酵母醇脱氢酶提纯及的应用的研究.docxVIP

摘要醇脱氢酶(ADH)存在于动物、植物及微生物细胞中，是生物体内重要的氧化还原催化剂，很多醇类代谢都是通过 ADH 完成的。ADH 在工业生产、分析测定、医学领域都有较广泛的应用，因此成为研究的热点之一。第一章概述了生物大分子的纯化方法，酶分析法以及反胶束技术的基本知识和应用研究。第二章探讨了“十六烷基三甲基溴化铵(CTAB)—辛烷—戊醇”反胶束体系萃取酵母 ADH。试验了表面活性剂和助溶剂的浓度以及 pH 值对正萃的影响，离子强度和 pH 值等因素对反萃的影响，确定了最佳萃取条件。第三章研究了有机分子对酶促反应“乙醇+NAD+←→乙醛+NADH+H+”的影响，确定了吡啶的抑制类型，考察了吡啶对 ADH 荧光光谱的影响。试验了共存组分对测定吡啶的干扰作用，用酶法测定了模拟水样中吡啶的含量。第四章探讨了反胶束固定化 ADH 的酶学性质，考察了含水量，CTAB，戊醇以及 pH 对固定化的影响。对游离和固定化 ADH 的酶学性质研究结果表明：酶促反应的最适 pH 值分别是 8.8 和 8.6，最适温度分别是 28℃和 20℃，对乙醇的米氏常数分别为 8.8mmol/L 和 5.5mmol/L。25℃时游离酶存放 2h 约失活 90%，而固定化酶仅失活 28%，表明反胶束固定化 ADH 具有较好的热稳定性。关键词：反胶束；萃取；固定化；醇脱氢酶；酶法测定1AbstractAlcohol dehydrogenase(ADH) is an important bio-catalyst that exist in the cellsof animals, plants and microorganisms.It has become one of the foci of study appliedin the aspects of industrial production, determination and medical field.In the first part the purification of biological macromolecules,the basicknowledge and applied research of enzymatic analysis and the technology of reversemicelle are summarized.In the second part the extraction of yeast ADH using reverse micelle system ofcetyl trimethyl ammonium bromide(CTAB)/octane/pentanol was investigated.Theeffect for extraction of the concentration of surfactant and cosolvent,pH value,ionicstrength were tested.The optimum conditions were established.In the third part the effect of organic molecules on the enzymatic reactions“C2H5OH+NAD ← → CH3CHO+NADH+H+” is studied.The inhibition type wasdetermined.The influence of pyridine on the fluorescence spectroscopy of ADH wasinvestigated. The interference of coexisting components was studied.The enzymaticmathod has been used to determine the concentration of pyridine in imitated samples.In the fourth part the characterization of immobilized ADH in reversed micellessystem are studied.The effect for the immobilization of W0,the concentration ofCTAB,pentanol and pH were investigated.The study on the kinetic property of thefree and immobilized enzyme indicates that optimum pH of the enzymatic r