CN100348302C - Method for preparing water-soluble affinity ultrafiltration carrier - Google Patents
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- CN100348302C CN100348302C CNB2004100271917A CN200410027191A CN100348302C CN 100348302 C CN100348302 C CN 100348302C CN B2004100271917 A CNB2004100271917 A CN B2004100271917A CN 200410027191 A CN200410027191 A CN 200410027191A CN 100348302 C CN100348302 C CN 100348302C
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- 238000000108 ultra-filtration Methods 0.000 title claims abstract description 36
- 238000000034 method Methods 0.000 title abstract description 11
- 108010064983 Ovomucin Proteins 0.000 claims abstract description 19
- 238000002360 preparation method Methods 0.000 claims abstract description 5
- 238000005406 washing Methods 0.000 claims abstract 2
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 34
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 20
- 239000000243 solution Substances 0.000 claims description 18
- 239000012153 distilled water Substances 0.000 claims description 14
- 229920002307 Dextran Polymers 0.000 claims description 13
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 10
- CDBYLPFSWZWCQE-UHFFFAOYSA-L Sodium Carbonate Chemical compound [Na+].[Na+].[O-]C([O-])=O CDBYLPFSWZWCQE-UHFFFAOYSA-L 0.000 claims description 10
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 10
- 239000000872 buffer Substances 0.000 claims description 8
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 claims description 6
- 239000007788 liquid Substances 0.000 claims description 6
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 5
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 claims description 5
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 5
- 239000011575 calcium Substances 0.000 claims description 5
- 229910052791 calcium Inorganic materials 0.000 claims description 5
- 235000019253 formic acid Nutrition 0.000 claims description 5
- 239000011591 potassium Substances 0.000 claims description 5
- 229910052700 potassium Inorganic materials 0.000 claims description 5
- 239000001103 potassium chloride Substances 0.000 claims description 5
- 235000011164 potassium chloride Nutrition 0.000 claims description 5
- 229910000029 sodium carbonate Inorganic materials 0.000 claims description 5
- 239000007853 buffer solution Substances 0.000 claims description 4
- 238000006243 chemical reaction Methods 0.000 claims description 4
- 238000012545 processing Methods 0.000 claims description 2
- 229920001503 Glucan Polymers 0.000 claims 3
- 239000012530 fluid Substances 0.000 claims 3
- LRWZZZWJMFNZIK-UHFFFAOYSA-N 2-chloro-3-methyloxirane Chemical compound CC1OC1Cl LRWZZZWJMFNZIK-UHFFFAOYSA-N 0.000 claims 2
- 230000008878 coupling Effects 0.000 claims 1
- 238000010168 coupling process Methods 0.000 claims 1
- 238000005859 coupling reaction Methods 0.000 claims 1
- 238000004821 distillation Methods 0.000 claims 1
- 108090000631 Trypsin Proteins 0.000 abstract description 25
- 102000004142 Trypsin Human genes 0.000 abstract description 25
- 239000012588 trypsin Substances 0.000 abstract description 25
- 108090000317 Chymotrypsin Proteins 0.000 abstract description 12
- 229960002376 chymotrypsin Drugs 0.000 abstract description 12
- 238000000746 purification Methods 0.000 abstract description 9
- BRLQWZUYTZBJKN-UHFFFAOYSA-N Epichlorohydrin Chemical compound ClCC1CO1 BRLQWZUYTZBJKN-UHFFFAOYSA-N 0.000 abstract description 8
- 238000011084 recovery Methods 0.000 abstract description 5
- 230000004913 activation Effects 0.000 abstract description 2
- 239000012465 retentate Substances 0.000 description 28
- 238000003756 stirring Methods 0.000 description 17
- 239000012528 membrane Substances 0.000 description 11
- 238000010828 elution Methods 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- 102000035195 Peptidases Human genes 0.000 description 3
- 108091005804 Peptidases Proteins 0.000 description 3
- 239000004365 Protease Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- -1 epichlorohydrin small molecules Chemical class 0.000 description 3
- 238000001179 sorption measurement Methods 0.000 description 3
- 230000000694 effects Effects 0.000 description 2
- 239000004615 ingredient Substances 0.000 description 2
- 229920002521 macromolecule Polymers 0.000 description 2
- 230000000717 retained effect Effects 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- 230000009286 beneficial effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000003760 magnetic stirring Methods 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 230000036632 reaction speed Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000013341 scale-up Methods 0.000 description 1
- 150000003384 small molecules Chemical class 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
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Abstract
本发明涉及一种水溶性亲和超滤载体的制备方法,采用环氧氯丙烷活化法,制备一种葡聚糖-卵粘蛋白水溶性的大分子载体,用于超滤过程。由于胰蛋白酶与胰凝乳蛋白酶的分子量相近,通过卵粘蛋白特异性的吸附胰蛋白酶,滤过胰凝乳蛋白酶,使胰蛋白酶与胰凝乳蛋白酶有效的分离开来,吸附的胰蛋白酶通过洗脱得以纯化,获得胰蛋白酶纯化倍数达到93倍,回收80%以上。The invention relates to a preparation method of a water-soluble affinity ultrafiltration carrier. An epichlorohydrin activation method is used to prepare a dextran-ovomucin water-soluble macromolecular carrier for the ultrafiltration process. Since the molecular weight of trypsin and chymotrypsin is similar, the trypsin is specifically adsorbed by ovomucin, the chymotrypsin is filtered, and the trypsin and chymotrypsin are effectively separated, and the adsorbed trypsin is washed by washing The removal was purified, the purification ratio of trypsin was 93 times, and the recovery was more than 80%.
Description
技术领域technical field
本发明涉及有机化工领域,具体是一种水溶性亲和超滤载体的制备方法。The invention relates to the field of organic chemical industry, in particular to a preparation method of a water-soluble affinity ultrafiltration carrier.
背景技术Background technique
目前超滤技术主要用于生物大分子的分离纯化,具有保持物质活性,容易工业放大的优点。但是单纯超滤分离技术对分子量相近的成分难以分开。针对此问题采用亲和-超滤技术,即合成一种水溶性大分子量亲和载体,特异吸附目标分子,使目标分子被滤膜截留,滤过杂质分子,最后目标分子通过洗脱获得纯化。本发明采用可溶性200万分子量的葡聚糖通过环氧氯丙烷活化,共价连接卵粘蛋白,形成可溶性大分子量亲和载体,应用于制备高纯度胰蛋白酶。At present, ultrafiltration technology is mainly used for the separation and purification of biological macromolecules, which has the advantages of maintaining the activity of substances and being easy for industrial scale-up. However, pure ultrafiltration separation technology is difficult to separate components with similar molecular weights. Affinity-ultrafiltration technology is used to solve this problem, that is, a water-soluble large-molecular-weight affinity carrier is synthesized to specifically adsorb target molecules, so that the target molecules are intercepted by the filter membrane, filter out impurity molecules, and finally the target molecules are purified by elution. The invention adopts soluble dextran with a molecular weight of 2 million to be activated by epichlorohydrin, covalently connects ovomucin to form a soluble large molecular weight affinity carrier, and is applied to prepare high-purity trypsin.
发明内容Contents of the invention
本发明的目的在于提供一种水溶性亲和超滤载体的制备方法,制备一种水可溶性大分子亲和载体,应用于亲和-超滤过程,使分子量相近的成分得以分开,使目标分子获得纯化。例如由于胰蛋白酶与胰凝乳蛋白酶的分子量相近,通过卵粘蛋白特异性的吸附胰蛋白酶,滤过胰凝乳蛋白酶,使胰蛋白酶与胰凝乳蛋白酶有效的分离开来,吸附的胰蛋白酶通过洗脱得以纯化,获得胰蛋白酶纯化倍数达到93倍,回收80%以上。The purpose of the present invention is to provide a method for preparing a water-soluble affinity ultrafiltration carrier, to prepare a water-soluble macromolecular affinity carrier, which is applied to the affinity-ultrafiltration process, so that components with similar molecular weights can be separated, and target molecules can be separated. Get purified. For example, due to the similar molecular weight of trypsin and chymotrypsin, trypsin is specifically adsorbed by ovomucin, and chymotrypsin is filtered to effectively separate trypsin from chymotrypsin, and the adsorbed trypsin passes through The elution was purified, the purification factor of trypsin was 93 times, and the recovery was more than 80%.
本发明的方法是采用环氧氯丙烷活化的方法,制备出一种葡聚糖-卵粘蛋白水溶性亲和超滤大分子载体。The method of the invention adopts the method of epichlorohydrin activation to prepare a dextran-ovomucin water-soluble affinity ultrafiltration macromolecule carrier.
本发明的水溶性亲和超滤载体的制备方法具体地包括以下步骤:The preparation method of the water-soluble affinity ultrafiltration carrier of the present invention specifically comprises the following steps:
第一步、在配料杯中加入葡聚糖和氢氧化钠,使葡聚糖浓度达到3.5~4.5%重量比,氢氧化钠浓度达到0.4~0.5mol/L,搅拌至溶解完全,处理30~40分钟;所述葡聚糖分子量150-300万,最佳是200万;The first step is to add dextran and sodium hydroxide to the ingredient cup, so that the concentration of dextran reaches 3.5-4.5% by weight, and the concentration of sodium hydroxide reaches 0.4-0.5mol/L, stir until it is completely dissolved, and process for 30- 40 minutes; the molecular weight of the dextran is 1.5-3 million, preferably 2 million;
第二步、加入4~5%体积环氧氯丙烷;The second step, add 4~5% epichlorohydrin by volume;
第三步、将第二步得到的料液放入55~60℃恒温摇床,摇床转速100~110r/min,避光反应1.8~2小时;The third step is to put the feed liquid obtained in the second step into a constant temperature shaker at 55-60°C, the speed of the shaker is 100-110r/min, and react in the dark for 1.8-2 hours;
第四步、将第三步得到的料液移至超滤膜分离装置中,膜截留分子量为10万,超滤除去溶液中剩余的环氧氯丙烷,氢氧化钠小分子;先用蒸馏水洗至pH7.8~8.2,再用0.1M,pH9.5碳酸钠缓冲液洗,至截留液体积为配料杯中原液体积的1.2±0.1倍;The fourth step, move the feed liquid obtained in the third step to the ultrafiltration membrane separation device, the molecular weight cut-off of the membrane is 100,000, ultrafiltration removes the remaining epichlorohydrin and small molecules of sodium hydroxide in the solution; first wash with distilled water To pH7.8~8.2, then wash with 0.1M, pH9.5 sodium carbonate buffer until the volume of the retained solution is 1.2±0.1 times the volume of the original solution in the ingredient cup;
第五步、将卵粘蛋白溶解在第四步得到的截留液,使卵粘蛋白浓度为6±0.1g/L;移至35~40℃恒温摇床,摇床转速100~110r/min,避光处理23~24小时;The fifth step, dissolve ovomucin in the retentate obtained in the fourth step, so that the concentration of ovomucin is 6±0.1g/L; move to a constant temperature shaker at 35-40°C, and the shaker speed is 100-110r/min, Protect from light for 23-24 hours;
第六步、处理后的溶液移至超滤膜分离装置中,膜截留分子量为14万,除去未被偶联的卵粘蛋白,先用蒸馏水洗,然后用0.1M甲酸-0.5M氯化钾,pH2.5甲酸洗,又用蒸馏水洗至pH6.2~6.5,最后用0.5M氯化钾-0.05M氯化钙-0.1M、pH7.8Tris-HCl缓冲液洗,至葡聚糖溶解在Tris-HCl缓冲液中,至截留液体积为配料杯中原液体积的1.2±0.1倍,搅拌1~1.5小时,获得葡聚糖-卵粘蛋白水溶性亲和超滤载体。Step 6: Move the treated solution to an ultrafiltration membrane separation device with a molecular weight cut-off of 140,000 to remove uncoupled ovomucin, wash it with distilled water, and then use 0.1M formic acid-0.5M potassium chloride , washed with formic acid at pH 2.5, washed with distilled water to pH 6.2-6.5, and finally washed with 0.5M potassium chloride-0.05M calcium chloride-0.1M, pH7.8 Tris-HCl buffer until the dextran was dissolved in In Tris-HCl buffer solution, until the volume of the retained solution is 1.2±0.1 times the volume of the original solution in the batching cup, stir for 1-1.5 hours to obtain the dextran-ovomucin water-soluble affinity ultrafiltration carrier.
本发明与现有技术相比,具有如下的优点和有益效果:解决了单纯超滤分离中对分子量相近的组分纯化效率低下的难题,利用卵粘蛋白对胰蛋白酶的特异亲和吸附作用,获得的卵粘蛋白-葡聚糖亲和超滤载体对粗胰蛋白酶进行纯化,获得很好的纯化效果。合成的水溶性亲和载体有许多优点:亲和载体与目标蛋白在均相体系中反应速度快,达到吸附或洗脱平衡的时间短,吸附容量大。Compared with the prior art, the present invention has the following advantages and beneficial effects: it solves the problem of low purification efficiency of components with similar molecular weights in simple ultrafiltration separation, and utilizes the specific affinity adsorption of ovomucin to trypsin, The obtained ovomucin-dextran affinity ultrafiltration carrier is used for purification of crude trypsin, and a good purification effect is obtained. The synthetic water-soluble affinity carrier has many advantages: the reaction speed between the affinity carrier and the target protein in a homogeneous system is short, the time to reach adsorption or elution equilibrium is short, and the adsorption capacity is large.
发明人对本发明——葡聚糖-卵粘蛋白亲和超滤载体的制备经过长期的创造性研究和实验,有许多成功的实施例,下面举几个具体的实施例。在实施过程中,超滤装置的膜截留分子量为10万和14万,超滤器规格由处理量大小决定。The inventor has gone through a long period of creative research and experiments on the preparation of the present invention - dextran-ovomucin affinity ultrafiltration carrier, and there are many successful examples. Here are some specific examples. During the implementation process, the membrane molecular weight cut-off of the ultrafiltration device is 100,000 and 140,000, and the specification of the ultrafilter is determined by the processing capacity.
具体实施方式Detailed ways
实施例1:Example 1:
把购买Sigma公司的分子量200万的葡聚糖1克装进烧杯中,加水15mL,用磁力搅拌器分散均匀,使葡聚糖完全溶解,然后加0.4克氢氧化钠,加水10mL,用磁力搅拌器搅拌35分钟;加入环氧氯丙烷1mL,迅速磁力搅拌分散均匀;将上述搅拌液迅速移至57±2℃恒温摇床中,摇床转速100~110r/min,避光反应1.8小时;将上述反应液移至超滤杯中,膜截留分子量为10万,除去剩余的氢氧化钠,环氧氯丙烷小分子。先用蒸馏水洗至pH8.0±0.2,且至截留液为40±5mL,然后再用0.1M,pH9.5碳酸钠缓冲液100±5mL洗至截留液为30±2.5mL,整个处理过程需避光;将卵粘蛋白180±2mg溶解在上述截留液中,磁力搅拌均匀,移至37±2℃的恒温摇床(100~110r/min)中,避光23小时;处理后的溶液超滤分离,膜截留分子量为14万,除去未被偶联的卵粘蛋白,先用140mL的蒸馏水洗,至截留液为40±5mL;然后用0.1M甲酸-0.5M氯化钾,pH2.5甲酸80mL洗,至截留液为35±5mL,再用蒸馏水洗至截留液pH为6.2~6.5,且截留液体积为30±5mL,最后用0.5M氯化钾-0.05M氯化钙-0.1M、pH7.8Tris-HCl缓冲液120mL洗,至截留液体积为30±2.5mL,停止超滤,倒出截留液,磁力搅拌1.5小时,获得葡聚糖-卵粘蛋白水溶性亲和超滤载体,至3.5~4.5℃冰箱中保存。Put 1 gram of dextran with a molecular weight of 2 million purchased from Sigma into a beaker, add 15 mL of water, disperse evenly with a magnetic stirrer, and completely dissolve the dextran, then add 0.4 g of sodium hydroxide, add 10 mL of water, and stir with a magnetic force Stir for 35 minutes; add 1mL of epichlorohydrin, and quickly magnetically stir to disperse evenly; quickly move the above stirring solution to a constant temperature shaker at 57±2°C, the shaker speed is 100-110r/min, and react in the dark for 1.8 hours; The above reaction solution was transferred to an ultrafiltration cup, the molecular weight cut-off of the membrane was 100,000, and the remaining sodium hydroxide and epichlorohydrin small molecules were removed. First wash with distilled water to pH8.0±0.2, and the retentate is 40±5mL, then wash with 0.1M, pH9.5 sodium carbonate buffer 100±5mL until the retentate is 30±2.5mL, the whole process needs Protect from light; dissolve 180±2mg of ovomucin in the above-mentioned retentate, stir evenly with magnetic force, move to a constant temperature shaker (100~110r/min) at 37±2°C, and protect from light for 23 hours; the treated solution exceeds Separation by filtration, the molecular weight cut-off of the membrane is 140,000, remove uncoupled ovomucin, first wash with 140mL of distilled water until the retentate is 40±5mL; then use 0.1M formic acid-0.5M potassium chloride, pH2.5 Wash with 80mL of formic acid until the retentate is 35±5mL, then wash with distilled water until the pH of the retentate is 6.2-6.5, and the volume of the retentate is 30±5mL, and finally wash with 0.5M potassium chloride-0.05M calcium chloride-0.1M , pH 7.8 Tris-HCl buffer solution 120mL, until the volume of the retentate is 30±2.5mL, stop the ultrafiltration, pour out the retentate, and stir magnetically for 1.5 hours to obtain the dextran-ovomucin water-soluble affinity ultrafiltration carrier , Store in a refrigerator at 3.5-4.5°C.
通过葡聚糖-卵粘蛋白水溶性亲和超滤载体的卵粘蛋白特异性的吸附胰蛋白酶,滤过胰凝乳蛋白酶,使胰蛋白酶与胰凝乳蛋白酶有效的分离开来,吸附的胰蛋白酶通过洗脱得以纯化,获得胰蛋白酶纯化倍数达到91倍,回收85%。Ovomucin specifically adsorbs trypsin through the dextran-ovomucin water-soluble affinity ultrafiltration carrier, and filters chymotrypsin to effectively separate trypsin from chymotrypsin, and the adsorbed trypsin The protease was purified by elution, the purification factor of trypsin was 91 times, and the recovery was 85%.
实施例2:Example 2:
把购买Pharmacia公司的分子量200万的葡聚糖秤量3克装进烧杯中,加水50mL,用磁力搅拌器分散均匀,且使葡聚糖完全溶解,然后加入氢氧化钠1.3克,水15mL,用磁力搅拌器搅拌30分钟;然后加入环氧氯丙烷3±0.2mL,迅速磁力搅拌分散均匀;将上述搅拌液迅速移至恒温摇床中,温度58±2℃,摇床转速100~110r/min,避光反应2小时;将上述反应液移至超滤杯中,膜截留分子量为10万,除去剩余的氢氧化钠,环氧氯丙烷小分子,先用蒸馏水洗至pH8.0±0.2,且至截留液为80±5mL,然后再用0.1M,pH9.5碳酸钠缓冲液300±5mL洗至截留液体积为80±5mL,整个处理过程需避光;将卵粘蛋白490±2mg溶解在上述截留液中,磁力搅拌均匀,移至37±2℃的恒温摇床(100~110r/min)中,避光24小时;处理后的溶液超滤分离,膜截留分子量为14万,除去未被偶联的卵粘蛋白,先用400mL的蒸馏水洗,至截留液为90±5mL。然后用0.1M甲酸-0.5M氯化钾,pH2.5甲酸240mL洗,至截留液为90±5mL,再用蒸馏水洗至截留液pH为6.2~6.5,且截留液体积为90±5mL,最后用0.5M氯化钾-0.05M氯化钙-0.1M、pH7.8Tris-HCl缓冲液350mL洗,至截留液体积为80±5mL,停止超滤,倒出截留液,磁力搅拌1.5小时,获得葡聚糖-卵粘蛋白水溶性亲和超滤载体,放至4℃冰箱中保存。Put 3 grams of dextran with a molecular weight of 2 million purchased from Pharmacia into a beaker, add 50 mL of water, disperse evenly with a magnetic stirrer, and completely dissolve the dextran, then add 1.3 grams of sodium hydroxide and 15 mL of water. Stir with a magnetic stirrer for 30 minutes; then add 3±0.2mL of epichlorohydrin, and quickly magnetically stir to disperse evenly; quickly move the above stirring solution to a constant temperature shaker, the temperature is 58±2°C, and the shaker speed is 100~110r/min , and react in the dark for 2 hours; move the above reaction solution to an ultrafiltration cup, the molecular weight cut-off of the membrane is 100,000, remove the remaining sodium hydroxide and epichlorohydrin small molecules, and wash with distilled water to pH8.0±0.2 first, And until the retentate is 80±5mL, then wash with 300±5mL of 0.1M, pH9.5 sodium carbonate buffer until the retentate volume is 80±5mL. The whole process needs to be protected from light; dissolve 490±2mg of ovomucin In the above-mentioned retentate, stir evenly with magnetic force, move it to a constant temperature shaker (100-110r/min) at 37±2°C, and keep away from light for 24 hours; the treated solution is separated by ultrafiltration, and the molecular weight cut-off of the membrane is 140,000. The uncoupled ovomucin was first washed with 400mL of distilled water until the retentate was 90±5mL. Then wash with 0.1M formic acid-0.5M potassium chloride, pH2.5 formic acid 240mL until the retentate is 90±5mL, then wash with distilled water until the retentate pH is 6.2~6.5, and the retentate volume is 90±5mL, finally Wash with 350mL of 0.5M potassium chloride-0.05M calcium chloride-0.1M, pH7.8 Tris-HCl buffer until the volume of the retentate is 80±5mL, stop ultrafiltration, pour out the retentate, and stir magnetically for 1.5 hours to obtain Dextran-ovomucin water-soluble affinity ultrafiltration carrier, stored in a refrigerator at 4°C.
通过葡聚糖-卵粘蛋白水溶性亲和超滤载体的卵粘蛋白特异性的吸附胰蛋白酶,滤过胰凝乳蛋白酶,使胰蛋白酶与胰凝乳蛋白酶有效的分离开来,吸附的胰蛋白酶通过洗脱得以纯化,获得胰蛋白酶纯化倍数达到90倍,回收90%。Ovomucin specifically adsorbs trypsin through the dextran-ovomucin water-soluble affinity ultrafiltration carrier, and filters chymotrypsin to effectively separate trypsin from chymotrypsin, and the adsorbed trypsin The protease was purified by elution, the purification factor of trypsin reached 90 times, and the recovery was 90%.
实施例3:Example 3:
把购买Pharmacia公司的分子量200万的葡聚糖1.5克装进烧杯中,加水25mL,用磁力搅拌器分散均匀,且使葡聚糖完全溶解,然后加入氢氧化钠0.6克,再加入水8mL,用磁力搅拌器搅拌40分钟;然后加入环氧氯丙烷1.5mL,磁力搅拌迅速分散均匀;将上述搅拌液迅速移至恒温摇床中,温度58±2℃,摇床转速100~110r/min,避光反应1.9小时;将上述反应液移至超滤杯中,膜截留分子量为10万,除去剩余的氢氧化钠,环氧氯丙烷小分子,先用蒸馏水洗至pH7.8±0.2,且至截留液为40±5mL,然后再用0.1M,pH9.5碳酸钠缓冲液150±5mL洗至截留液体积为40±2mL,整个处理过程需避光;将卵粘蛋白238±2mg溶解在上述截留液中,磁力搅拌均匀,移至37±2℃的恒温摇床(100~110r/min)中,避光23~24小时;处理后的溶液超滤分离,膜截留分子量为14万,除去未被偶联的卵粘蛋白,先用180mL的蒸馏水洗,至截留液为40±5mL。然后用0.1M甲酸-0.5M氯化钾,pH2.5甲酸100mL洗,至截留液为45±5mL,再用蒸馏水洗至截留液pH为6.2~6.5,且截留液体积为40±5mL,最后用0.5M氯化钾-0.05M氯化钙-0.1M、pH7.8Tris-HCl缓冲液180mL洗,至截留液体积为40±2mL,停止超滤,倒出截留液,磁力搅拌1.5小时,得葡聚糖-卵粘蛋白水溶性亲和超滤载体,放至3.5~4℃冰箱中保存。Put 1.5 grams of dextran with a molecular weight of 2 million purchased from Pharmacia into a beaker, add 25 mL of water, disperse evenly with a magnetic stirrer, and completely dissolve the dextran, then add 0.6 grams of sodium hydroxide, then add 8 mL of water, Stir with a magnetic stirrer for 40 minutes; then add 1.5mL of epichlorohydrin, and disperse quickly and evenly with magnetic stirring; quickly move the above stirring solution to a constant temperature shaker at a temperature of 58±2°C and a shaker speed of 100-110r/min. Protect from light and react for 1.9 hours; transfer the above reaction solution to an ultrafiltration cup, the molecular weight cut-off of the membrane is 100,000, remove the remaining sodium hydroxide and epichlorohydrin small molecules, first wash with distilled water to pH7.8±0.2, and until the retentate is 40±5mL, then wash with 0.1M, pH9.5 sodium carbonate buffer 150±5mL until the volume of retentate is 40±2mL, the whole process needs to be protected from light; dissolve ovomucin 238±2mg in In the above-mentioned retentate solution, magnetically stir evenly, move to a constant temperature shaker (100-110r/min) at 37±2°C, and keep away from light for 23-24 hours; the treated solution is separated by ultrafiltration, and the molecular weight cut-off of the membrane is 140,000. To remove uncoupled ovomucin, first wash with 180mL of distilled water until the retentate is 40±5mL. Then wash with 0.1M formic acid-0.5M potassium chloride, pH2.5 formic acid 100mL, until the retentate is 45±5mL, then wash with distilled water until the retentate pH is 6.2~6.5, and the retentate volume is 40±5mL, finally Wash with 180mL of 0.5M potassium chloride-0.05M calcium chloride-0.1M, pH7.8 Tris-HCl buffer until the volume of the retentate is 40±2mL, stop the ultrafiltration, pour out the retentate, and stir magnetically for 1.5 hours to obtain Dextran-ovomucin water-soluble affinity ultrafiltration carrier, stored in a refrigerator at 3.5-4°C.
通过葡聚糖-卵粘蛋白水溶性亲和超滤载体的卵粘蛋白特异性的吸附胰蛋白酶,滤过胰凝乳蛋白酶,使胰蛋白酶与胰凝乳蛋白酶有效的分离开来,吸附的胰蛋白酶通过洗脱得以纯化,获得胰蛋白酶纯化倍数达到93倍,回收82%。Ovomucin specifically adsorbs trypsin through the dextran-ovomucin water-soluble affinity ultrafiltration carrier, and filters chymotrypsin to effectively separate trypsin from chymotrypsin, and the adsorbed trypsin The protease was purified by elution, the purification ratio of trypsin was 93 times, and the recovery was 82%.
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