产气肠杆菌α-乙酰乳酸脱羧酶基因的克隆和表达及影响重组酶活性的因素  

Cloning and Expression of α acetolactate Decarboxylase Gene for Enterobacter aerogenes and Factors Influencing the Activity of the Recombinant Enzyme

作　　者：尹东[1] 曾庆华[1] 卢大宁[1] 黄百渠[1] 李彦舫[2] 

机构地区：[1]东北师范大学遗传与细胞研究所,长春130024 [2]中国人民解放军农牧大学,长春130062

出　　处：《生物工程学报》1999年第4期501-506,共6页Chinese Journal of Biotechnology

摘　　要：用 Ｐ Ｃ Ｒ 方法从产气肠杆菌（ Ｅｎｔｅｒｏｂａｃｔｅｒ ａｅｒｏｇｅｎｅｓ） 中克隆出０９ｋｂ 的 Ｄ Ｎ Ａ 片段，经 Ｄ Ｎ Ａ 测序证明是α乙酰乳酸脱羧酶（αａｃｅｔｏｌａｃｔａｔｅ ｄｅｃａｒｂｏｘｙｌａｓｅ ，α Ａ Ｌ Ｄ Ｃ） 基因。将α Ａ Ｌ Ｄ Ｃ基因重组到质粒ｐ Ｂ Ｖ２２０ 后，转化大肠杆菌，经筛选获得高效表达的重组子菌株。重组α Ａ Ｌ Ｄ Ｃ 基因表达量占菌体总蛋白量的２７ ％ 。酶活检测表明重组子细胞表达的α Ａ Ｌ Ｄ Ｃ 活性是产气肠杆菌的１２０００ 倍。另外，粗提的重组α Ａ Ｌ Ｄ Ｃ 的最适ｐ Ｈ 为６５ ～７０ ，ｐ Ｈ 稳定范围为５５ ～８０ ，适合的温度为３５ ～４５ ℃。 Ｂａ２ ＋ 、 Ｃｄ２ ＋ 、 Ｆｅ２ ＋ 、 Ｃｏ２ ＋ 、 Ｍｎ２ ＋ 、 Ｓｎ２ ＋ 可提高重组α Ａ Ｌ Ｄ Ｃ 的活性。氨基酸修饰剂可降低其活性。acetolactate decarboxylase (α ALDC) gene was cloned from E.aerogenes by PCR amplification.The gene was inserted into the vector pBV220 to construct an expression plasmid pBVYⅡ,and high level of expression of α ALDC in E.coil DH5α was observed.The expressed α ALDC accounted for 27% of the total protein in the recombinant E.coil cell.The α ALDC activity of recombinant bacterium was 12000 fold higher than that of E.aerogenes. In addition,the partially purified α ALDC was optimal activity at pH 6.5～7.0 and at a temperature of 35～45℃.Its activity was stability at pH5 5～8 0.Ba 2+ ,Cd 2+ ,Fe 2+ ,Co 2+ ,Mn 2+ and Sn 2+ had a stimulatory effect on the enzyme activity.Aminoacid modifiers could inhibit differently its activity.

关 键 词：产气肠杆菌 乙酰乳酸脱羧酶 基因克隆表达 酶活 

分 类 号：Q939.121[生物学—微生物学]