摘要
以ｐＵＣ１９质粒为载体，以Ｅ． ｃｏｌｉ ＪＭ１０９为受体，构建了含α一乙酰乳酸脱羧酶（α－ＡＬＤＣ）基因的地衣芽孢杆菌 Ｂａｃｉｌｌｕｓ ｌｉｃｈｅｎｉｆｏｒｍｉｓ ＡＳ１０１０６的基因文库，得到４８００个重组转化子中均含有４～１０ｋｂ的外源插入ＤＮＡ片段。从基因文库中筛选到６个阳性克隆，对其中１个克隆的α－乙酰乳酸脱羧酶基因片段进行亚克隆分析表明，该α－乙酰乳酸脱酶基因位于１．６ｋｂ的ＢａｍＨＩ－ＥｃｏＲＩ片段上。对其研究发现，α－乙酰乳酸脱酶基因在大肠杆菌中表达产生的乙酰乳酸脱羧酶在最适反应温度和ｐＨ值等与供体菌的酶活相当。利用大肠杆菌和酵母菌穿梭质粒ｐＹＥＳ２为载体，可以将α－乙酰乳酸脱羧酶基因引入酿酒酵母中，从而使重组酵母菌株具有降解啤酒发酵中生成的α－乙酰乳酸，减少双乙酰含量的能力。
A genomic library of B. licheniformis AS10106 that contained the a --acetolactate decarboxylase gene(a--ALDC) was constructed with vector pUC19 and host E. coli JM109 strain. The inserted fragments of foreign DNA ranged from 4 to 10kb in the 4800 clones thus obtained. Six positive clones were detected after screening the plated library by the method of clony coloration. Subcloning of the DNA fragment containing the α-acetolactate decarboxylase gene showed that the acetolactate decarboxylase gene was on an l.6kb BamH I --EcoRI fragment. Preliminary analysis of the enzyme expressed from one recombinant plasmid pGEA showed that the properties of the recombinant enzyme, such as the optimal temperature and pH of reaction, were identical to those of the native enzyme. Using yeast--E. coli shuttle vector pYES2, an exprssion recombinant plasmid pYEA containing B. licheniformis AS10106 α--acetolactate decarboxylase gene was constructed. S. cerevisiae H158 transformed with pYEA had expressed α--acetolactate decarboxylase activity and shown the ability to reduce the formation of diacetyl during beer fermentation.
