【作者】 黄淑芳；

【导师】 姜忠义；

【作者基本信息】 天津大学 ， 化学工艺， 2004， 硕士

【摘要】 传统溶胶-凝胶(sol-gel)法进行酶的固定化具有酶活损失大和传质阻力大的缺点。本文以聚乙二醇（PEG）为多功能试剂对传统sol-gel过程进行改进，以提高固定化甲醇脱氢酶(ADH)的活性。以PEG为模板剂修饰凝胶，使凝胶的孔径由 7.8nm增大到 9.3nm，比表面积也由 463 m2﹒g-1增加到 534m2﹒g-1，底物在凝胶孔中的有效扩散系数提高到原来的 1.4 倍。用修饰后的凝胶固定化的ADH的活性比传统凝胶固定化的ADH的活性高。此外，利用活化PEG(SC-PEG)为修饰剂对ADH进行修饰，以提高ADH在固定化后的活性，ADH的修饰度约 90％。对修饰后的ADH进行固定化，ADH的活性比未经修饰的ADH的活性有明显提高。由甲醛转化为甲醇的酶促反应比较两条提高酶活途径下的四种固定化的ADH的活性：未修饰凝胶固定化修饰ADH（固定化ADHⅢ）活性> 修饰凝胶固定化修饰ADH（固定化ADHⅣ）活性> 修饰凝胶固定化未修饰ADH（固定化ADHⅡ）活性> 传统固定化方法下的ADH（固定化ADHⅠ）活性。而且固定化ADHⅡ、ADHⅢ、ADHⅣ的稳定性均高于固定化ADHⅠ的稳定性。可见凝胶的修饰以及ADH的修饰均可提高固定化ADH的活性和稳定性，但是ADH的修饰较凝胶的修饰对提高ADH的活性效果明显。 另外，为了比较吸附法与传统 sol-gel 包埋法固定化 ADH 的优劣，对两种方法固定化的 ADH 的活性进行了比较。采用大孔硅胶对 ADH 进行吸附，得到吸附动力学曲线和吸附等温线，ADH 在硅胶上的吸附等温线可以用 Langmuir方程拟合。并且分析了硅胶孔径对 ADH 吸附量的影响，得到大孔径的硅胶对ADH 有着更大吸附量的结论。由甲醛转化为甲醇的酶促反应动力学实验测得的反应初速度及拟合得到的米氏常数表明吸附法固定化 ADH 表现出比 sol-gel 法固定化 ADH 高的催化活性。更多还原

【Abstract】 Enzyme immobilization by traditional sol-gel approach suffers from thedrawbacks of relatively high enzyme activity loss and high mass transfer resistance,solutions to this problem have been tentatively exploited herein, PEGs wereemployed as multifunctional additives to enhance immobilized enzyme activities: 1.Tetraethylorthosilicate (TEOS) derived gel is modified in the presence ofpolyethylene glycol (PEG, molecular weight 600), the effects of PEG addition to theprecursor solution on the microstructure of the gel are studied, the size of pores isincreased in the resultant gels on the bridge-linkage between PEG and precursor,thus diffusion rates is comparably increased to1.4 times. 2. PEG SuccinimidylCarbonate (SC-PEG, molecular weight 5,000) is used to modify alcoholdehydrogenase (ADH) before being immobilized in the gel in order to avoid thedamage of enzyme in sol-gel process and also improve the properties of gels such ashydrophilicity. The activities of diverse immobilized ADH have been investigated bythe enzymatic reaction of converting formaldehyde into methanol: unmodified gelimmobilized modified ADH（Immobilized ADHⅢ）>modified gel immobilizedmodified ADH（Immobilized ADHⅣ）>modified gel immobilized unmodified ADH（ Immobilized ADH Ⅱ ） > unmodified gel immobilized unmodified ADH（ImmobilizedADHⅠ）.And the stabilities of immobilizedADHⅡ、ADHⅢ、ADHⅣ are higher than that of immobilized ADHⅠ. This result indicated that enzymeactivities after the modification of gel and the modification of ADH respectivelysignificantly increased compared to the native immobilized ADH, but modificationon ADH is more effective. In addition, in order to compare the enzyme immobilization differencesbetween adsorption immobilization process and sol-gel encapsulation process. ADHis respectively immobilized onto macroporous silica carrier by adsorption and intomicroporous silica gel by sol-gel encapsulation. Through the study of adsorptionkinetics and adsorption equilibrium, the kinetics curve and adsorption isotherm areobtained. The adsorption isotherm is fitted by Langmuir equation. The effect of poresize on the adsorption capacity is examined. The sol-gel encapsulated ADH isprepared with silica matrix. The catalytic activities of the above-mentioned<WP=5>immobilized ADHs towards the conversion of formaldehyde into methanol arecompared. The initial rates of enzymatic reaction catalyzed by adsorptionimmobilized ADH is higher than that catalyzed by sol-gel encapsulated ADH,whereas the Michaelis constants of the former are less than that of the latter. Theseresults indicate that the catalytic activity of adsorption immobilized ADH is higherthan that of sol-gel encapsulated ADH.更多还原