【作者】 郝建荣；

【导师】 许平； 崔行；

【作者基本信息】 山东大学 ， 生物化学与分子生物学， 2006， 博士

【摘要】 丙酮酸(pyruvic acid)是一种重要的医药、化工产品，其生产一直是生物化工领域的研究热点。酶催化法生产丙酮酸具有生产成本低、造成的污染少等优点，成为关注的焦点。乳酸是生产丙酮酸的原材料之一，目前为止人们发现自然界存在三种可以催化乳酸生成丙酮酸的酶类，分别是：乳酸脱氢酶(lactate dehydrogenase，LDH)，乳酸氧化酶(lactate oxidase，LOX)和细胞色素氧化酶b2(flavocytochrome b2，FCB2)。其中FCB2研究较少，未见用于生产丙酮酸的报道。利用乳酸氧化酶生产丙酮酸已有报道，但由于在催化乳酸生成丙酮酸的过程中有过氧化氢的产生，导致丙酮酸的稳定性降低，所以使该酶在丙酮酸生产中的应用受到限制。催化乳酸生成丙酮酸的脱氢酶是NAD~+非依赖性的乳酸脱氢酶，该酶催化乳酸生成丙酮酸，不依赖NAD~+为辅酶，同时催化过程没有过氧化氢的生成，因此利用该酶生产丙酮酸具有广阔的应用前景。 酶催化法生产丙酮酸的核心是筛选具有高催化活性的菌株，该菌株不仅具有催化合成丙酮酸的高活性的酶或酶系，同时要满足催化条件简便易行，副产物少，后续加工程序简单等特性。研究该菌株的催化活性，确定关键酶的性质和催化机理，确定参与的代谢途径，对优化转化条件、提取纯化关键酶蛋白、构建相关的基因工程菌具有重要指导意义，并有助于最终达到提高转化效率，提高丙酮酸产量的目的。 经典的研究代谢途径的方法是推测和验证相结合，随着蛋白质组学的迅速发展，利用蛋白质组学研究代谢途径已经逐步代替了以往的方法，双向电泳技术自1975年问世以来已经成为一种分离蛋白质的有效工具，伴随着基质辅助激光解吸飞行时间质谱分析技术(matrix assisted laser desorption／ionization time of flight mass spectrometry，MALDI-TOF MS)的广泛应用，使得蛋白质鉴定变得较可行。并在一定条件下对蛋白质的表达进行分析为研究代谢网络的组成及功能提供了重要的信息；另外酶活性的测定也是探讨代谢通路的途径之一。 Pseudomonas stutzeri SDM由本实验室从土壤中筛选得到，DL-乳酸是该菌的最佳碳源，在含有DL-乳酸为唯一碳源的培养基上培养并收集菌体作为休止细胞更多还原

【Abstract】 Pyruvate is an important starting material widely used as an effective precursor in the chemical, drug and agrochemical industries. At present time pyruvate can be produced by both fermentation and enzyme-catalyzed reaction. Nowadays more and more attentions were focused on the enzyme-catalyzed reaction for environmental reasons and the cost of the raw material. It is very important to find an enzyme which has high catalyzing efficiency utilized for pyruvate producing. There are three kinds of enzymes that can catalyze lactate to pyruvate. One is named flavocytochrome b2 which was seldom used to produce pyruvate from lactate. Another is lactate oxidase (LOX) which were found in many species. The last one is lactate dehydrogenase (LDH) which was divided into two types as follows: nicotinamide adenine dinucleotide-linked LDH (nLDH) and nicotinamide adenine dinucleotide-independent LDH (iLDH).Fortunately we found a useful strain of Pseudomonas stutzeri SDM which can grow well at simple mediums containing lactate as sole carbon source. It is the first time that this bacterial was found to be able to produce pyruvate. However the enzymes responsible for the conversion of lactate to pyruvate have not been identified and a little was known about the metabolic mechanism of this strain.This paper mainly focused on:1. P. stutzeri SDM which was newly isolated from soil could grow in a simple medium containing lactate as sole carbon source. When it was cultured on lactate-containing medium the free cells had the capacity of coupling the oxidation of D- and L-lactate and producing pyruvate. To get clearly insight into the lactate-utilization mechanisms two-dimensional gel electrophoresis (2DE) were carried out on total cell lysates. High performance liquid chromatography (HPLC) was performed to detect tricarboxylic acid cycle intermediate and several metabolites including malic acid, pyruvic acid, lactic acid, acetic acid, α-ketoglutaric acid and更多还原