Study On The Characteristics And Application Of Alcohol Dehydrogenase

Alcohol dehydrogenase(ADH) is a zinc-containing enzyme that exist in the cells of animals, plants and microorganisms.It is used as a catalyst for the oxidation of primary alcohol and reduction of the aldehyde and as a specific indicator for diagnosis of liver cell in the medical analysis. In recent years,the study on the application of the enzyme is in full swing with the development of oil phase catalysis and membrane reactor in the aspects as biological sensor used in analysis of the concentration of ethanol and as a catalyst for producing raw materials or intermediate in chemical industry and as a experimental material in the immobilization of enzyme as well as new reagent for decomposing ethanol of wine.It has become one of the foci of study applied in the aspects of medical analysis and industrial production,etc.The thesis is composed of four parts.In the first part the mechanism of the inhibition on ADH was introduced as well as was the method of quantitative analysis of ethanol in addition with the theory and application of the immobilization of enzyme.In the seconed part the impact of inhibitor on the catalysis of ADH was investigated.The inhibition of cadmiun ion and thiocyanate on the enzymatic reaction under the optimum conditions based on the catalytic reaction of alcohol and nicotinamide adenine dinucleotide oxidized form(NAD) by ADH was studied.The experiment of elimination of interference of the inhibitors was done by adding the ligand of metal ion—ethylene dinamine tetraacetic acid (EDTA).The result indicates that the interference of certain concentration's cadmium ion for determining substrate by the catalytic reaction of ADH can be eliminated effectively.The influence of cadmiun ion and thiocyanate on the fluorescent spectra of ADH was investigated and the phenomenon of the fluorescence of ADH quenching statically was found.That interaction of ADH with cadmium ion and thiocyanate by using the technique of the synchronous fluorescence was explored. In the third part the determination of ethanol by enzymatic kinetic fluorescent method was studied.Based on the reaction catalyzed by ADH: C2H5OH+NAD→CH3CHO +NADH.The changing rate of fluorescence intensity of nicotinamide adenine dinucleotide reduced form(NADH) was determined for calculating the velocity of the enzymatic reaction and the calibration curve of ethanol was drawn.The ethanol in samples was determined by the calibration curve.The method has been used to determine the concentration of ethanol in calf serum and drinks with satisfactory.In the fourth part the preperation and enzymatic characterizations of immobilized ADH on chitosan was investigated.Chitosan was used as the carrier and glutaraldehyde as the crosslinking reagent.ADH was immobilized by covalently bonding.The influence on the immobilization for ADH of the concentration of glutaraldehyde,binding time of enzyme and pH value of the solution was discussed. The study on the enzymatic characterizations of free and immobilized ADH indicates that optimum pH of the enzymatic reaction were 8.4 and 8.8,the optimum temperatures were 31℃and 26℃,the Km for ethanol were 28.2 and 8.1mmol/L respectively.Compared with the free enzyme,the immobilized enzyme has better thermo-stability, storage stability and reusability.