CN101892216A - Preparation method of polyamide-amine dendritic molecule as carrier material for aminoacylase immobilization - Google Patents
Preparation method of polyamide-amine dendritic molecule as carrier material for aminoacylase immobilization Download PDFInfo
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- CN101892216A CN101892216A CN2009100844979A CN200910084497A CN101892216A CN 101892216 A CN101892216 A CN 101892216A CN 2009100844979 A CN2009100844979 A CN 2009100844979A CN 200910084497 A CN200910084497 A CN 200910084497A CN 101892216 A CN101892216 A CN 101892216A
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- silica gel
- polyamide
- aminoacylase
- carrier material
- immobilized enzyme
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- 108010003977 aminoacylase I Proteins 0.000 title claims abstract description 15
- ULGJWNIHLSLQPZ-UHFFFAOYSA-N 7-[(6,8-dichloro-1,2,3,4-tetrahydroacridin-9-yl)amino]-n-[2-(1h-indol-3-yl)ethyl]heptanamide Chemical compound C1CCCC2=NC3=CC(Cl)=CC(Cl)=C3C(NCCCCCCC(=O)NCCC=3C4=CC=CC=C4NC=3)=C21 ULGJWNIHLSLQPZ-UHFFFAOYSA-N 0.000 title claims abstract description 14
- 239000012876 carrier material Substances 0.000 title claims abstract description 10
- 238000002360 preparation method Methods 0.000 title claims description 8
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical class O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 claims abstract description 20
- 108010093096 Immobilized Enzymes Proteins 0.000 claims abstract description 19
- 238000000034 method Methods 0.000 claims abstract description 12
- 239000000412 dendrimer Substances 0.000 claims abstract description 11
- 229920000736 dendritic polymer Polymers 0.000 claims abstract description 11
- 238000004132 cross linking Methods 0.000 claims abstract description 9
- 239000000741 silica gel Substances 0.000 claims abstract description 8
- 229910002027 silica gel Inorganic materials 0.000 claims abstract description 8
- SXRSQZLOMIGNAQ-UHFFFAOYSA-N Glutaraldehyde Chemical compound O=CCCCC=O SXRSQZLOMIGNAQ-UHFFFAOYSA-N 0.000 claims abstract description 5
- 239000003446 ligand Substances 0.000 claims abstract description 5
- BAPJBEWLBFYGME-UHFFFAOYSA-N Methyl acrylate Chemical compound COC(=O)C=C BAPJBEWLBFYGME-UHFFFAOYSA-N 0.000 claims description 6
- 125000003277 amino group Chemical group 0.000 claims description 4
- 238000007259 addition reaction Methods 0.000 claims description 3
- 238000007112 amidation reaction Methods 0.000 claims description 3
- WYTZZXDRDKSJID-UHFFFAOYSA-N (3-aminopropyl)triethoxysilane Chemical compound CCO[Si](OCC)(OCC)CCCN WYTZZXDRDKSJID-UHFFFAOYSA-N 0.000 claims description 2
- PIICEJLVQHRZGT-UHFFFAOYSA-N Ethylenediamine Chemical compound NCCN PIICEJLVQHRZGT-UHFFFAOYSA-N 0.000 claims description 2
- 238000006243 chemical reaction Methods 0.000 claims description 2
- 239000004952 Polyamide Substances 0.000 claims 1
- 150000001412 amines Chemical group 0.000 claims 1
- 239000003153 chemical reaction reagent Substances 0.000 claims 1
- 229920002647 polyamide Polymers 0.000 claims 1
- 125000006850 spacer group Chemical group 0.000 claims 1
- 238000010189 synthetic method Methods 0.000 claims 1
- 239000000758 substrate Substances 0.000 abstract description 2
- 238000001308 synthesis method Methods 0.000 abstract description 2
- XUYPXLNMDZIRQH-UHFFFAOYSA-N N-acetylmethionine Chemical compound CSCCC(C(O)=O)NC(C)=O XUYPXLNMDZIRQH-UHFFFAOYSA-N 0.000 abstract 1
- 238000000926 separation method Methods 0.000 abstract 1
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 18
- 239000000243 solution Substances 0.000 description 5
- 230000015572 biosynthetic process Effects 0.000 description 4
- 238000003756 stirring Methods 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- 239000008367 deionised water Substances 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- 108090000790 Enzymes Proteins 0.000 description 2
- 102000004190 Enzymes Human genes 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 2
- 239000000203 mixture Substances 0.000 description 2
- 238000010992 reflux Methods 0.000 description 2
- 239000000377 silicon dioxide Substances 0.000 description 2
- LYLHVKVAISPJHX-UHFFFAOYSA-N CO.C(CN)N Chemical compound CO.C(CN)N LYLHVKVAISPJHX-UHFFFAOYSA-N 0.000 description 1
- 230000006229 amino acid addition Effects 0.000 description 1
- 239000007864 aqueous solution Substances 0.000 description 1
- 239000011942 biocatalyst Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 230000003197 catalytic effect Effects 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 239000003814 drug Substances 0.000 description 1
- 230000007613 environmental effect Effects 0.000 description 1
- 239000000706 filtrate Substances 0.000 description 1
- 125000000524 functional group Chemical group 0.000 description 1
- 229920001002 functional polymer Polymers 0.000 description 1
- 239000000499 gel Substances 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 229920002521 macromolecule Polymers 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000007935 neutral effect Effects 0.000 description 1
- 239000008055 phosphate buffer solution Substances 0.000 description 1
- 229920000962 poly(amidoamine) Polymers 0.000 description 1
- 238000012805 post-processing Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 239000000376 reactant Substances 0.000 description 1
- 230000035945 sensitivity Effects 0.000 description 1
- 239000007787 solid Substances 0.000 description 1
- DUZHCFGUSIJGMU-UHFFFAOYSA-N toluene;3-triethoxysilylpropan-1-amine Chemical compound CC1=CC=CC=C1.CCO[Si](OCC)(OCC)CCCN DUZHCFGUSIJGMU-UHFFFAOYSA-N 0.000 description 1
- 238000005406 washing Methods 0.000 description 1
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- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
Abstract
本发明涉及一种新型的固定化酶载体材料的制备方法，特别是以树枝状分子表面修饰硅胶为基体，以氨基酰化酶作为交联配体的固定化酶载体材料的制备方法。采用发散合成的方法，将树枝状分子键合到硅胶表面，制得树枝状分子表面修饰硅胶。通过戊二醛交联法实现树枝状分子表面修饰硅胶与氨基酰化酶的交联，并应用于N-乙酰-DL-蛋氨酸的分离。The invention relates to a method for preparing a novel immobilized enzyme carrier material, in particular to a method for preparing an immobilized enzyme carrier material with dendritic molecule surface-modified silica gel as a substrate and aminoacylase as a cross-linking ligand. Dendritic molecules are bonded to the surface of silica gel by divergent synthesis method to prepare dendritic molecular surface modified silica gel. The cross-linking of dendrimer surface-modified silica gel and aminoacylase was realized by glutaraldehyde cross-linking method, and applied to the separation of N-acetyl-DL-methionine.
Description
技术领域technical field
本发明涉及一种新型的固定化酶载体材料的制备方法，特别是以树枝状分子表面修饰硅胶为基体，以氨基酰化酶作为交联配体的固定化酶载体材料的制备方法。The invention relates to a method for preparing a novel immobilized enzyme carrier material, in particular to a method for preparing an immobilized enzyme carrier material with dendritic molecule surface-modified silica gel as a substrate and aminoacylase as a cross-linking ligand.
背景技术Background technique
树枝状大分子(Dendrimer)是一类新型的功能高分子化合物，它的特点是可以在分子水平进行分子的大小、形状和功能基团的设计。由于其独特结构，在医学、材料科学、生命科学及分析化学等领域中有着广泛的应用。把树枝状大分子引到硅胶表面，可以大大增加硅胶的表面积、容量和选择性。固定化酶具有稳定性好、可重复并连续使用等优点，并且可以提高产物的纯度和过程效率，克服游离生物催化剂对环境敏感、性质不稳定、易失活或死亡等缺点。载体对固定化酶有较大的影响，要制得理想的固定化酶，既要选用合理有效的固定化方法，同时又要有良好的载体。因此借助现代技术合成性能优良的固定化酶载体是固定化酶研究领域的主要研究内容。Dendrimer is a new type of functional polymer compound, which is characterized by the design of molecular size, shape and functional groups at the molecular level. Due to its unique structure, it has a wide range of applications in the fields of medicine, material science, life science and analytical chemistry. Introducing dendrimers to the surface of silica can greatly increase the surface area, capacity and selectivity of silica. Immobilized enzymes have the advantages of good stability, reproducibility and continuous use, and can improve product purity and process efficiency, and overcome the shortcomings of free biocatalysts such as environmental sensitivity, unstable properties, easy inactivation or death. The carrier has a great influence on the immobilized enzyme. To obtain an ideal immobilized enzyme, it is necessary to choose a reasonable and effective immobilization method and a good carrier. Therefore, the synthesis of immobilized enzyme carriers with excellent performance by means of modern technology is the main research content in the field of immobilized enzyme research.
发明内容Contents of the invention
本发明的目的是研究一种新型的固定化酶载体材料的制备方法，将树枝状分子应用到固定化氨基酰化酶的制备中，可以大大提高固定化酶的稳定性、重复性和寿命，简化后处理，提高收率与选择性，从而提高固定化酶的使用寿命，降低反应条件对固定化酶的影响，提高催化效率。在树枝状大分子的端氨基上交联氨基酰化酶，使其能够通过与N-乙酰-L-氨基酸发生反应，达到分离N-乙酰-DL-氨基酸的目的。The purpose of the present invention is to study a novel preparation method of immobilized enzyme carrier material, and the application of dendrimers to the preparation of immobilized aminoacylase can greatly improve the stability, repeatability and lifespan of immobilized enzyme. Simplify post-processing, improve yield and selectivity, thereby increasing the service life of immobilized enzymes, reducing the impact of reaction conditions on immobilized enzymes, and improving catalytic efficiency. The aminoacylase is cross-linked on the terminal amino group of the dendritic macromolecule, so that it can react with N-acetyl-L-amino acid to achieve the purpose of separating N-acetyl-DL-amino acid.
本发明的要点是利用聚酰胺-胺树枝状分子的结构和功能特点，先将其键合到硅胶表面，制得聚酰胺-胺表面修饰硅胶，并继续在其端氨基上交联氨基酰化酶配体。其具体制备过程包括：The gist of the present invention is to use the structure and functional characteristics of polyamide-amine dendrimers to first bond it to the surface of silica gel to prepare polyamide-amine surface-modified silica gel, and continue to cross-link amino acylation on its terminal amino group Enzyme ligand. Its specific preparation process includes:
(1)树枝状分子表面修饰硅胶的合成：以3-氨丙基三乙氧基硅烷做硅烷化试剂，采用发散合成法，以丙烯酸甲酯和乙二胺为反应物，通过多次重复的马氏加成反应、酰胺化反应，得到了聚酰胺-胺表面修饰的硅胶。(1) Synthesis of dendrimer surface-modified silica gel: 3-aminopropyltriethoxysilane is used as a silylating agent, and a divergent synthesis method is adopted, with methyl acrylate and ethylenediamine as reactants, through repeated steps Markov addition reaction, amidation reaction, obtained polyamide-amine surface modified silica gel.
(2)氨基酰化酶的交联：利用戊二醛交联法将氨基酰化酶交联至树枝状大分子聚酰胺-胺修饰的硅胶的端氨基上，合成以聚酰胺-胺为间隔臂，氨基酰化酶为配体的新型固定化酶。(2) Cross-linking of aminoacylase: use glutaraldehyde cross-linking method to cross-link aminoacylase to the terminal amino group of dendrimer polyamide-amine modified silica gel, and synthesize polyamide-amine as the interval Arm, a novel immobilized enzyme with aminoacylase as a ligand.
附图说明Description of drawings
1、图1聚酰胺-胺树枝状分子表面修饰硅胶的合成过程1. Figure 1 Synthesis process of polyamide-amine dendrimers surface-modified silica gel
2、图2氨基酰化酶的交联过程2. Figure 2 Cross-linking process of aminoacylase
具体实施方式Detailed ways
实施例1Example 1
树枝状分子表面修饰硅胶的合成：Synthesis of Dendrimer Surface Modified Silica Gel:
将硅胶置于50ml圆底烧瓶中，加入1mol·L-1的盐酸水溶液，电磁搅拌下100℃回流反应6小时；滤出固体，用去离子水反复冲洗至水洗液为中性，室温下真空干燥至恒重。然后与甲苯混合搅拌10分钟，之后后将硅胶、10％的3-氨丙基三乙氧基硅烷的甲苯溶液加入置于50ml圆底烧瓶中，在室温搅拌24小时，再在120℃回流搅拌2.5小时，冷却至室温后抽滤，滤出物用甲醇洗涤数次，在47℃真空干燥24h。Put the silica gel in a 50ml round-bottomed flask, add 1mol·
将上步的硅胶与甲醇混合搅拌10分钟，然后缓慢加入20％的丙烯酸甲酯甲醇溶液，在25℃恒温反应12.5小时，之后用甲醇冲洗3-4次。在47℃真空干燥24小时，得到0.5代的聚酰胺-胺表面修饰硅胶。将键合0.5代聚酰胺-胺的硅胶与甲醇混合搅拌10分钟，然后缓慢加入20％的乙二胺甲醇溶液，在40℃恒温反应19.5小时，然后用甲醇冲洗3-4次。在47℃真空干燥24小时，得到1.0代的聚酰胺-胺表面修饰硅胶。重复以上马氏加成反应和酰胺化反应，分别合成1.5代、2.0代、2.5代、3.0代的聚酰胺-胺表面修饰硅胶。Mix the silica gel in the previous step with methanol and stir for 10 minutes, then slowly add 20% methanol solution of methyl acrylate, react at a constant temperature of 25°C for 12.5 hours, and then rinse with methanol for 3-4 times. Vacuum-dried at 47° C. for 24 hours to obtain a 0.5-generation polyamide-amine surface-modified silica gel. The silica gel bonded with 0.5-generation polyamidoamine was mixed with methanol and stirred for 10 minutes, then slowly added 20% ethylenediamine methanol solution, reacted at 40°C for 19.5 hours at a constant temperature, and then washed with methanol for 3-4 times. Vacuum-dried at 47° C. for 24 hours to obtain a 1.0-generation polyamide-amine surface-modified silica gel. Repeat the above Markov addition reaction and amidation reaction to synthesize 1.5-generation, 2.0-generation, 2.5-generation, and 3.0-generation polyamide-amine surface-modified silica gels, respectively.
实施例2：Example 2:
氨基酰化酶的交联：Cross-linking of aminoacylases:
以经过3.0代聚酰胺-胺表面修饰的硅胶在常温下与1％的戊二醛交联4小时，过滤后用去离子水反复冲洗去除过量的戊二醛，在30℃真空干燥12小时；之后将反应后的硅胶加入到0.2mol·L-1的氨基酰化酶溶液中(溶液用pH＝7.0的磷酸缓冲液配制，静置30分钟后使用)，在常温下反应2小时，过滤后用去离子水冲洗5-6次，封口备用。Use 3.0-generation polyamide-amine surface-modified silica gel to cross-link with 1% glutaraldehyde at room temperature for 4 hours, filter and rinse with deionized water repeatedly to remove excess glutaraldehyde, and vacuum-dry at 30°C for 12 hours; Afterwards, the reacted silica gel was added to a 0.2 mol L -1 aminoacylase solution (the solution was prepared with a phosphate buffer solution with pH = 7.0 and used after standing for 30 minutes), reacted at room temperature for 2 hours, and filtered Rinse with deionized water 5-6 times, seal and set aside.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102161758A (en) * | 2011-01-26 | 2011-08-24 | 大连理工大学 | Preparation method of novel silica gel-based hyperbranched PAMAM (polyamidoamine) chelating resin |
| CN104558628A (en) * | 2013-10-17 | 2015-04-29 | 中国石油化工股份有限公司 | Magnetic nanoscale resin compound as well as preparation method and application thereof |
| CN110183667A (en) * | 2019-06-24 | 2019-08-30 | 南京大学 | A kind of preparation method and applications of the without phosphorus broom shaped polymer of silica gel load |
| CN110394148A (en) * | 2019-07-05 | 2019-11-01 | 重庆科技学院 | A kind of composite material preparation method and product and application of silica gel modified with methyl acrylate |
-
2009
- 2009-05-20 CN CN2009100844979A patent/CN101892216A/en active Pending
Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102161758A (en) * | 2011-01-26 | 2011-08-24 | 大连理工大学 | Preparation method of novel silica gel-based hyperbranched PAMAM (polyamidoamine) chelating resin |
| CN104558628A (en) * | 2013-10-17 | 2015-04-29 | 中国石油化工股份有限公司 | Magnetic nanoscale resin compound as well as preparation method and application thereof |
| CN110183667A (en) * | 2019-06-24 | 2019-08-30 | 南京大学 | A kind of preparation method and applications of the without phosphorus broom shaped polymer of silica gel load |
| CN110183667B (en) * | 2019-06-24 | 2021-07-16 | 南京大学 | A kind of preparation method of silica gel-supported phosphorus-free broom-like polymer and application thereof |
| CN110394148A (en) * | 2019-07-05 | 2019-11-01 | 重庆科技学院 | A kind of composite material preparation method and product and application of silica gel modified with methyl acrylate |
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Application publication date: 20101124 |