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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 PDF

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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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杨屹
高翔
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Beijing University of Chemical Technology
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Beijing University of Chemical Technology
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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

一种聚酰胺-胺树枝状分子用于氨基酰化酶固定化的载体材料的制备方法 Preparation method of polyamide-amine dendritic molecule as carrier material for aminoacylase immobilization

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·L - 1 hydrochloric acid aqueous solution, and reflux at 100°C for 6 hours under electromagnetic stirring; filter out the solid, rinse repeatedly with deionized water until the washing liquid is neutral, and vacuum at room temperature Dry to constant weight. Then mix with toluene and stir for 10 minutes, then add silica gel and 10% 3-aminopropyltriethoxysilane toluene solution into a 50ml round bottom flask, stir at room temperature for 24 hours, and then reflux at 120°C After 2.5 hours, it was cooled to room temperature and filtered with suction. The filtrate was washed several times with methanol and dried in vacuum at 47°C for 24 hours.

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.

Claims (3)

1.一种新型的固定化酶载体材料的制备方法,其特征在于: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,并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,得到以聚酰胺-胺为间隔臂,氨基酰化酶为配体的新型固定化酶。1. A kind of preparation method of novel immobilized enzyme carrier material is characterized in that: do silylating reagent with 3-aminopropyltriethoxysilane, adopt divergent synthetic method, take methyl acrylate and ethylenediamine as reaction The polyamide-amine surface-modified silica gel was obtained through repeated Markov addition reactions and amidation reactions, and the aminoacylase was cross-linked to the dendrimer polyamide by glutaraldehyde cross-linking method. On the terminal amino group of silica gel modified with amine, a new type of immobilized enzyme with polyamide-amine as spacer arm and aminoacylase as ligand is obtained. 2.制备权利要求1所述新型固定化酶载体材料的制备,其特征在于:将树枝状分子应用到固定化酶载体材料的聚酰胺-胺表面修饰硅胶的制备过程。2. The preparation of the novel immobilized enzyme carrier material according to claim 1, characterized in that: the preparation process of the polyamide-amine surface-modified silica gel applied to the immobilized enzyme carrier material with dendrimers. 3.根据权利要求1所述新型固定化酶载体材料的制备,其特征在于:在所得的聚酰胺-胺表面修饰的硅胶上可以进一步键合氨基酰化酶。3. The preparation of the novel immobilized enzyme carrier material according to claim 1, characterized in that aminoacylase can be further bonded to the obtained polyamide-amine surface-modified silica gel.
CN2009100844979A 2009-05-20 2009-05-20 Preparation method of polyamide-amine dendritic molecule as carrier material for aminoacylase immobilization Pending CN101892216A (en)

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Cited By (4)

* Cited by examiner, † Cited by third party
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

Cited By (5)

* Cited by examiner, † Cited by third party
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