Study On The Selection And The Kinetic Characteristic Of Alcohol Dehydrogenase Bacteria

Alcohol dehydrogenase(ADH) is the key enzyme of the primary short-chain alcohol metabolism in many organisms which has attracted great attention because of its peculiar catalysis. At present reports about the application of ADH increased gradually. However the commercial ADH was obtained from livers of animals mostely which was costly and limited by their sourse.Therefore it is an important way to separate microorganisum which has high ADH activity to realize the scale production of ADH and the way has a good foreground.It is necessary to industrialize the ADH-production-strain to realize the scale production of ADH .So enough knowledge to the growth and substrat-consume kinetic feature ADH-production-strain is needed. Research on this respect was seldom reported. In this article a strain of ethanol-consuming bacteria producing ADH identified as Acetobacter was separated. The reaction kinetics of Acetobacter and some of the ADH biochemical properties were studied. The research not only provided some reliable reference and message for the continuous culture then large-scale production of Acetobacte Z127 research but also found basis for the further study and application of Acetobacte Z127 ADH.Here shows the main methods, content and conculsion of the research.1) The selection and identification of bacteria producing ADH. The bacteria sample gotten was cultured in the medium with ethanol as its sole carbon sourse. Separate the bacteria being able to utilize ethanol efficiently by grads-training method (to increase ethanol consistence gradually in the culture medium). Then identify the separated bacteria according to its shape feature and the physiological biochemical reaction results. As the result a strain of bacteria which could consume ethanol efficiently was selected and identified as Acetobacter. It is a new strain of Acetobacter because its physiological and biochemical characteristic didn't match to that of any exiting strain of Acetobacter genus. Therefore it was named Acetobacter Z127 in the project. The ethanol consistence AcetobacterZ127 could utilize was less than 0.6mol/L. The optimal ethanol consistence for the growth of AcetobacterZ127 was 0.4mol/L.2) Reaction kinetics of Acetobacter Z127. The mechanism of cell growth was studied with Monod model based on the theory of biochemical reaction kinetics.The biodynamic formula was formed to describe the consume course of ethanol .It accorded with the substrat-consume model which includes the endogenesis metabolism.Related parameters in the two modle were tested.The proof-test showed that the two model formed could reflect the law of Acetobacter Z127 growth and ethanol degradation. 3) The extraction and purification of Acetobacter Z127 ADH. Four methods were employed to isolate ADH from Acetobacter Z127 just to find bacteriolytic solution could break cell efficiently and maintain ADH activity simultaneously and thus it was suitable for the extraction of Acetobacter Z127 ADH. ADH was pured 5.49 fold with a final yield of 48.09% by bacteriolytic solution crude extract, (NH4)2SO4 fractionation, sephadexG100 chromatography. The molecular weigt of Acetobacter Z127 ADH was 28.0 KDa judged by SDS-PAGE which was similar to the Entamoeba histolytica ADH(31.0 KDa) reported.4) Research on parts of ADH properties. Incubate ADH solution in buffer system with different pH value at 37℃ for 20min then measure the activity.The optimum pH ADH oxidate ethanol is 7.0. And Incubate ADH solution in the same buffer system at different temperature just to find Acetobacte Z127 ADH could tolerance thermal and the optimum temperature was 60℃. Metal ion had effect on Acetobacte Z127 ADH activity. Kalium ion inhanced ADH activity but bivalent metal ion restrained its activity at different degree. Zine ion didn't affect ADH activity just because it was a cofacter of ADH. ADH was denatured terribly in Fe3+ solution.