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Study on the Purification and Application of Alcohol Dehydrogenase from Yeast

Author: ZhangXiaoJun
Tutor: LiuChangXian
School: Central South University for Nationalities
Course: Analytical Chemistry
Keywords: Reversed micelles Extraction Immobilization Alcohol dehydro- genase Enzymatic determination
CLC: TQ464.8
Type: Master's thesis
Year: 2011
Downloads: 30
Quote: 0
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Abstract


Alcohol dehydrogenase(ADH) is an important bio-catalyst that exist in the cells of animals, plants and microorganisms.It has become one of the foci of study applied in the aspects of industrial production, determination and medical field.In the first part the purification of biological macromolecules,the basic knowledge and applied research of enzymatic analysis and the technology of reverse micelle are summarized.In the second part the extraction of yeast ADH using reverse micelle system of cetyl trimethyl ammonium bromide(CTAB)/octane/pentanol was investigated.The effect for extraction of the concentration of surfactant and cosolvent,pH value,ionic strength 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 was determined.The influence of pyridine on the fluorescence spectroscopy of ADH was investigated. The interference of coexisting components was studied.The enzymatic mathod has been used to determine the concentration of pyridine in imitated samples.In the fourth part the characterization of immobilized ADH in reversed micelles system are studied.The effect for the immobilization of W0,the concentration of CTAB,pentanol and pH were investigated.The study on the kinetic property of the free and immobilized enzyme indicates that optimum pH of the enzymatic reaction were 8.8 and 8.6, the optimum temperature were 28℃and 20℃,the michaelis constant for alcohol were 8.8mmol/L and 5.5mmol/L respectively.The enzymes were deposited for 120min at 25℃,the activities were lost 90% and 28% for the free and immobilized enzyme respectively.It shows that the immobilized ADH in reversed micelles has better thermal stability.

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