Study On The Screening And Identification Of High Producing Alcohol Dehydrogenase Strain And Enzymatic Characteriatics

The alcohol dehydrogenase （ADH， E.C.1.1.1.1） is widely present in the plant,microorganisms and animals. The main research conclusions are as follows:1. Screening of ADH strain.Using Acid-producing flat, hydrolysis of calcium carbonatetransparent circle method and ethanol as the only carbon source to screen out a strain TDY1which can tolerance7%ethanol. After the analysis of morphological, physiological andbiochemical tests and16S rDNA sequence, the strain was identified as Gluconacetobacter,named Gluconacetobacter sp. TDY1.2．Study on the growth and enzyme production conditions of strain TDY1.Single factorexperiment to optimize the culture medium of1.5%glucose,2%complex nitrogen source （yeastextract+beef paste）,0.4%calcium chloride and0.4%organic acid; the best growth conditionswere the initial pH6.0, inoculation amount8%, Volume30mL/300mL,25℃for20h. ThroughBox-Behnken design, The optimal conditions of enzyme producing were glucose1.54%,complex nitrogen source2.02%, liquid volume of48.70mL and lactic acid0.42%, TDY1enzyme activity reached7191U/mg.3. Extraction and purification the intracellular ADH of strain TDY1.Four different cell walldisruption methods，such as ultrasonic treatment，repeated freezing，lysozyme digestion and celllysis solution were used to treat strain TDY1cells respectively. Comparing the cell walldisruption rates and the activities of the intracellular alcohol dehydrogenase to choose the mostappropriate method for cell wall disruption of TDY1. The ultrasonic treatment collected theactivity of4004U/mg and its cell wall disruption rates was97.3%，significantly different fromthree others （P <0.01）. The optimum conditions of ultrasonic treatment were ultrasonic time9s, Breaking time75s, ultrasonic power95W.Using the ultrasonication method, ammoniumsulfate salting-out precipitation, dialysis and DEAE-52anion exchange chromatographythree-step process, purified by8.7times, the enzyme activity yield of38.7%. The subunitmolecular weight of purified ADH was48KDa that identified by SDS-PAGE.4. Study on some ADH properties of the strain TDY1. The optimal oxidation reaction pH waspH9.0,stable at the range of pH8-9. The optimum reaction temperature was70℃, stable at therange of10-60℃.The optimum substrate was n-propanol. There was a big difference betweenthe effects of different metal ions on the activity of NAD⁺-ADH, in which Mn²⁺had a promoting effect; Fe³⁺didn’t promote or inhibit the enzyme activity; K⁺, Na⁺, Mg²⁺and other metal ionshad different degrees of inhibition; Ba²⁺ion made ADH complete loss of activity.