摘要
目的:选育乙醇脱氢酶(Alcohol dehydrogenase,ADH)高产的菌株,研究ADH的提取方法、酶学性质等,建立ADH解酒作用模型,为解酒产品的开发提供理论依据。
研究的主要内容及结论如下:
1)菌株的筛选及鉴定。通过醋醅在无糖液体培养基中富集培养,筛选出一株可以快速将乙醇转化为乙酸的菌株,经过形态观察和16SrDNA测序及进化分析鉴定为醋酸杆菌。
2)诱变育种和发酵工艺优化。以筛选得到的菌株为出发菌株,用紫外线诱变的方法对该菌株进行诱变,从192个突变株中筛选得到了利用乙醇能力更强的醋酸杆菌C-118菌株(C-118),并对C-118的发酵工艺进行了优化,最终确定了C-118的最适发酵条件,研究发现C-118液体发酵的最适乙醇浓度为9%,最适温度为35℃,最佳发酵时间为36h。
3) ADH的提纯及酶学性质研究。用4种不同的方法提取ADH,比较后发现溶菌酶法破碎菌体得到的ADH活性较高,C-118经发酵、收集菌体、溶菌酶破壁、硫酸按盐析、透析和Sephadex -G100过滤层析得到了纯化的ADH,聚丙烯酰胺凝胶电泳证实该酶的分子量约为60KDa。ADH的酶学性质研究发现,最适pH值为8.0左右,最适温度60℃,最适底物为正丙醇,金属离子对ADH活性的影响存在比较大的差异,其中K+、Mg2+、Al3+等对ADH基本没有影响,Ag+、Cu2+、Hg2+等重金属离子对ADH的抑制作用强烈。
4)ADH解酒作用初步研究。通过与中药提取物的相互作用研究,筛选出了几味能够在体外促进C-118 ADH活性的药材,如人参、赤小豆等,为复合型解酒保肝药物的开发提供了药理依据;通过构建ADH对小鼠耐乙醇作用的影响药效学模型,初步考察了C-118 ADH的解酒作用,结果发现C-118 ADH对小鼠耐乙醇能力有明显改善,可以起到解酒的效果,为ADH解酒制剂的开发提供了药效学依据。
研究的主要意义在于:
本课题通过筛选和紫外诱变得到了一株可以快速利用乙醇产醋酸的菌株,并从该菌株中提取纯化了ADH,丰富了乙醇脱氢酶基础理论研究的内容。
在对ADH特性进行研究的基础上,进一步对其解酒作用进行了研究,为以ADH为主要活性成分的解酒产品的开发进行了有益的探索。
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.
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