass concentration is 1%, being added to filler is in the cellulosic anion-exchange column of DEAE-52, earlier with pH=5.0 acetate-sodium acetate buffer solution wash-out, use the NaCl aqueous solution wash-out of 0-2mol/L gradient again, elutriant (anticoagulant active is stronger) be concentrated into effluent volume 1/3, dialysis, reconcentration to dialysis back liquid long-pending 1/3;
4. gel permeation chromatography: the concentrated solution that 3. step is obtained is splined on the SephadexG-100 gel-filtration column, further separation and purification, and the effluent liquid of collection is concentrated into 1/4 of effluent volume, and the concentrated solution lyophilize gets high-purity glycosaminoglycan;
(5) antibacterial peptide preparation: with the liquid concentration of described relative molecular weight 10,000 following components to 1/2 of this liquid volume, be splined on the SephadexG-100 gel-filtration column, with 0.2mol/L pH=7.0 aqueous sodium acetate solution wash-out, elutriant is concentrated into 1/2 of this effluent volume, concentrated solution is splined on the SephadexG-25 gel-filtration column, 0.2mol/L pH=7.0 aqueous sodium acetate solution wash-out, elutriant is concentrated into 1/5 of this effluent volume, and the concentrated solution lyophilize gets the pure product of antibacterial peptide.
By the check, the glycosaminoglycan purity of preparation reaches 92.8%, yield be raw material heavy 0.28%; Antibacterial peptide purity reaches 92.5%, yield be raw material heavy 0.07%.
Embodiment 3
A kind of method for preparing Urechis uniconctus glycosaminoglycan and antibacterial peptide simultaneously comprises the steps:
(1) raw material is handled: after the raw material Urechis uniconctus was cleaned, homogenate was made in homogenate;
(2) enzymolysis: in described homogenate, add bacillus subtilis neutral proteinase, the addition of bacillus subtilis neutral proteinase is 0.9% of a homogenate quality, and enzymolysis 5h under 45 ℃ of water bath condition is after enzymolysis solution boils the enzyme that goes out, the centrifugal 15min of 5000r/min gets supernatant liquor;
(3) ultrafiltration: with the hollow fiber filter membrane ultra-filtration and separation of described supernatant liquor by relative molecular mass 10,000, obtain relative molecular mass 10,000 below with the liquid of the two kind components of relative molecular mass more than 10,000;
(4) preparation of glycosaminoglycan
1. isolating protein: with the liquid concentration of the component of described relative molecular mass more than 10,000 to 1/4 of this liquid volume, transfer pH to 2.0 with the HCl aqueous solution earlier, the centrifugal precipitation of removing, supernatant liquor is transferred pH to 7.0 with the NaOH aqueous solution, the centrifugal precipitation of removing, it is 4% that supernatant liquor adding trichoroacetic acid(TCA) makes the final quality concentration of trichoroacetic acid(TCA), the centrifugal protein precipitation of removing;
2. alcohol precipitation, washing: the supernatant liquor that 1. step is obtained under agitation adds ethanol, and making the alcoholic acid volumetric concentration is 80%, leaves standstill 20 hours under 4 ℃ of conditions, the centrifugal 15min of 5000r/min, remove supernatant liquor, precipitation is used acetone and absolute ethanol washing 2 times successively, slightly put forward glycosaminoglycan;
3. anion exchange chromatography: will slightly put forward glycosaminoglycan and be dissolved in the distilled water, making its mass concentration is 2%, be added in the anion-exchange column that filler is Q-Sepharose Fast Flow, earlier with pH=5.3 acetate-sodium acetate buffer solution wash-out, use the NaCl aqueous solution wash-out of 0-2mol/L gradient again, elutriant (anticoagulant active is stronger) be concentrated into effluent volume 1/2, dialysis, reconcentration to dialysis back liquid long-pending 1/3;
4. gel permeation chromatography: the concentrated solution that 3. step is obtained is splined on the SephadexG-100 gel-filtration column, further separation and purification, and the effluent liquid of collection is concentrated into 1/5 of effluent volume, and the concentrated solution lyophilize gets high-purity glycosaminoglycan;
(5) antibacterial peptide preparation: with the liquid concentration of described relative molecular weight 10,000 following components to 1/3 of this liquid volume, be splined on the SephadexG-100 gel-filtration column, with 0.2mol/L pH=7.0 aqueous sodium acetate solution wash-out, elutriant is concentrated into 1/3 of this effluent volume, concentrated solution is splined on the SephadexG-25 gel-filtration column, 0.2mol/L pH=7.0 aqueous sodium acetate solution wash-out, elutriant is concentrated into 1/4 of this effluent volume, and the concentrated solution lyophilize gets the pure product of antibacterial peptide.
By the check, the glycosaminoglycan purity of preparation reaches 94.8%, yield be raw material heavy 0.31%; Antibacterial peptide purity reaches 91.8%, yield be raw material heavy 0.059%.
A kind of method for preparing Urechis uniconctus glycosaminoglycan and antibacterial peptide simultaneously comprises the steps:
(1) raw material is handled: after the raw material Urechis uniconctus was cleaned, homogenate was made in homogenate;
(2) enzymolysis: add bacillus subtilis neutral proteinase in described homogenate, the addition of bacillus subtilis neutral proteinase is 1% of a homogenate quality, enzymolysis 4h under 47 ℃ of water bath condition, and after enzymolysis solution boiled the enzyme that goes out, the centrifugal 13min of 6000r/min got supernatant liquor;
(3) ultrafiltration: with the hollow fiber filter membrane ultra-filtration and separation of described supernatant liquor by relative molecular mass 10,000, obtain relative molecular mass 10,000 below with the liquid of the two kind components of relative molecular mass more than 10,000;
(4) preparation of glycosaminoglycan
1. isolating protein: with the liquid concentration of the component of described relative molecular mass more than 10,000 to 1/4 of this liquid volume, transfer pH to 2.0 with the HCl aqueous solution earlier, the centrifugal precipitation of removing, supernatant liquor is transferred pH to 7.0 with the NaOH aqueous solution, the centrifugal precipitation of removing, it is 5% that supernatant liquor adding trichoroacetic acid(TCA) makes the final quality concentration of trichoroacetic acid(TCA), the centrifugal protein precipitation of removing;
2. alcohol precipitation, washing: the supernatant liquor that 1. step is obtained under agitation adds ethanol, and making the alcoholic acid volumetric concentration is 70%, leaves standstill 28 hours under 4 ℃ of conditions, the centrifugal 10min of 8000r/min, remove supernatant liquor, precipitation is used acetone and absolute ethanol washing 4 times successively, slightly put forward glycosaminoglycan;
3. anion exchange chromatography: will slightly put forward glycosaminoglycan and be dissolved in the distilled water, making its mass concentration is 3%, be added in the anion-exchange column that filler is Q-Sepharose Fast Flow, earlier with pH=5.5 acetate-sodium acetate buffer solution wash-out, use the NaCl aqueous solution wash-out of 0-2mol/L gradient again, elutriant (anticoagulant active is stronger) be concentrated into effluent volume 1/4, dialysis, reconcentration to dialysis back liquid long-pending 1/4;
4. gel permeation chromatography: the concentrated solution that 3. step is obtained is splined on the SephadexG-100 gel-filtration column, further separation and purification, and the effluent liquid of collection is concentrated into 1/6 of effluent volume, and the concentrated solution lyophilize gets high-purity glycosaminoglycan;
(5) antibacterial peptide preparation: with the liquid concentration of described relative molecular weight 10,000 following components to 1/4 of this liquid volume, be splined on the SephadexG-100 gel-filtration column, with 0.2mol/L pH=7.0 aqueous sodium acetate solution wash-out, elutriant is concentrated into 1/3 of this effluent volume, concentrated solution is splined on the SephadexG-25 gel-filtration column, 0.2mol/L pH=7.0 aqueous sodium acetate solution wash-out, elutriant is concentrated into 1/6 of this effluent volume, and the concentrated solution lyophilize gets the pure product of antibacterial peptide.
By the check, the glycosaminoglycan purity of preparation reaches 94.3%, yield be raw material heavy 0.29%; Antibacterial peptide purity reaches 93.4%, yield be raw material heavy 0.071%.
Embodiment 5
Urechis uniconctus glycosaminoglycan anticoagulation:
1 materials and methods
1.1 experiment material
Laboratory animal is selected New Zealand white rabbit for use, and glycosaminoglycan (GAG) is from Urechis uniconctus (embodiment 1 preparation).Laboratory apparatus: LG-PABER type thrombin analyser.
1.2 experimental technique
Adopt the experiment in vitro method.
(1) mensuration of prothrombin time (PT) and thrombin time (TT):
New Zealand white rabbit, vetanarcol anesthesia, heart extracting blood, 3.8% Sodium Citrate anti-freezing (1: 9), behind the mixing with 3000r/min, centrifugal 15min gets supernatant, isolates platelet poor plasma (PPP), get PPP 100 μ l, add glycosaminoglycan 10 μ l, add PT and the TT reagent of pre-temperature 15min behind the pre-temperature 5min, with platelet aggregation thrombin analysis-e/or determining setting time.
(2) mensuration of activatory partial thromboplastin time (APTT):
Top method is isolated platelet poor plasma (PPP), get PPP 100 μ l, add 10 μ l glycosaminoglycan and 100 μ l APTT reagent, add the CaCl2100 μ l of pre-temperature 15min behind the pre-temperature 5min, with platelet aggregation thrombin analysis-e/or determining setting time.
2 results and analysis
It is blank that physiological saline is established in experiment, 0.005mg/ml and the heparin sodium of 0.01mg/ml is done positive control, the glycosaminoglycan setting is an initial concentration with the peak concentration of heparin, 0.01mg/ml, 0.2mg/ml, 0.4mg/ml, five concentration groups of 0.6mg/ml, 0.8mg/ml, carry out external anticoagulation test, the results are shown in Figure 1,2,3, find out from three figure, the Urechis uniconctus glycosaminoglycan can obviously prolong PT and APTT, and effect is weaker than heparin slightly, for TT certain prolongation effect is arranged also.
The anti-microbial activity of Urechis uniconctus antibacterial peptide:
1 experimental technique
Adopt the Oxford agar diffusion method.The concentration of Vibrio parahaemolyticus and Aeromonas hydrophila is 2.3 * 10
6Cfu/ml, the concentration of intestinal bacteria and streptococcus aureus is 1.2 * 10
6Cfu/ml, in each Oxford cup, add 200 μ l antibacterial peptides (embodiment 1 preparation), do positive control with penicillin, aqua sterilisa is done negative control, 4 ℃ change constant incubator over to after leaving standstill 24h, and intestinal bacteria and streptococcus aureus are cultivated 24h for 37 ℃, and Vibrio parahaemolyticus and Aeromonas hydrophila are cultivated 24h for 30 ℃, cultivate the back and measure antibacterial circle diameter, and judge the power of bacteriostatic activity according to its diameter with vernier callipers.
2 experimental results
The results are shown in Figure 4, as can be seen from Figure 4, antibacterial peptide all has tangible bacteriostatic activity to Vibrio parahaemolyticus, Aeromonas hydrophila, intestinal bacteria and streptococcus aureus, and is wherein the strongest to the Vibrio parahaemolyticus bacteriostatic activity, Aeromonas hydrophila is taken second place, the most weak to the streptococcus aureus bacteriostatic activity.
Claims (2)
1. a method for preparing Urechis uniconctus glycosaminoglycan and antibacterial peptide simultaneously is characterized in that comprising the steps:
⑴ raw material is handled: after the raw material Urechis uniconctus was cleaned, homogenate was made in homogenate;
⑵ enzymolysis: in described homogenate, add bacillus subtilis neutral proteinase, enzymolysis 4-6h under 43 ℃ of-47 ℃ of water bath condition, after enzymolysis solution boiled the enzyme that goes out, the centrifugal 10min-15min of 5000r-8000r/min got supernatant liquor; The addition of described bacillus subtilis neutral proteinase is the 0.8%-1% of described homogenate quality;
⑶ ultrafiltration: with the hollow fiber filter membrane ultra-filtration and separation of described supernatant liquor by relative molecular mass 10,000, obtain relative molecular mass 10,000 below with the liquid of the two kind components of relative molecular mass more than 10,000;
⑷ the preparation of glycosaminoglycan
1. isolating protein: with the liquid concentration of the component of described relative molecular mass more than 10,000 1/3-1/4 to this liquid volume, transfer pH to 2.0 with the HCl aqueous solution earlier, the centrifugal precipitation of removing, supernatant liquor is transferred pH to 7.0 with the NaOH aqueous solution, the centrifugal precipitation of removing, it is 3%-5% that supernatant liquor adding trichoroacetic acid(TCA) makes the final quality concentration of trichoroacetic acid(TCA), the centrifugal protein precipitation of removing;
2. alcohol precipitation, washing: the supernatant liquor that 1. step is obtained under agitation adds ethanol, making the alcoholic acid volumetric concentration is 70%-80%, under 4 ℃ of conditions, left standstill 20-28 hour, the centrifugal 10min-15min of 5000r-8000r/min, remove supernatant liquor, precipitation is used acetone and absolute ethanol washing 2-4 time successively, must slightly put forward glycosaminoglycan;
3. anion exchange chromatography: will slightly put forward glycosaminoglycan and be dissolved in the distilled water, making its mass concentration is 1%-3%, be added in the anion-exchange column, earlier with pH=5.0-5.5 acetate-sodium acetate buffer solution wash-out, use the NaCl aqueous solution wash-out of 0-2mol/L gradient again, elutriant is concentrated into the 1/2-1/4, dialysis, reconcentration of effluent volume to the long-pending 1/3-1/4 of dialysis back liquid;
4. gel permeation chromatography: the concentrated solution that 3. step is obtained is splined on the SephadexG-100 gel-filtration column, further separation and purification, and the effluent liquid of collection is concentrated into the 1/4-1/6 of effluent volume, and the concentrated solution lyophilize gets high-purity glycosaminoglycan;
⑸ antibacterial peptide preparation: with the liquid concentration of described relative molecular weight 10,000 following components 1/2-1/4 to this liquid volume, be splined on the SephadexG-100 gel-filtration column, with 0.2mol/L pH=7.0 aqueous sodium acetate solution wash-out, elutriant is concentrated into the 1/2-1/3 of this effluent volume, concentrated solution is splined on the SephadexG-25 gel-filtration column, 0.2mol/L pH=7.0 aqueous sodium acetate solution wash-out, elutriant is concentrated into the 1/4-1/6 of this effluent volume, and the concentrated solution lyophilize gets the pure product of antibacterial peptide.
2. a kind of method for preparing Urechis uniconctus glycosaminoglycan and antibacterial peptide simultaneously according to claim 1, the filler that it is characterized in that described anion-exchange column are DEAE-52 Mierocrystalline cellulose or Q-Sepharose Fast Flow.
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| CN118324892B (en) * | 2024-06-12 | 2024-09-06 | 中国科学院烟台海岸带研究所 | Antibacterial peptide derived from urechis unicinctus, mutant and application thereof |
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