| Alcohol dehydrogenase can catalyze ethanol into acetaldehyde by using nicotinamide adenine dinucleotide as coenzyme. Then the acetaldehyde can be converted into acetic acid by the catalysis of aldehyde dehydrogenase, and ultimately generating carbon dioxide and water. Acetobacter used in the industrial production of vinegar can produce a large number of alcohol dehydrogenase and aldehyde dehydrogenase to catalyze acetic acid into ethanol. In order to enhance the production of aldehyde dehydrogenase by Acetobacter , this issue did the C118 strain selection and fermentation process of mutagenesis strain AbI-9-3. Initially established industrial production methods for extraction of alcohol dehydrogenase, and stability formulation largely prompting the activity of enzyme and optimization of anti-inebriation effects of ADH model animal testing conducted on the basis of research to understand the effect of liquor for the enzyme products of research and development has laid a good foundation.Experiment used as the starting Acetobacter strain C118 by UV mutagenesis, chemical mutagenesis and high concentrations of ethanol and other domesticated combination of UV-mutagenesis approach to acid production capacity per unit volume and wet cell yield as an index filter to be 4 Strains, select two of which are excellent indicators of a bacteria strain, as the purpose and known as AbI-9-3. Through the optimization of culture conditions Acetobacter identified AbI-9-3 strain medium and fermentation conditions. The best medium: yeast extract 3%, glucose 1%, calcium carbonate 0.6%, 5% ethanol. In the inoculum was 10%, temperature 35℃, speed 200 to / min, when a 24-hour fermentation inducers ethanol, fermented for 48 hours, the largest fermentation production of 24.03g / L, ADH activity for a maximum 93000U / g, than the C118 increased 3.04 times. This study also explored the acid-base indicator methyl red in the application of screening experiments, according to colony size and the indicator changes color is very easy to select high-yield strains, and the mutation breeding Acetobacter provide a simple, efficient experimental methods of screening.Find a simple and efficient extraction method ADH - organic solvent method to broken cells, the conditions of -20℃for rapid cooling of the acetone treatment, home refrigerators -20℃standing for 2 hours, the results of the effect of organic solvents acetone broken a good recovery rate of activity of lysozyme law has reached 90%. ADH at the same time, the essence of technology to come to 1~1.5 times the acetone precipitation can be effectively ADH protein and enzyme activity in the retention rate over 80%, impurity removal rate is also higher range. By Sephadex-G100 gel column chromatography was purified ADH.Investigated salt, sugar, alcohol, acid and antioxidants on the ADH activity under normal temperature and under high-temperature protection. Finally, to identify four kinds of material and to determine their concentration in the dosage of the best of 0.03M glycine, 0.5M sorbitol, and the 50~100mM of KCl. 20mM~50mM of calcium carbonate for the basic enzyme stabilizing. Orthogonal test after study of their interaction. Eventually arrive at 1 mM KCl, 0.03 M glycine, 1 M sorbitol, calcium carbonate is greater than 20 mM has a protective effect on ADH.Through screening and modeling, the creation of a better evaluation of the effect of ADH Jiejiu animal model, and the model was applied to pre-test and formal tests have been good results. To determine the initial effect of the ADH concentration 15000U/Kg, maximum tolerated dose of 20g/Kg, agents for the development of anti-inebriation ADH provides the basis of pharmacodynamics. |
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