Extraction Of The High Activity Of Acetobacter Breeding And Alcohol Dehydrogenase

Purpose:Select a strain of bacteria which has a great capability to produce alcohol dehydrogenase(ADH),study on the extract method of ADH and its enzymatic properties.Establish dealcoholic model of ADH. provide a theoretical basis for the development of ADH products.The main content and conclusions are as follows:1) The screening and identification of target strain. Using high concentrations of ethanol in the medium , through the enrichment culture, selected a strain of bacteria which can convert ethanol into acetic acid quickly. After morphology and 16 SrDNA sequencing,this strain of bacteria was identified as Acetobacter.2) Mutation breeding and fermentation process optimization.By UV mutagenesis on starting strain 192 mutants were screened.Finaly the C-118 strain was selected and it has a greater ability about the use of alcohol than the starting strain. the fermentation process of C-118 was optimized and the optimum fermentation conditions as follows: ethanol concentration is 9%, temperature is 35℃and time for 36 hours.3) The purification of C-118 ADH and enzymatic properties. Four methods were employed to isolate ADH from C-118 .The findings demonstrate that use lysozyme could break cell efficiently and maintain ADH activity simultaneously and thus it was suitable for the extraction of C-118 ADH. C-118 ADH was purified by using fermentation and collection, lysozyme crude extract, (NH4)2SO4 fractionation, dialysis and sephadex G-100 chromatography. The molecular weigt of C-118 ADH was 60 KDa judged by SDS-PAGE.Study on some of C-118 ADH properties indicate that:the optimum pH of C-118 alcohol dehydrogenase for the oxidation of ethanol is 8.0, the optimum temperature is 60℃and the optimum substrate is n-propanol. Metal ions had different effect on the activity of C-118 ADH. K+,Mg2+,Al3+showed no influence while heavy metal ions such as Ag+,Cu2+,Hg2+ have a great inhibit ability on the enzyme. 4) Preliminary studies of ADH on dealcoholic action showed several Traditional Chinese Medicine extracts screened out,such as Panax ginseng, Phaseolus calcaratus etc.,can enhanced in vitro. Provided pharmacodynamics basis to the development of compound drugs for liver protection.Estabilished an ethanol resistance pharmacodynamics model of mice for studing the C-118 ADH dealcoholic effects. The results showed that C-118 ADH can enhance ethanol resistance significantly for mice.These indicate that C-118 ADH can be used for dealcoholic drugs.The values of these studies lies in:In this article a strain of ethanol-fast-consuming bacteria was separated from solid fermentation of vinegar,and then ADH is extracted and purified from this strain. Enriching basic theoretical value of alcohol dehydrogenase.At the same time a preliminary study on dealcoholic was performed. These results provide theoretical basis for the developping of dealcoholic products on ADH.All of these studies have a high value both on theoretical and on practical respects.