辣根过氧化物酶在双功能环氧载体和胺基载体上的固定化及固定化酶催化氧化性能研究
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摘要
酶是一种高效、高度专一的生物催化剂。随着科学技术的发展，酶的应用越来越广泛。但是，酶的稳定性差，对高温、有机溶剂或极端pH敏感，限制了酶的利用。酶固定化技术的出现，为酶的应用开辟了广阔的应用前景。酶的固定化研究和应用在工业生产、化学分析、医药和环境工程等众多领域都备受关注。
辣根过氧化物酶（HRP）是一种含有血红素辅基（铁原卟啉）的糖蛋白复合酶，具有特殊催化氧化性能，可以在较为宽泛的条件下催化氧化芳香类化合物，尤其对水介质中的酚类及苯胺类化合物的氧化反应具有良好的催化作用。含酚工业废水对水环境的生态平衡和人类的健康都造成严重的危害，采用固定化的辣根过氧化物酶来处理酚类废水，在环境保护和酶工程领域都是重要的研究课题。研究制备性能良好的载体，高效地固定辣根过氧化物酶，并将固定化辣根过氧化物酶应用于含酚废水的催化氧化降解处理，是重要的环境治理与环境保护技术。
本课题通过分子设计，研究制备了两类新型的双功能环氧载体，深入研究了对辣根过氧化物酶的固定化特性与固定化机理，也采用胺基载体对辣根过氧化物酶实施了固定化，研究了酶学性质，并将固定化辣根过氧化物酶用于苯酚的催化氧化，考察研究了催化氧化特性。本课题的研究在酶固定化领域具有明显的科学意义，对于酶催化技术在环境工程中的应用，具有重要的参考价值。
首先，采用“接出法”将甲基丙烯酸缩水甘油酯（glycidyl methacrylate，GMA）接枝聚合在硅胶表面，制得了接枝微粒PGMA/SiO_2，又通过环氧基团的开环反应，将乙二胺（EDA）键合在接枝微粒表面，制备了阳离子型双功能复合载体EDA-PGMA/SiO_2（第一种载体），共价偶联法实施了辣根过氧化物酶的固定化；重点考察了两种次价键力(静电相互作用与疏水相互作用)在共价键合法固定HRP过程中的作用，探索研究了作用机理。研究结果表明，在水介质中，复合载体EDA-PGMA/SiO_2表面胺基的质子化作用，使载体微粒的zeta电位在较大的pH范围内保持正值；当介质的pH=8.5，大于HRP的等电点（等电点为8）时，酶蛋白与载体之间所产生的强静电相互作用，会显著促进HRP的固定化；EDA的键合率在30%附近的载体，静电相互作用对固酶的促进作用最强，固定化酶的偶联率与比活力具有最高值，分别为57.85%和0.257U g~(-1)·min~(-1)。疏水相互作用对化学法固定辣根过氧化物酶，也会产生明显的作用，当以接枝微粒PGMA/SiO_2为载体时，增大溶液中NaCl的浓度，可有效促进酶蛋白与载体之间的疏水相互作用，提高固定化酶的偶联率与比活力。
接着，以对羟基苯磺酸钠（SHBS）为试剂，使接枝大分子PGMA的部分环氧基团发生开环反应，制备了阴离子型双功能复合载体SHBS-PGMA/SiO_2（第二种载体），并采用共价偶联法实施了辣根过氧化物酶的固定化；重点考察了HRP与载体SHBS-PGMA/SiO_2之间的静电相互作用在共价固定HRP过程中的作用，并探索研究了作用机理。研究结果表明，复合载体SHBS-PGMA/SiO_2表面大量的磺酸根离子，使其zeta电位在较大的pH范围内显示很大的负值，即SHBS-PGMA/SiO_2表面携带高密度的负电荷。当pH=6.0，HRP分子荷正电，酶蛋白分子与载体之间会产生很强的静电相互作用，显著促进HRP的固定化；当载体表面SHBS的键合率在18%附近时，静电相互作用对固酶的促进作用最强，固定化酶的偶联率与比活力达最高值，分别为63.64%和0.27U g~(-1)·min~(-1)。
催化氧化实验表明，以上述两种环氧载体固定的辣根过氧化物酶催化活性不高，于是又采用胺基载体（二乙烯三胺改性的交联聚丙烯腈微球，DETA-CPAN），以戊二醛为偶联剂，实施了辣根过氧化物酶的固定化，考察了最适酶固定化条件。研究结果指出，最适固定化条件为：温度为30℃，固定化时间为8h，戊二醛浓度为0.02mol L-1，pH值为8.0，本实验体系的适宜酶溶液量为10mL。在最适条件下，HRP的偶联率和固定化酶的比活力分别可达到41.96%和0.32U g~(-1)·min~(-1)。
最后，对最适条件下所制得的固定化酶的酶学性质进行了研究，并将其用于苯酚的催化氧化，考察了其催化性能。酶学性质研究结果表明，固定化酶的最适pH为7.0，且具有较好的pH稳定性；最适温度为30℃，且在20-50℃范围内，固定化酶都保持有较高的活性；游离酶和固定化酶的表观米氏常数Km值分别为1.35×10-3mol·L-1、4.05×10-2mol·L~(-1)，固定化酶的米氏常数高于游离酶的米氏常数，表明固定化酶与底物之间的亲和力比游离酶要低，同于大多数固定化酶的情况。苯酚溶液催化氧化降解实验结果表明，催化氧化体系中底物苯酚与氧化剂H_2O_2的比例为1:1时，催化效果最好；在本研究体系中加入少量（0.5g）固定化酶，25℃下进行苯酚的催化氧化降解实验2h，苯酚的去除率可达到28.57%。随着给酶量的增加，苯酚的去除率也在增加，当加入2.0g固定化酶时，去除率可达到56%。循环使用5次后，固定化酶仍具有较高的酶活性，表明固定化酶具有较好的重复循环使用性能。
Enzyme is a kind of highly efficient, highly specific biological catalyst. With thedevelopment of science and technology, enzyme is used widely. However, the poor stability,sensitive to high temperatures, organic solvents and extreme pH, the use of enzymes islimited. The emergence of enzyme immobilization technology opens up a new prospect forthe application of enzyme. Study and application of enzyme immobilization are concernedin the industrial production, chemical analysis, medical, environmental engineering, andmany other field.
Horseradish peroxidase (HRP) is a kind of glycoprotein compound enzyme containingprotoheme prosthetic group (iron protoporphyrin), and possessing special catalytic oxidationproperties. It can catalyze oxidation the aromatic compounds under relatively broad condition.Especially, the catalytic effect is better for the oxidation reaction of phenol and anilinecompounds in water system. Industrial waste water containing phenol has serious harm to theecological balance of water environment and human health. Immobilized horseradishperoxidase used to treat phenolic wastewater is an important research subject inenvironmental protection and enzyme engineering field. Researching and preparingimmobilized horseradish peroxidase carrier with good performance, and applied to catalyticoxidation of wastewater containing phenol degradation process, is an important environmentalmanagement and environmental protection technology.
In this paper, based on molecular designing, two kinds of novel difunctional epoxycarrier are prepared, and the properties of immobilized horseradish peroxidase andimmobilization mechanism are studied deeply. The immobilization of horseradish peroxidaseused amino carrier is carried out, and enzymology properties are studied. The immobilized horseradish peroxidase is used for catalytic oxidation of phenol, and the catalytic oxidationproperties are researched. This topic has significant scientific significance in the enzymeimmobilization field, and important reference value for the enzyme catalysis technologyapplication in environmental engineering.
Firstly, the glycidylmethacrylate (GMA) is grafted on silica surface using the "graftingfrom" method, and grafted particles PGMA/SiO_2are prepared. By ring-opening reaction ofethylenediamine (EDA) and epoxy groups, ethylenediamine are bonded on the particlesurface, resulting in preparation of the difunctional composite carrier EDA-PGMA/SiO_2.The immobilization of horseradish peroxidase on the difunctional on composite carrierEDA-PGMA/SiO_2is carried out using covalent coupling method. The effect of twosecondary valence bond forces (electrostatic interaction and hydrophobic interaction) in theimmobilization process of HRP is studied focused, and mechanism is researched. Theresults show that the zeta potential keeps positive value in larger range because of theprotonation effect of amino groups on the surface of EDA-PGMA/SiO_2composite carrier.The electrostatic interaction between EDA-PGMA/SiO_2and horseradish peroxidase has asignificant role to promote the enzyme immobilization when pH of8.5. When bonding rateof ethylenediamine is30%, the immobilized enzyme coupling ratio and the specific activityhas the highest,57.85%and0.257U g~(-1)·min~(-1), respectively. Hydrophobic interaction canproduce significant effect on immobilized horseradish peroxidase using chemical method.When the grafted PGMA/SiO_2particles as the carrier, adding NaCl electrolyte in solutioncan effectively promote the hydrophobic interactions between enzyme protein and thecarrier, and enhance coupling rate and specific activity of immobilized enzyme.
Secondly, graft particles PGMA/SiO_2is chemically modified using sodium4-hydroxybenzenesulfonate (SHBS), and anionic difunctional composite carrierSHBS-PGMA/SiO_2is prepared. The immobilization of horseradish peroxidase on thedifunctional on composite carrier SHBS-PGMA/SiO_2is carried out using covalent couplingmethod. The effect of electrostatic interaction in the immobilization process of HRP isstudied focused, and mechanism is researched. The results show that the zeta potential keeps negative value in larger range because of abundant sulfonic acid ions on the surfaceof SHBS-PGMA/SiO_2composite carrier. The electrostatic interaction betweenSHBS-PGMA/SiO_2and horseradish peroxidase has a significant role to promote theenzyme immobilization when pH of6.0. When the bonding rate of SHBS is18%, theimmobilized enzyme coupling ratio and the specific activity has the highest,63.64%and0.27U g~(-1)·min~(-1), respectively.
Catalytic oxidation experiments show that, the catalytic activity of two kinds of epoxycarrier immobilized horseradish peroxidase is not high. So, the amino carrier(diethylenetriamine modified crosslinked polyacrylonitrile microsphere, DETA-CPAN)modified using glutaraldehyde as coupling agent, is used to immobilize horseradishperoxidase. The optimal enzyme immobilization conditions are investigated. The resultsindicate that the optimal immobilized conditions as follows: the temperature is30℃,immobilization time is8h, glutaraldehyde concentration is0.02mol L-1, pH value is8.0,and the appropriate amount of enzyme solution is10ml. In optimal conditions, the couplingratio of HRP and the specific activity of immobilized enzyme can reach41.96%and0.32U g~(-1)·min~(-1), respectively.
Finally, the enzymatic properties of immobilized enzyme prepared at the optimalconditions are studied. The catalytic oxidation performance to phenol is investigated. Theenzymology properties results show that the optimal pH of the immobilized enzyme is7.0,and the immobilized enzyme has good pH stability. The optimum temperature is30°C, andthe immobilized enzyme remains higher activity within20°C to50℃. The apparentmichaelis constant of free enzyme and immobilized enzyme are1.35×10-3mol L-1,4.05×10-2mol L-1, respectively. This indicates that the affinity between immobilized enzymeand the substrate is lower than that of free affinity between enzyme and substrates. Theexperimental results of catalytic oxidation degradation of phenol show that the catalyticeffect is the best as the ratio of oxidant H_2O_2and substrate phenol of1:1. The removal rateof phenol can reach28.57%at temperature of25°C and time of2h when a small amount(0.5g) of immobilized enzyme used. The removal rate of phenol increases with the increase of enzyme amount. The removal rate of phenol can reach56%when2g of immobilizedenzyme used. The immobilized enzyme still has high enzyme activity after5cycle use andthe immobilized enzyme can be used in recirculation.
辣根过氧化物酶（HRP）是一种含有血红素辅基（铁原卟啉）的糖蛋白复合酶，具有特殊催化氧化性能，可以在较为宽泛的条件下催化氧化芳香类化合物，尤其对水介质中的酚类及苯胺类化合物的氧化反应具有良好的催化作用。含酚工业废水对水环境的生态平衡和人类的健康都造成严重的危害，采用固定化的辣根过氧化物酶来处理酚类废水，在环境保护和酶工程领域都是重要的研究课题。研究制备性能良好的载体，高效地固定辣根过氧化物酶，并将固定化辣根过氧化物酶应用于含酚废水的催化氧化降解处理，是重要的环境治理与环境保护技术。
本课题通过分子设计，研究制备了两类新型的双功能环氧载体，深入研究了对辣根过氧化物酶的固定化特性与固定化机理，也采用胺基载体对辣根过氧化物酶实施了固定化，研究了酶学性质，并将固定化辣根过氧化物酶用于苯酚的催化氧化，考察研究了催化氧化特性。本课题的研究在酶固定化领域具有明显的科学意义，对于酶催化技术在环境工程中的应用，具有重要的参考价值。
首先，采用“接出法”将甲基丙烯酸缩水甘油酯（glycidyl methacrylate，GMA）接枝聚合在硅胶表面，制得了接枝微粒PGMA/SiO_2，又通过环氧基团的开环反应，将乙二胺（EDA）键合在接枝微粒表面，制备了阳离子型双功能复合载体EDA-PGMA/SiO_2（第一种载体），共价偶联法实施了辣根过氧化物酶的固定化；重点考察了两种次价键力(静电相互作用与疏水相互作用)在共价键合法固定HRP过程中的作用，探索研究了作用机理。研究结果表明，在水介质中，复合载体EDA-PGMA/SiO_2表面胺基的质子化作用，使载体微粒的zeta电位在较大的pH范围内保持正值；当介质的pH=8.5，大于HRP的等电点（等电点为8）时，酶蛋白与载体之间所产生的强静电相互作用，会显著促进HRP的固定化；EDA的键合率在30%附近的载体，静电相互作用对固酶的促进作用最强，固定化酶的偶联率与比活力具有最高值，分别为57.85%和0.257U g~(-1)·min~(-1)。疏水相互作用对化学法固定辣根过氧化物酶，也会产生明显的作用，当以接枝微粒PGMA/SiO_2为载体时，增大溶液中NaCl的浓度，可有效促进酶蛋白与载体之间的疏水相互作用，提高固定化酶的偶联率与比活力。
接着，以对羟基苯磺酸钠（SHBS）为试剂，使接枝大分子PGMA的部分环氧基团发生开环反应，制备了阴离子型双功能复合载体SHBS-PGMA/SiO_2（第二种载体），并采用共价偶联法实施了辣根过氧化物酶的固定化；重点考察了HRP与载体SHBS-PGMA/SiO_2之间的静电相互作用在共价固定HRP过程中的作用，并探索研究了作用机理。研究结果表明，复合载体SHBS-PGMA/SiO_2表面大量的磺酸根离子，使其zeta电位在较大的pH范围内显示很大的负值，即SHBS-PGMA/SiO_2表面携带高密度的负电荷。当pH=6.0，HRP分子荷正电，酶蛋白分子与载体之间会产生很强的静电相互作用，显著促进HRP的固定化；当载体表面SHBS的键合率在18%附近时，静电相互作用对固酶的促进作用最强，固定化酶的偶联率与比活力达最高值，分别为63.64%和0.27U g~(-1)·min~(-1)。
催化氧化实验表明，以上述两种环氧载体固定的辣根过氧化物酶催化活性不高，于是又采用胺基载体（二乙烯三胺改性的交联聚丙烯腈微球，DETA-CPAN），以戊二醛为偶联剂，实施了辣根过氧化物酶的固定化，考察了最适酶固定化条件。研究结果指出，最适固定化条件为：温度为30℃，固定化时间为8h，戊二醛浓度为0.02mol L-1，pH值为8.0，本实验体系的适宜酶溶液量为10mL。在最适条件下，HRP的偶联率和固定化酶的比活力分别可达到41.96%和0.32U g~(-1)·min~(-1)。
最后，对最适条件下所制得的固定化酶的酶学性质进行了研究，并将其用于苯酚的催化氧化，考察了其催化性能。酶学性质研究结果表明，固定化酶的最适pH为7.0，且具有较好的pH稳定性；最适温度为30℃，且在20-50℃范围内，固定化酶都保持有较高的活性；游离酶和固定化酶的表观米氏常数Km值分别为1.35×10-3mol·L-1、4.05×10-2mol·L~(-1)，固定化酶的米氏常数高于游离酶的米氏常数，表明固定化酶与底物之间的亲和力比游离酶要低，同于大多数固定化酶的情况。苯酚溶液催化氧化降解实验结果表明，催化氧化体系中底物苯酚与氧化剂H_2O_2的比例为1:1时，催化效果最好；在本研究体系中加入少量（0.5g）固定化酶，25℃下进行苯酚的催化氧化降解实验2h，苯酚的去除率可达到28.57%。随着给酶量的增加，苯酚的去除率也在增加，当加入2.0g固定化酶时，去除率可达到56%。循环使用5次后，固定化酶仍具有较高的酶活性，表明固定化酶具有较好的重复循环使用性能。
Enzyme is a kind of highly efficient, highly specific biological catalyst. With thedevelopment of science and technology, enzyme is used widely. However, the poor stability,sensitive to high temperatures, organic solvents and extreme pH, the use of enzymes islimited. The emergence of enzyme immobilization technology opens up a new prospect forthe application of enzyme. Study and application of enzyme immobilization are concernedin the industrial production, chemical analysis, medical, environmental engineering, andmany other field.
Horseradish peroxidase (HRP) is a kind of glycoprotein compound enzyme containingprotoheme prosthetic group (iron protoporphyrin), and possessing special catalytic oxidationproperties. It can catalyze oxidation the aromatic compounds under relatively broad condition.Especially, the catalytic effect is better for the oxidation reaction of phenol and anilinecompounds in water system. Industrial waste water containing phenol has serious harm to theecological balance of water environment and human health. Immobilized horseradishperoxidase used to treat phenolic wastewater is an important research subject inenvironmental protection and enzyme engineering field. Researching and preparingimmobilized horseradish peroxidase carrier with good performance, and applied to catalyticoxidation of wastewater containing phenol degradation process, is an important environmentalmanagement and environmental protection technology.
In this paper, based on molecular designing, two kinds of novel difunctional epoxycarrier are prepared, and the properties of immobilized horseradish peroxidase andimmobilization mechanism are studied deeply. The immobilization of horseradish peroxidaseused amino carrier is carried out, and enzymology properties are studied. The immobilized horseradish peroxidase is used for catalytic oxidation of phenol, and the catalytic oxidationproperties are researched. This topic has significant scientific significance in the enzymeimmobilization field, and important reference value for the enzyme catalysis technologyapplication in environmental engineering.
Firstly, the glycidylmethacrylate (GMA) is grafted on silica surface using the "graftingfrom" method, and grafted particles PGMA/SiO_2are prepared. By ring-opening reaction ofethylenediamine (EDA) and epoxy groups, ethylenediamine are bonded on the particlesurface, resulting in preparation of the difunctional composite carrier EDA-PGMA/SiO_2.The immobilization of horseradish peroxidase on the difunctional on composite carrierEDA-PGMA/SiO_2is carried out using covalent coupling method. The effect of twosecondary valence bond forces (electrostatic interaction and hydrophobic interaction) in theimmobilization process of HRP is studied focused, and mechanism is researched. Theresults show that the zeta potential keeps positive value in larger range because of theprotonation effect of amino groups on the surface of EDA-PGMA/SiO_2composite carrier.The electrostatic interaction between EDA-PGMA/SiO_2and horseradish peroxidase has asignificant role to promote the enzyme immobilization when pH of8.5. When bonding rateof ethylenediamine is30%, the immobilized enzyme coupling ratio and the specific activityhas the highest,57.85%and0.257U g~(-1)·min~(-1), respectively. Hydrophobic interaction canproduce significant effect on immobilized horseradish peroxidase using chemical method.When the grafted PGMA/SiO_2particles as the carrier, adding NaCl electrolyte in solutioncan effectively promote the hydrophobic interactions between enzyme protein and thecarrier, and enhance coupling rate and specific activity of immobilized enzyme.
Secondly, graft particles PGMA/SiO_2is chemically modified using sodium4-hydroxybenzenesulfonate (SHBS), and anionic difunctional composite carrierSHBS-PGMA/SiO_2is prepared. The immobilization of horseradish peroxidase on thedifunctional on composite carrier SHBS-PGMA/SiO_2is carried out using covalent couplingmethod. The effect of electrostatic interaction in the immobilization process of HRP isstudied focused, and mechanism is researched. The results show that the zeta potential keeps negative value in larger range because of abundant sulfonic acid ions on the surfaceof SHBS-PGMA/SiO_2composite carrier. The electrostatic interaction betweenSHBS-PGMA/SiO_2and horseradish peroxidase has a significant role to promote theenzyme immobilization when pH of6.0. When the bonding rate of SHBS is18%, theimmobilized enzyme coupling ratio and the specific activity has the highest,63.64%and0.27U g~(-1)·min~(-1), respectively.
Catalytic oxidation experiments show that, the catalytic activity of two kinds of epoxycarrier immobilized horseradish peroxidase is not high. So, the amino carrier(diethylenetriamine modified crosslinked polyacrylonitrile microsphere, DETA-CPAN)modified using glutaraldehyde as coupling agent, is used to immobilize horseradishperoxidase. The optimal enzyme immobilization conditions are investigated. The resultsindicate that the optimal immobilized conditions as follows: the temperature is30℃,immobilization time is8h, glutaraldehyde concentration is0.02mol L-1, pH value is8.0,and the appropriate amount of enzyme solution is10ml. In optimal conditions, the couplingratio of HRP and the specific activity of immobilized enzyme can reach41.96%and0.32U g~(-1)·min~(-1), respectively.
Finally, the enzymatic properties of immobilized enzyme prepared at the optimalconditions are studied. The catalytic oxidation performance to phenol is investigated. Theenzymology properties results show that the optimal pH of the immobilized enzyme is7.0,and the immobilized enzyme has good pH stability. The optimum temperature is30°C, andthe immobilized enzyme remains higher activity within20°C to50℃. The apparentmichaelis constant of free enzyme and immobilized enzyme are1.35×10-3mol L-1,4.05×10-2mol L-1, respectively. This indicates that the affinity between immobilized enzymeand the substrate is lower than that of free affinity between enzyme and substrates. Theexperimental results of catalytic oxidation degradation of phenol show that the catalyticeffect is the best as the ratio of oxidant H_2O_2and substrate phenol of1:1. The removal rateof phenol can reach28.57%at temperature of25°C and time of2h when a small amount(0.5g) of immobilized enzyme used. The removal rate of phenol increases with the increase of enzyme amount. The removal rate of phenol can reach56%when2g of immobilizedenzyme used. The immobilized enzyme still has high enzyme activity after5cycle use andthe immobilized enzyme can be used in recirculation.
引文
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