| 题名 | TiO2纳米纤维的制备及在固定化酶中的应用 |
| 作者 | 王欣然 |
| 学位类别 | 硕士 |
| 答辩日期 | 2011-05-24 |
| 授予单位 | 中国科学院研究生院 |
| 导师 | 张松平 |
| 关键词 | 纳米纤维 固定化酶 糖酯 胆汁酸检测 辅酶再生 |
| 其他题名 | Preparation of TiO2 nanofiber and its application in enzyme immobilization |
| 学位专业 | 生物化工 |
| 中文摘要 | 纳米纤维比表面积大、制备简单以及宏观呈膜形态等特性，使其做为固定化酶的载体具有独特的优势和良好的工业应用前景。目前用于固定化酶的纳米纤维多为有机高分子材料，而生物相容性更好的无机材料，如基于TiO2、SiO2的纳米纤维尚未被广泛应用。本论文结合溶胶凝胶-静电纺丝技术制备出TiO2纳米纤维，并将其用于固定化碱性蛋白酶Protex 6L，在有机相中催化重要的生物表面活性剂脂肪酸糖酯的合成反应；同时还首次尝试用于包括辅酶的多酶体系的固定化，进行胆汁酸的分析检测。具体研究包括如下几个方面： (1) 结合溶胶凝胶法和静电纺丝技术，制备TiO2纳米纤维。考察了电压和电纺液流量对纳米纤维平均直径及形貌的影响。在电场强度1.8 kV cm-1，电纺液流速为500 μL h-1时，所制备出的纳米纤维平均直径为125 nm且分布均一。 (2) 利用碱性蛋白酶Protex 6L，在叔戊醇/二甲基亚砜混合溶剂中催化月桂酸乙烯酯与蔗糖的转酯化反应制备月桂酸蔗糖酯。首先考察了底物加入方式对反应速率的影响，蔗糖以粉末形式最后直接加入反应体系中时，反应效率最高。采用响应面法对影响糖酯合成的主要因素：温度、溶剂中水含量以及Protex 6L的pH值进行优化，得到最佳实验条件为：43 oC、3.4%的含水量和pH 10.0的Protex 6L冻干粉，在此条件下响应面的预测和验证试验得到的糖酯合成初速率分别为8.7 mg.mL-1.h-1和8.66 mg.mL-1.h-1。在最佳实验条件下， 1.5 mg.mL-1的游离的Protex 6L在 9 h内可以将98%的蔗糖转化为糖酯。这一结果远高于其他常用于蔗糖酯合成的固定化脂肪酶，包括Novozym 435，Lipase AK, Lipase Candida Rugosa，以及蛋白酶Proleather。 (3) 利用TiO2纳米纤维固定化Protex 6L并用于月桂酸糖酯的合成，对固定化条件和合成反应条件进行了优化，载酶量达到201mg.g-1时，比活力最高为2.45μmol.h-1.mg-1；在50 oC和5%水含量的反应条件下，经过36 h的反应，基于蔗糖的转化率为97%，产率为Novozym 435的34倍。考察固定化Protex 6L的重复使用性表明，酶膜重复使用10次，剩余活性为52.4%。 (4) 本论文构建了可用于胆汁酸检测的包括3a-羟基类固醇脱氢酶、黄递酶和辅酶NADH的多酶偶联体系，并首次尝试将纳米纤维用于该多酶体系的固定化。采用2,6-二氯酚靛酚作为黄递酶的底物，胆汁酸浓度为10 mmol.L-1和50 mmol.L-1，反应2 h后体系在600 nm下的吸光度值改变分别为0.017和0.05。初步证实了多酶偶联反应的进行，但是反应效率还有待进一步提高。 |
| 英文摘要 | Nanofiber have huge potential on enzyme immobilization and industry application due to their promising characteristics involving high surface-volume ratio, easy preparation and non-woven membrane appearance. Nowadays, nanofibers used for enzyme immobilization are mostly made from polymeric material. However, inorganic nanofiber, such as TiO2 and SiO2 based nanofibers which have better biocompability, has not been widely applied. In this work, TiO2 nanofiber prepared through sol-gel process and electrospinning technology was applied for an alkaline protease Protex 6L immobilization. The nanofibrous Protex 6L was then applied to synthesize an important biosurfactant sugar ester in organic solvent. Additionally, a multi-enzyme system involving co-enzyme regeneration was established and for first time immobilized on TiO2 nanofiber for choleric acid assay. The thesis includes of the following four parts: (1) TiO2 nanofiber was prepared through combined sol-gel process and electrospinning technology. Effect of electric field strength and flow rate of electrospinning solution on nanofiber diameter and morphology was investigated. The optimal condition for electrospinning was electric field strength 1.8 KV/cm and the flow rate 500 mL/h, under which uniform TiO2 nanofiber was produced with the average diameter 125nm. (2) Protex 6L was applied to catalyze the synthesis of sucrose laurate from vinyl laurate and sucrose in DMSO/two-methyl-two-butanol solvent mixture. Substrate feeding protocol was investigated and the highest efficiency was achieved when the sucrose powder was added to reaction system ultimately. Temperature, water content in solvent mixture and pH of Protex 6L, which are the three key factors controlling sugar ester synthesis, were optimized by response surface analysis and the optimal condition was 43 oC, 3.4% water content and Protex 6L powder lyophilized from buffer of pH10.0. Under the optimized condition, the estimated and practical initial rate of sugar synthesis was 8.7 mg.mL-1.h-1 and 8.66 mg.mL-1.h-1 respectively, and 98% sucrose was converted to sugar ester by 1.5 mg.mL-1 free Protex 6L within 9h. Such sugar ester synthesis efficiency was far higher than that of other tested enzymes including immobilized lipase Novozym 435, Lipase AK, Lipase Candida Rugosa and protease Proleather. (3) TiO2 nanofiber immobilized Protex 6L was applied on sucrose laurate synthesis. Conditions for Protex 6L immobilization and sucrose laurate synthesis reaction were optimized, and the optimal enzyme loading was 201mg.g-1 and the specific activity was 2.45μmol.h-1.mg-1. Under the optimized reaction condition, 50 oC and 5% water content, the sucrose conversion was 97% within 36h, which is 34-time higher than that of Novozym 435. After 10 cycles, the residual enzyme activity was 52.4%. (4) This paper established a multi-enzyme system containing 3a-hydroxysteroid dehydrogenase(3a-HSD), diaphorase and NADH, and for first time immobilized this multi-enzyme system on TiO2 nanofiber for choleric acid assay. Using 2,6-dichlorophenolindophenol (DCPIP) as the substrate of diaphorase, changes in absorbance at 600nm was 0.017 and 0.05 with choleric acid concentration of 10 mmol.L-1 and 50 mmol.L-1, respectively. This proved the activity of immobilized multi-enzyme and the regeneration of NADH. However, the enzyme activity need to be further improved. |
| 语种 | 中文 |
| 公开日期 | 2013-09-24 |
| 页码 | 91 |
| 内容类型 | 学位论文 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1730]  |
| 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 | 王欣然. TiO2纳米纤维的制备及在固定化酶中的应用[D]. 中国科学院研究生院. 2011. |
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