【摘要】 苹果酸-乳酸酶是苹果酸-乳酸发酵过程中负责苹果酸转化为乳酸的功能酶。在进行酒酒球菌SD2a的苹果酸-乳酸酶基因(mleA)克隆测序基础上,以PGK1强启动子和ADH1终止子为调控元件,以大肠杆菌-酵母菌穿梭质粒YEp352为载体,构建了重组表达质粒并转化酿酒酵母YS58。酵母转化子用SD/Ura平板筛选鉴定。斑点杂交检测表明目的基因mleA转化到受体菌中,SDSPAGE检测表明获得的转化子表达了约60kDa的目标蛋白。获得的转化子在添加了L苹果酸的培养基中培养4d;取培养液上清用HPLC检测L苹果酸及L乳酸含量,采用t检验进行差异显著性分析,结果表明mleA基因进行了功能性的表达,将L苹果酸转化成L乳酸,L苹果酸和L乳酸含量分别与对照差异极显著和显著,苹果酸的相对降低率平均为20.95%。在有选择压力条件下,重组质粒相对稳定,而在无选择压力条件下,传代培养10d后大约有65%的重组质粒丢失。更多还原

【Abstract】 Malo-lactic enzyme is the function enzyme to turn L-malic acid into L-lactic acid during malolactic fermentation (MLF). mleA gene encoding for Malo-lactic enzyme from pLmleA, PGK1 promoter from plasmid pVC727-6 and ADH1 terminator from plasmid pEA-1 were ligated and inserted into YEp352, resulted the expression plasmid. Yeast transformants were screened on SD/-Ura (YNB medium containing leucine, histidine and tryptophan). The target gene was detected in the yeast transformants by dot blotting hybridization. The target protein was expressed by SDS-PAGE detecting. After transformants were cultured in media containing L-malate for 4d, the culture supernatant was collected and L-malate and L-lactic acid content were detected by HPLC. The result indicated that functional expression of mleA gene was achieved in recombinant S. cerevisiae, turning L-malic acid into L-lactic acid. L-malate contents of the transformants show extra significant difference with the control ones in t test, while L-lactic contents of the transformants show significant difference with the control ones. Average amount of 1064mg/L L-lactic acids were detected and the comparative average drop rates of L-malate were 20.95%, while no L-lactic was detected from control transformants. The yeast transformants containing the foreign gene of mleA were stable relatively under selective pressure, but about 65% of the recombined plasmid were lost in 10 days subculture.更多还原