含酶薄膜涂层的生物催化与应用研究
文献类型：学位论文
| 作者 | 张立挺 |
| 学位类别 | 博士 |
| 答辩日期 | 2014-03 |
| 授予单位 | 中国科学院研究生院 |
| 导师 | 王平 |
| 关键词 | 固定化酶 界面催化 自清洁涂层 聚乙二醇修饰 POTC |
| 其他题名 | Biocatalysis of Enzyme Immobilized in Thin-film Coating and Its Applications |
| 学位专业 | 生物化工 |
| 中文摘要 | 随着工业生物技术的发展，固定化酶不仅有效降低了传统酶催化合成的生产成本，而且有力拓展了酶催化的应用领域。含酶薄膜涂层由于其固定化酶效率高、载酶量大和酶活稳定性好等优点，因而在有毒有害物质降解，抗菌抗生物污渍，生物传感器和生物燃料电池等领域有大量应用报道。然而，现有含酶薄膜涂层主要以水基材料为骨架，其成膜性和机械强度及其化学稳定性较差，无法满足日常接触性表面的实际应用需要。本次研究以溶剂基聚氨酯为涂层骨架材料，利用薄膜涂层固定化酶技术，将淀粉酶和蛋白酶分别与聚氨酯复合在金属表面成膜，从而获得具有良好机械强度的自清洁功能涂层；并结合热喷墨打印技术，在滤纸表面制备了基于含酶薄膜涂层的生物检测Point of care testing (POCT)装置。 本文首先通过直接乳化溶剂基聚氨酯前体和酶蛋白水溶液，制备出具有自清洁蛋白质、多糖等生物基污渍的含酶涂层，将固定化酶应用拓展至自清洁智能涂层领域。然而研究发现天然酶蛋白分子与溶剂基聚氨酯涂层存在相容性差的问题，其主要表现为：酶分子在聚氨酯涂层中大量团聚，其团聚体直径为4-6 微米；且分布极不均匀，颗粒间的距离可达20至200 微米不等，表面酶分子容易被快速洗脱而导致涂层活力快速下降，经过4次每次3分钟的水溶液冲洗后，涂层无明显活力剩余。为了改善酶蛋白在涂层中的分布，并使涂层具有更加稳定的表面活力和更长的自清洁功效期，我们分别尝试了葵酰氯和聚乙二醇（PEG）化学共价修饰，甲基-β-环糊精包结和表面活性剂离子配对物理修饰。实验发现PEG修饰可以明显改善酶蛋白在涂层中的分布，甲基-β-环糊精包结和离子配对修饰能显著延缓表面酶分子洗脱。此结果启示我们可以调节酶的PEG修饰度，在改善蛋白质分布的同时，缓释表面酶分子，从而达到优化分布和延长涂层功效期的目的。 选用直链小分子量PEG-1K-NHS为酶蛋白修饰剂，5倍和10倍量PEG修饰后的酶蛋白在涂层中以亚微米级颗粒分布，并具有显著的涂层活力（70和37 mU/cm2）和较稳定的重复使用性，连续水洗10次后涂层活力依旧能达到自清洁最低活力要求。室温下涂层活力半衰期可达350天，80 oC高温下为20天。PEG修饰不仅提高了α-淀粉酶在溶液中的稳定性，而且将最佳活力温度从40 oC升高至50 oC，通过圆二色光谱扫描发现PEG修饰对蛋白质二级结构未产生影响，说明修饰后酶蛋白活力下降，是由PEG修饰引起的酶分子表面空间位阻造成。PEG修饰促进酶分子在油水界面的吸附，并使酶蛋白具有更好的界面稳定性，此吸附可能引起油包水乳液颗粒变小，从而改善酶蛋白在溶剂基聚氨酯涂层中分布的现象。利用多功能8-arm-PEG-10K琥珀酰亚胺乙酸酯交联酶分子并制备涂层，可以获得220 mU/cm2的初始活力和更好的稳定性，反复使用10次涂层剩余活力依旧高于30%。 基于薄膜涂层空间限制作用能使酶蛋白具有更高的稳定性的特定，我们借助热喷墨打印技术，在滤纸上制备聚苯乙烯反应阵列和含葡萄糖氧化酶和乳酸氧化酶的薄膜涂层，从而获得使用便捷、制备简单、稳定性较好的生化分析POCT试纸装置。含酶涂层单孔反应阵列能对浓度为0-10 mM的葡萄糖溶液，和0-5 mM的乳酸溶液进行定量分析。当分析物浓度在0-3 mM的浓度范围时，试纸颜色深度与分析物浓度呈现良好的线性关系，分析物浓度高于4 mM时由于色彩饱和影响逐渐显著，检测灵敏度逐渐下降。在低温和室温下保存，试纸具有稳定的检测效果，即使在50oC下经过30天保存葡萄糖检测效果依旧稳定，乳酸检测试纸可以稳定12-15天。双通道检测试纸可同时对含有葡萄糖和乳酸的人工血液样品进行定量分析，并利用手机对显色结果进行RGB色彩分析，发现红，绿，蓝三个通道对分析物浓度都呈现良好的线性关系，其中绿色光通道分析灵敏度最好。纸质界面淀粉涂层中固定的葡萄糖氧化酶的Km与溶液中的自由酶接近，但是kcat降至溶液中的三分之一，催化效率的降低可以归因于滤纸反应载体和淀粉涂层引起的底物传质速率的下降。在溶液体系中和淀粉涂层固相体系中葡萄糖氧化酶的失活活化能分别约为64 KJ.mol-1和100 KJ.mol-1，说明涂层中的固定化酶具有更高的热稳定性。 |
| 英文摘要 | With the development of industrial biotechnology, the enzyme immobilizationnot only limited the cost of the enzymatic synthesis, but also expanded the applications of enzymatic catalysis. Due to higher enzyme loading,immobilizationefficiency, and better stability compared with other methods, enzyme containing thin-film coatings have been wildly used in the degradation of toxic and hazardous compounds, anti-bacterial anti-biological stains, biosensors and biofuel cellareas. However, the present thin-film matrixes are mainly water-based and do not have enough mechanical strength to make them applicable on touchable surfaces in our daily life. Based on the enzymatic thin-film coating technology, a novel self-cleaning coating with good mechanical strength was developed through the incorporation of hydrolytic enzymes into solvent-born polyurethane matrix.Furthermore, when the thermo ink-jet printing was adapted to develop the enzymatic thin-film coatings, a point of care testing (POCT) paper device was fabricated for the monitoring of glucose and lactate in human blood. The self-cleaning coating was prepared by directemulsification the solvent-born polyurethane and hydrolyticenzyme (protease TC160 or α-Amylase) solutions, which could effectively remove the bio-based stains, such as proteins and polysaccharides. This research expanded the application of enzyme immobilization into smart coating field. However, the native enzymes are not compatible with solvent-born polyurethane, thus lead to the significant enzymeaggregation and inhomogeneous distribution in coating matrix. The enzyme exposed on coating surface was susceptible to be eluted and resulted in rapid surface activity decline that no obvious coating activity left after 4 times of 3 minutes washing.To optimize the enzyme distribution and prolong the coating function period, the covalent binding of polyethylene glycol (PEG) or decanal chloride on protein surface and physical methyl-β-cyclodextrin complexation or ion paring was adapted. The PEG modification showed obvious enzyme distribution improvement and methyl-β-cyclodextrin complexation effectivelyalleviated the enzyme elution from the coating surface, which inspired us to develop a moderate PEG modified enzyme coating with controlled release enzyme from coating surface. The enzyme modified by 5-fold and 10-fold PEG-1K NHS ester was still aggregated with sub-micrometer diameter in coatings, which initially had 70 and 37 mU/cm2 surface activity and they were still higher than the minimal requirement for self-cleaning functionality even after 10 times of re-testing. Meanwhile, the coating half-life time could reach 350 days at room temperature and 20 days in 80 oC environment. The PEG modification will not affect the secondary structure as demonstrated by circular dichroism, indicating that the pegylationinduced enzyme activity decrease could be attributed to the steric hindrance from PEG-side chains. Pegylation also promoted the enzyme adsorption and stabilization in oil-water interface, therefore could cause smaller emulsion particles and improve the enzyme distribution in coating matrix. 8-arm-PEG-10K NHS ester was also used as cross-linker to pre-crosslink the enzyme and generate a coating activity as high as 220 mU/cm2, meanwhile, more than 30% surface activity was retained after 10 times of repetive testing. Considering the enzyme stabilization induced bythin-film coating spatial confinement,a POCT device was developed by the thermo inkjet printing technology, which flexibly printed the enzymes, coating materials and polystyrene onto filter paper according to our pre-designed pattern. Single-well array with enzymeembedded thin-film could quantitatively analysis 0-10 mM glucose or 0-5 mM lactate solution, and it showed a good linear relationship between the paper color intensity and analyte concentration from 0 to 3 mM. When the analyte concentration is higher than 4 mM, the sensitivity decreased because of the colo |
| 语种 | 中文 |
| 公开日期 | 2015-07-08 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/15514]  |
| 专题 | 过程工程研究所_研究所（批量导入） |
| 推荐引用方式 GB/T 7714 | 张立挺. 含酶薄膜涂层的生物催化与应用研究[D]. 中国科学院研究生院. 2014. |
入库方式： OAI收割
来源：过程工程研究所
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