基于双功能高分子设计制备有机-无机杂化固定化酶载体
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摘要
设计和制备有机-无机杂化载体是包括酶工程在内的诸多领域的研究热点和前沿。本文借鉴生物矿化思想,以充分发掘和合理拓展高分子材料的功能为出发点,筛选、设计并制备了同时具有成型功能和诱导矿化功能的高分子,基于双功能高分子制备不同结构的有机-无机杂化载体,并用于固定化酶研究。
利用吸附-包埋相结合的方法制备碳酸钙-海藻酸杂化凝胶用于葡萄糖醛酸苷酶(GUS)的固定。将吸附GUS的碳酸钙颗粒包埋于海藻酸凝胶中,可有效抑制酶泄漏。同时海藻酸可调控无机组分碳酸钙的形貌,抑制其晶型转变,使之维持中孔结构和较强的吸附能力。固定于碳酸钙-海藻酸杂化凝胶的GUS显示出良好的循环使用稳定性和储存稳定性。
选择生物高分子精蛋白(Pro),模拟硅藻细胞的细胞膜(高分子)-细胞壁(无机)结构,构建精蛋白-聚苯乙烯磺酸钠-氧化硅((PSS/Pro)n-1/Pro/Silica)杂化微囊用于固定化过氧化氢酶(CAT)。Pro在杂化微囊构建过程中实现两种功能:在CaCO3颗粒表面与PSS通过层层自组装(LbL)形成高分子囊膜;诱导仿生硅化,在最外层形成氧化硅外壳。研究发现,将LbL自组装与仿生硅化相结合制备的杂化微囊机械强度高,酶泄漏率低,是理想的固定化载体。
通过氧化-氨化-还原系列反应对海藻酸进行氨化改性,合成双功能高分子Ⅰ型胺化海藻酸(NH2-Alg(Ⅰ))并诱导仿生硅化,制备Ⅰ型胺化海藻酸/氧化硅(NH2-Alg(Ⅰ)/silica)杂化凝胶固定化CAT。NH2-Alg(Ⅰ)在水溶液中自发形成胶束,改变合成条件(氧化剂比例、氨化剂类型)可调控胶束直径,并最终决定杂化凝胶的尺寸和形貌。通过改变pH值、NH2-Alg(Ⅰ)浓度、前驱体浓度、离子强度等条件可调控NH2-Alg(Ⅰ)/silica的生成量。包埋于杂化凝胶中的CAT对高温和极端酸碱环境的耐受能力提高,同时储存稳定性也增强。
通过碳二亚胺接枝反应对海藻酸进行氨化改性,合成双功能高分子Ⅱ型胺化海藻酸(NH2-Alg(Ⅱ))并诱导仿生硅化,制备Ⅱ型胺化海藻酸/氧化硅(NH2-Alg(Ⅱ)/silica)杂化凝胶固定化醇脱氢酶(YADH)。通过在高分子主链上接枝多胺支链使NH2-Alg(Ⅱ)具备诱导仿生硅化的功能,加速前驱体的缩聚。氨化改性使海藻酸的凝胶化功能增强,NH2-Alg(Ⅱ)/silica杂化凝胶在磷酸盐缓冲液中的溶胀度从268%降低到50%。固定化酶的温度、pH、重复使用和储存稳定性得到全面提高。
Design and preparation of organic-inorganic hybrid carrier have become hot issue and research frontier in many areas including enzyme engineering. Inspired by biomineralization, aiming at fully exploring and rationally extend the function of polymer materials, in this study, a kind of polymer with shape-forming and biomimetic mineralization inducing ability was screened, designed and synthesized. Employing these bifunctional polymers, different structured organic-inorganic carriers were fabricated and utilized for enzyme immobilization.
β-Glucuronidase (GUS) was immobilized in CaCO3-Alg hybrid gel by an adsorption-encapsulation method. Alginate showed a strong shape-forming ability and its hydrogel matrix effectively prevented the leakage of enzyme. Meanwhile, alginate helped to maintain the mesoporous structure and adsorption ability of CaCO3 through inhibiting the recrystallization process. As a result, GUS loading efficiency, recycling and storage stability were all improved significantly.
Inspired by the delicate structure of diatom cell, a novel and facile method for preparing organic-inorganic composite microcapsules was developed by a synergy of layer-by-layer (LBL) self-assembly and biomimetic mineralization. Protamine (Pro), a natural bifunctional polymer, was chosen as both a positive layer component and an inducer for silicification, providing a simple and efficient approach to form the cell membrane-like multilayer with a complete, uniform and robust cell wall-like silica shell. Catalase (CAT) is captured in the CaCO3 templates via co-precipitation and encapsulated in the composite capsules followed by dissolution of the templates. The encapsulation efficiency, harsh condition tolerance and long-term storage stability of the encapsulated enzyme were all notably improved due to the shielding effect of the inorganic shell.
A novel bifunctional polymer, NH2-Alg(Ⅰ), was synthesized through an oxidation-amination-reduction process. NH2-Alg(Ⅰ) was then employed to induce the biomimetic mineralization from a silica precursor, Na2SiO3. The size and morphology of the resultant NH2-Alg(Ⅰ)/silica hybrid hydrogel could be controlled by the reaction conditions of co-precipitation process. Next, NH2-Alg(Ⅰ)/silica hybrid hydrogel was utilized for CAT immobilization. The encapsulated CAT exhibited an encapsulating efficiency as high as 100% as well as significantly improved thermal, pH, storage stability.
Another novel bifunctional polymer with both a shape-forming function and a silicification-inducing functions, NH2-Alg(Ⅱ), was synthesized through a carbodiimide process using EDC and NHS. NH2-Alg(Ⅱ) could form hydrogel and induce silicification simultaneously, generating a robust hybrid hydrogel with dense structure and homogenously dispersed silica particles. The impregnated silica particles remarkably reduced the swelling of alginate hydrogel from 268% to 50%. The enzyme YADH after encapsulation in the hybrid hydrogel exhibited improved temperature, pH, recycling and storage stability.
利用吸附-包埋相结合的方法制备碳酸钙-海藻酸杂化凝胶用于葡萄糖醛酸苷酶(GUS)的固定。将吸附GUS的碳酸钙颗粒包埋于海藻酸凝胶中,可有效抑制酶泄漏。同时海藻酸可调控无机组分碳酸钙的形貌,抑制其晶型转变,使之维持中孔结构和较强的吸附能力。固定于碳酸钙-海藻酸杂化凝胶的GUS显示出良好的循环使用稳定性和储存稳定性。
选择生物高分子精蛋白(Pro),模拟硅藻细胞的细胞膜(高分子)-细胞壁(无机)结构,构建精蛋白-聚苯乙烯磺酸钠-氧化硅((PSS/Pro)n-1/Pro/Silica)杂化微囊用于固定化过氧化氢酶(CAT)。Pro在杂化微囊构建过程中实现两种功能:在CaCO3颗粒表面与PSS通过层层自组装(LbL)形成高分子囊膜;诱导仿生硅化,在最外层形成氧化硅外壳。研究发现,将LbL自组装与仿生硅化相结合制备的杂化微囊机械强度高,酶泄漏率低,是理想的固定化载体。
通过氧化-氨化-还原系列反应对海藻酸进行氨化改性,合成双功能高分子Ⅰ型胺化海藻酸(NH2-Alg(Ⅰ))并诱导仿生硅化,制备Ⅰ型胺化海藻酸/氧化硅(NH2-Alg(Ⅰ)/silica)杂化凝胶固定化CAT。NH2-Alg(Ⅰ)在水溶液中自发形成胶束,改变合成条件(氧化剂比例、氨化剂类型)可调控胶束直径,并最终决定杂化凝胶的尺寸和形貌。通过改变pH值、NH2-Alg(Ⅰ)浓度、前驱体浓度、离子强度等条件可调控NH2-Alg(Ⅰ)/silica的生成量。包埋于杂化凝胶中的CAT对高温和极端酸碱环境的耐受能力提高,同时储存稳定性也增强。
通过碳二亚胺接枝反应对海藻酸进行氨化改性,合成双功能高分子Ⅱ型胺化海藻酸(NH2-Alg(Ⅱ))并诱导仿生硅化,制备Ⅱ型胺化海藻酸/氧化硅(NH2-Alg(Ⅱ)/silica)杂化凝胶固定化醇脱氢酶(YADH)。通过在高分子主链上接枝多胺支链使NH2-Alg(Ⅱ)具备诱导仿生硅化的功能,加速前驱体的缩聚。氨化改性使海藻酸的凝胶化功能增强,NH2-Alg(Ⅱ)/silica杂化凝胶在磷酸盐缓冲液中的溶胀度从268%降低到50%。固定化酶的温度、pH、重复使用和储存稳定性得到全面提高。
Design and preparation of organic-inorganic hybrid carrier have become hot issue and research frontier in many areas including enzyme engineering. Inspired by biomineralization, aiming at fully exploring and rationally extend the function of polymer materials, in this study, a kind of polymer with shape-forming and biomimetic mineralization inducing ability was screened, designed and synthesized. Employing these bifunctional polymers, different structured organic-inorganic carriers were fabricated and utilized for enzyme immobilization.
β-Glucuronidase (GUS) was immobilized in CaCO3-Alg hybrid gel by an adsorption-encapsulation method. Alginate showed a strong shape-forming ability and its hydrogel matrix effectively prevented the leakage of enzyme. Meanwhile, alginate helped to maintain the mesoporous structure and adsorption ability of CaCO3 through inhibiting the recrystallization process. As a result, GUS loading efficiency, recycling and storage stability were all improved significantly.
Inspired by the delicate structure of diatom cell, a novel and facile method for preparing organic-inorganic composite microcapsules was developed by a synergy of layer-by-layer (LBL) self-assembly and biomimetic mineralization. Protamine (Pro), a natural bifunctional polymer, was chosen as both a positive layer component and an inducer for silicification, providing a simple and efficient approach to form the cell membrane-like multilayer with a complete, uniform and robust cell wall-like silica shell. Catalase (CAT) is captured in the CaCO3 templates via co-precipitation and encapsulated in the composite capsules followed by dissolution of the templates. The encapsulation efficiency, harsh condition tolerance and long-term storage stability of the encapsulated enzyme were all notably improved due to the shielding effect of the inorganic shell.
A novel bifunctional polymer, NH2-Alg(Ⅰ), was synthesized through an oxidation-amination-reduction process. NH2-Alg(Ⅰ) was then employed to induce the biomimetic mineralization from a silica precursor, Na2SiO3. The size and morphology of the resultant NH2-Alg(Ⅰ)/silica hybrid hydrogel could be controlled by the reaction conditions of co-precipitation process. Next, NH2-Alg(Ⅰ)/silica hybrid hydrogel was utilized for CAT immobilization. The encapsulated CAT exhibited an encapsulating efficiency as high as 100% as well as significantly improved thermal, pH, storage stability.
Another novel bifunctional polymer with both a shape-forming function and a silicification-inducing functions, NH2-Alg(Ⅱ), was synthesized through a carbodiimide process using EDC and NHS. NH2-Alg(Ⅱ) could form hydrogel and induce silicification simultaneously, generating a robust hybrid hydrogel with dense structure and homogenously dispersed silica particles. The impregnated silica particles remarkably reduced the swelling of alginate hydrogel from 268% to 50%. The enzyme YADH after encapsulation in the hybrid hydrogel exhibited improved temperature, pH, recycling and storage stability.
引文
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