介孔氧化硅/碳材料的合成及酶固定化
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摘要
介孔材料由于具有较高的比表面积、孔容以及可调的孔径结构、形貌等特点，在酶分子固定领域获得广泛的关注和应用。介孔氧化硅在材料制备、孔结构及形貌的调节等方面的研究已经比较深入，但是在酶固定方面的系统研究还不够全面。介孔碳由于具有较好的机械强度、化学稳定性和导电性，正在成为介孔材料制备及酶分子固定化领域新的研究热点，而磁性介孔碳材料的获得，可实现固定化药物及酶分子的导向应用及简易分离。本论文围绕介孔氧化硅和介孔碳材料的制备展开，考察了反应条件对所得材料结构的影响，并采用一步法制备了磁性含钴（Co）有序介孔碳。针对所得材料的孔结构特点，将其应用于不同尺寸酶分子的固定化，对酶固定化过程中的吸附行为以及固定化酶的催化行为进行了考察。本论文主要研究成果如下：
采用水热合成法，以嵌段聚合物P123为模板剂，正硅酸乙酯(TEOS)为硅源，通过改变晶化温度、搅拌状态及添加有机助剂制备了具有不同孔径的介孔氧化硅。随着晶化温度从100℃升高到200℃，介孔氧化硅的孔径会逐渐增大，最大孔径达到29.20nm。加入扩孔剂三甲苯(TMB)以后，可得到孔容为1.57cm3/g，孔径为21.63nm孔径的氧化硅。减少搅拌时间至10min，孔径将降低到7.60nm。对漆酶固定化的结果表明，由于孔径匹配作用，孔径为7.60nm的氧化硅比其他样品对漆酶具有更好的保护性。以制备的介孔氧化硅为模板，制备了有序介孔碳。将其用于溶菌酶固定化发现，溶菌酶在介孔碳上的吸附量在pH=11时最大，吸附行为符合Langmuir等温线并遵循准二级动力学。
以间苯二酚和甲醛为碳源前驱体，正硅酸乙酯为硅源，F127为模板剂，在酸性条件下制备了介孔Si-C复合材料。通过在80℃清洗可以去除掉其中的Si元素，得到具有高比表面积（602m2/g）和孔容（0.58cm3/g）的介孔碳。溶菌酶在介孔材料上的固定化符合Freundlich模型及准一级动力学模型。介孔碳焙烧温度的增加会引起其中微孔的增加及其向介孔的转化，从而引起比表面积和孔容的增加，但是介孔碳在空气中的耐热温度最高不超过450℃。经过不同温度焙烧的介孔碳对溶菌酶的吸附符合Langmuir吸附拟合和准二级动力学拟合。
采用软模板法，以间苯三酚-甲醛为碳源前驱体，F127为模板剂，在强酸性水相溶液中实现了介孔碳的快速合成。反应体系中温度以及盐酸浓度的改变会对介孔碳的孔结构产生明显影响。随着反应温度从30℃上升到50℃，所得介孔碳的孔径呈减小的趋势，比表面积和孔容在40℃时具有最大值。反应体系中盐酸的浓度在从0.5M增加到2.5M的过程中，所得介孔碳的孔径从7.6nm逐渐降低到5.6nm，且比表面积和孔容也逐渐减小。介孔材料固定化辣根过氧化酶(HRP)的结果表明，固定化HRP在介孔碳中保持了其蛋白质二级结构。与游离HRP相比，固定化酶的热稳定性、pH稳定性都有了明显的提高。经重复使用6次以后，固定化酶仍然保持了超过50%的活性。
采用简单的一步法，以甲阶酚醛树脂为碳源，草酸钴为磁性粒子前驱体，合成了含钴(Co)磁性有序介孔碳材料。Co含量为0.02时，介孔碳的比表面积和孔容从未掺杂磁性粒子的552m2/g和0.40cm3/g分别增加到621m2/g和0.48cm3/g，随着Co含量的继续增加，样品的比表面积和孔容逐渐减小。所得材料的饱和磁化强度(Ms)、剩余磁化轻度(Mr)和矫顽力(Hc)可以通过改变Co含量而改变。较低的Ms、Mr和Mr/Ms表明所制备材料表现出典型的铁磁性特征。通过对微过氧化物酶(MP-11)的固定化研究表明，MP-11在磁性介孔材料上的固定化符合Freundlich拟合和准二级动力学拟合。固定化酶的热稳定性和pH稳定性较游离酶都有所提高，且经过10次重复操作后，其仍然能保持60%的催化活性。
Mesoporous materials have attracted great attention in enzyme immobilization dueto their high specific surface area, pore volume and tunable pore size. In contrast tothe extensive investigation on the preparation and characterization of mesoporoussilica, less attention has been paid, to the best of our knowledge, to the systematicapplication in enzyme immobilization. Mesoporous carbon materials are of greatinterest owning to the advantage of high mechanical strength, chemical resistance andelectroconductibility. Magnetic mesoporous carbon with cobalt nanoparticles confinedin pore channels can act as adsorbent of enzyme because of the easy separation andcontrolled placement realized by means of an external magnetic field. This thesisfocuses on the synthesis of mesoporous silica and mesoporus carbon materials andtheir application in enzyme immobilization. Afacile “one-pot” strategy was utilized toprepare magnetic mesostructured Co/ordered mesoporous carbon(OMC). Theobtained mesoporous materials with different pore size were used to immobilizeenzyme with various size. The adsorption behavior of enzyme and the catalyticactivity of immobilized enzyme were also investigated. This thesis includes thefollowing sections.
Mesoporous silica with various pore sizewas synthesized using triblock copolymerP123as template tetraethyl orthosilicate(TEOS) as precursor andtrimethylbenzene(TMB) as swelling agent at various synthesis temperatures andunder different stir conditions. With the increase of temperature from100℃to200℃,the pore size of mesoporus silica increases gradually, reaching the largest porediameter of29.20nm at200℃. Disordered mesocellular silica foam with a high porevolume of1.57cm3/g and a large pore size of21.63nm was obtained by the addition ofTMB. The shrinkage of stir time does not affect the mesostructure except that the poresize reduces to7.6nm. Laccases immobilized on mesoporous silica with a pore size of7.6nm exhibit higher stability than those on other samples due to the matching ofenzyme size and pore diameter. The mesoporous silica was also used as hard templateto prepare ordered mesoporous carbon and the obtained materials were applied toimmobilize lysozyme. The adsorption amount of lysozyme reaches the largest valueat a pH of11. The adsorption process can be described by the Langmuir isotherm andpseudo-second-order kinetic.
Mesoporous silica/carbon composite materials were prepared under acidicconditions by using resorcinol/formaldehyde as carbon precursor, TEOS as silicaprecursor and F127as template. Mesoporous carbon with a high specific surface areaof602m2/gand a pore volume of0.58cm3/gcan be obtained by dissolving silica inNaOH solution at80℃. The adsorption process of lysozyme immobilized onmesoporous carbon can be described by the Freundlich isotherm andpseudo-first-order kinetic. The increase of calcination temperature leads to an increaseof the amount of micropore volume and an evolution from micropores to mesopores,resulting in an increase of surface area and pore volume, but mesoporous carbonshould be used below450℃.The adsorption of lysozyme on carbon materialscalcined at different temperatures can be described by the Langmuir isotherm andpseudo-second-order kinetic.
Mesoporous carbons have been rapidly synthesized using an aqueous strategy bythe polymerization of phloroglucinol and formaldehyde in the presence of triblockcopolymer F127under acidic conditions. The synthesis temperatures and theconcentrations of hydrochloric acid play an important role in the formation of the porestructure of mesoporous carbon. The pore size decreases with increasing synthesistemperatures, reaching7.6nm with the maximum surface area and pore volume at40℃. With the concentration ofhydrochloric acid increasing from0.5mol/L to2.5mol/L, the pore size of mesoporous carbon decreases from7.6nm to5.6nm, thesurface area from788to691m2/g, and the pore volume from0.38to0.25cm3/g.Mesoporous carbon was used to immobilize Horseradish Peroxidase(HRP). Theresults show that immobilized HRP remains intact and the thermal stability, pHstability and storage stability of immobilized HRP have been improved significantlyin comparison with free HRP. The immobilized HRP retain more than50%relativeactivity after recycling for6times.
A facile “one-pot” strategy was used to synthesize a mesostructured magneticCo/ordered mesoporous carbon(OMC) composite associated with a directcarbonization process from resol, cobalt oxalate and triblock copolymer F127.When the content of Co is0.02, the specific surface area and pore volume ofmagnetic mesoporous carbon increase from552m2/g and0.40cm3/g to621m2/g and0.48cm3/g, respectively, compared to the mesoporous carbon without Co,but the surface area and pore volume tend to decrease gradually with furtherincrease of Co content. The saturation magnetization(Ms), remanent magnetization(Mr) and coercive force(Hc) can be adjusted by varying the Cocontent. The low Ms, Mr and Ms/Mr indicate the ferromagnetism property ofmesoporous carbon. The prepared magnetic mesoporus carbon was used toimmobilize microperoxidome MP-11. The adsorption of MP-11can bedescribed by the Freundlich isotherm and comply with pseudo-second-order kinetic.The thermal stability, pH stability and storage stability of immobilized enzyme havebeen improved significantly in comparison with free MP-11. The immobilized MP-11retains more than60%relative activity after recycling for10times.
采用水热合成法，以嵌段聚合物P123为模板剂，正硅酸乙酯(TEOS)为硅源，通过改变晶化温度、搅拌状态及添加有机助剂制备了具有不同孔径的介孔氧化硅。随着晶化温度从100℃升高到200℃，介孔氧化硅的孔径会逐渐增大，最大孔径达到29.20nm。加入扩孔剂三甲苯(TMB)以后，可得到孔容为1.57cm3/g，孔径为21.63nm孔径的氧化硅。减少搅拌时间至10min，孔径将降低到7.60nm。对漆酶固定化的结果表明，由于孔径匹配作用，孔径为7.60nm的氧化硅比其他样品对漆酶具有更好的保护性。以制备的介孔氧化硅为模板，制备了有序介孔碳。将其用于溶菌酶固定化发现，溶菌酶在介孔碳上的吸附量在pH=11时最大，吸附行为符合Langmuir等温线并遵循准二级动力学。
以间苯二酚和甲醛为碳源前驱体，正硅酸乙酯为硅源，F127为模板剂，在酸性条件下制备了介孔Si-C复合材料。通过在80℃清洗可以去除掉其中的Si元素，得到具有高比表面积（602m2/g）和孔容（0.58cm3/g）的介孔碳。溶菌酶在介孔材料上的固定化符合Freundlich模型及准一级动力学模型。介孔碳焙烧温度的增加会引起其中微孔的增加及其向介孔的转化，从而引起比表面积和孔容的增加，但是介孔碳在空气中的耐热温度最高不超过450℃。经过不同温度焙烧的介孔碳对溶菌酶的吸附符合Langmuir吸附拟合和准二级动力学拟合。
采用软模板法，以间苯三酚-甲醛为碳源前驱体，F127为模板剂，在强酸性水相溶液中实现了介孔碳的快速合成。反应体系中温度以及盐酸浓度的改变会对介孔碳的孔结构产生明显影响。随着反应温度从30℃上升到50℃，所得介孔碳的孔径呈减小的趋势，比表面积和孔容在40℃时具有最大值。反应体系中盐酸的浓度在从0.5M增加到2.5M的过程中，所得介孔碳的孔径从7.6nm逐渐降低到5.6nm，且比表面积和孔容也逐渐减小。介孔材料固定化辣根过氧化酶(HRP)的结果表明，固定化HRP在介孔碳中保持了其蛋白质二级结构。与游离HRP相比，固定化酶的热稳定性、pH稳定性都有了明显的提高。经重复使用6次以后，固定化酶仍然保持了超过50%的活性。
采用简单的一步法，以甲阶酚醛树脂为碳源，草酸钴为磁性粒子前驱体，合成了含钴(Co)磁性有序介孔碳材料。Co含量为0.02时，介孔碳的比表面积和孔容从未掺杂磁性粒子的552m2/g和0.40cm3/g分别增加到621m2/g和0.48cm3/g，随着Co含量的继续增加，样品的比表面积和孔容逐渐减小。所得材料的饱和磁化强度(Ms)、剩余磁化轻度(Mr)和矫顽力(Hc)可以通过改变Co含量而改变。较低的Ms、Mr和Mr/Ms表明所制备材料表现出典型的铁磁性特征。通过对微过氧化物酶(MP-11)的固定化研究表明，MP-11在磁性介孔材料上的固定化符合Freundlich拟合和准二级动力学拟合。固定化酶的热稳定性和pH稳定性较游离酶都有所提高，且经过10次重复操作后，其仍然能保持60%的催化活性。
Mesoporous materials have attracted great attention in enzyme immobilization dueto their high specific surface area, pore volume and tunable pore size. In contrast tothe extensive investigation on the preparation and characterization of mesoporoussilica, less attention has been paid, to the best of our knowledge, to the systematicapplication in enzyme immobilization. Mesoporous carbon materials are of greatinterest owning to the advantage of high mechanical strength, chemical resistance andelectroconductibility. Magnetic mesoporous carbon with cobalt nanoparticles confinedin pore channels can act as adsorbent of enzyme because of the easy separation andcontrolled placement realized by means of an external magnetic field. This thesisfocuses on the synthesis of mesoporous silica and mesoporus carbon materials andtheir application in enzyme immobilization. Afacile “one-pot” strategy was utilized toprepare magnetic mesostructured Co/ordered mesoporous carbon(OMC). Theobtained mesoporous materials with different pore size were used to immobilizeenzyme with various size. The adsorption behavior of enzyme and the catalyticactivity of immobilized enzyme were also investigated. This thesis includes thefollowing sections.
Mesoporous silica with various pore sizewas synthesized using triblock copolymerP123as template tetraethyl orthosilicate(TEOS) as precursor andtrimethylbenzene(TMB) as swelling agent at various synthesis temperatures andunder different stir conditions. With the increase of temperature from100℃to200℃,the pore size of mesoporus silica increases gradually, reaching the largest porediameter of29.20nm at200℃. Disordered mesocellular silica foam with a high porevolume of1.57cm3/g and a large pore size of21.63nm was obtained by the addition ofTMB. The shrinkage of stir time does not affect the mesostructure except that the poresize reduces to7.6nm. Laccases immobilized on mesoporous silica with a pore size of7.6nm exhibit higher stability than those on other samples due to the matching ofenzyme size and pore diameter. The mesoporous silica was also used as hard templateto prepare ordered mesoporous carbon and the obtained materials were applied toimmobilize lysozyme. The adsorption amount of lysozyme reaches the largest valueat a pH of11. The adsorption process can be described by the Langmuir isotherm andpseudo-second-order kinetic.
Mesoporous silica/carbon composite materials were prepared under acidicconditions by using resorcinol/formaldehyde as carbon precursor, TEOS as silicaprecursor and F127as template. Mesoporous carbon with a high specific surface areaof602m2/gand a pore volume of0.58cm3/gcan be obtained by dissolving silica inNaOH solution at80℃. The adsorption process of lysozyme immobilized onmesoporous carbon can be described by the Freundlich isotherm andpseudo-first-order kinetic. The increase of calcination temperature leads to an increaseof the amount of micropore volume and an evolution from micropores to mesopores,resulting in an increase of surface area and pore volume, but mesoporous carbonshould be used below450℃.The adsorption of lysozyme on carbon materialscalcined at different temperatures can be described by the Langmuir isotherm andpseudo-second-order kinetic.
Mesoporous carbons have been rapidly synthesized using an aqueous strategy bythe polymerization of phloroglucinol and formaldehyde in the presence of triblockcopolymer F127under acidic conditions. The synthesis temperatures and theconcentrations of hydrochloric acid play an important role in the formation of the porestructure of mesoporous carbon. The pore size decreases with increasing synthesistemperatures, reaching7.6nm with the maximum surface area and pore volume at40℃. With the concentration ofhydrochloric acid increasing from0.5mol/L to2.5mol/L, the pore size of mesoporous carbon decreases from7.6nm to5.6nm, thesurface area from788to691m2/g, and the pore volume from0.38to0.25cm3/g.Mesoporous carbon was used to immobilize Horseradish Peroxidase(HRP). Theresults show that immobilized HRP remains intact and the thermal stability, pHstability and storage stability of immobilized HRP have been improved significantlyin comparison with free HRP. The immobilized HRP retain more than50%relativeactivity after recycling for6times.
A facile “one-pot” strategy was used to synthesize a mesostructured magneticCo/ordered mesoporous carbon(OMC) composite associated with a directcarbonization process from resol, cobalt oxalate and triblock copolymer F127.When the content of Co is0.02, the specific surface area and pore volume ofmagnetic mesoporous carbon increase from552m2/g and0.40cm3/g to621m2/g and0.48cm3/g, respectively, compared to the mesoporous carbon without Co,but the surface area and pore volume tend to decrease gradually with furtherincrease of Co content. The saturation magnetization(Ms), remanent magnetization(Mr) and coercive force(Hc) can be adjusted by varying the Cocontent. The low Ms, Mr and Ms/Mr indicate the ferromagnetism property ofmesoporous carbon. The prepared magnetic mesoporus carbon was used toimmobilize microperoxidome MP-11. The adsorption of MP-11can bedescribed by the Freundlich isotherm and comply with pseudo-second-order kinetic.The thermal stability, pH stability and storage stability of immobilized enzyme havebeen improved significantly in comparison with free MP-11. The immobilized MP-11retains more than60%relative activity after recycling for10times.
引文
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