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adhB和pdc是运动发酵单胞菌产乙醇途径的关键基因,分别编码乙醇脱氢酶和丙酮酸脱羧酶,将添加有聚球藻PCC7942rbcLS基因RBS序列的adhB和pdc基因插入pUC18载体,经双重菌液PCR检验和酶切检验得到分别含有pUC-adhB、pUC-pdc和pUC-adhB-pdc载体的3个重组菌株。活性检测实验表明聚球藻PCC7942的rbcLS基因的RBS序列能有效介导运动发酵单胞菌的adhB和pdc基因在大肠杆菌中表达,摇瓶发酵实验表明重组大肠杆菌的产乙醇能力较出发菌株大幅提升。鉴于乙醛指示平板法存在着对希夫试剂的要求较高、易产生较强的背景色等缺点,对定性检测丙酮酸脱羧酶和乙醇脱氢酶表达菌株的方法做了改进,即:将菌液诱导表达,然后分别添加对应于两种酶的底物,让酶与底物反应0.5至1小时,之后再加希夫试剂进行显色反应,结果表明改进后的方法比乙醛指示平板法更加简便、快速、可靠。
adhB and pdc are the key genes of Z. mobilis ethanol production pathway, encoding alcohol dehydrogenase and pyruvate decarboxylase respectively. The adhB and pdc genes with RBS sequence of Synechococcus sp.PCC7942rbcLS were inserted into pUC18 vector, The three recombinant strains containing the pUC-adhB, pUC-pdc and pUC-adhB-pdc vectors, respectively, were obtained by liquid PCR and digestion. The activity test showed that the RBS sequence of rbcLS gene of Synechococcus PCC7942 could effectively mediate the expression of adhB and pdc genes of Z. mobilis in E. coli. The shake flask fermentation experiments showed that the ethanol production capacity of recombinant E. coli was significantly higher than that of the original strain Enhance. In view of acetaldehyde indicating that the plate method has higher requirements for the Schiff reagent, easy to produce a strong background color and other shortcomings, the qualitative detection of pyruvate decarboxylase and alcohol dehydrogenase expression strains improved method, namely: Then, the substrates corresponding to the two enzymes were added to allow the enzyme to react with the substrate for 0.5 to 1 hour, and then the Schiff reagent was used for the color reaction. The results showed that the improved method is more effective than the acetaldehyde indicating plate Method is more simple, fast, reliable.