力复霉素产生菌地中海拟无枝菌酸菌U32丙氨酸脱氢酶基因克隆及表达  

Cloning and Characterization of Alanine Dehydrogenase Gene( ald ) from Amycolatopsis mediterranei U32

作　　者：姚玉峰[1] 卢捷[1] 俞浩[1] 赵国屏[1] 姜卫红[1] 焦瑞身[1] 

机构地区：[1]中国科学院上海植物生理生态研究所微生物次生代谢分子调控研究实验室,上海200032

出　　处：《微生物学报》2003年第2期206-213,共8页Acta Microbiologica Sinica

基　　金：国家自然科学基金重点项目资助 (1 0 0 3 963 0 0 1 0 )~~

摘　　要：丙氨酸脱氢酶 (EC 1 4 1 1 )可逆催化丙氨酸脱氨生成丙酮酸和NADH。它是生物体内的氨基酸代谢和氨同化途径的关键酶。在地中海拟无枝菌酸菌 (Amycolatopsismediterranei)U32中 ,丙氨酸脱氢酶的活力与力复霉素的生物合成有负相关现象 ,其活力受KNO3全局效应的调控。根据结核分枝杆菌 (Mycobacteriumtuberculosis)和天蓝链霉菌 (Streptomycescoelicolor)的丙氨酸脱氢酶氨基酸的保守序列和地中海拟无枝菌酸菌U32对氨基酸密码子的使用偏好 ,设计一对简并PCR引物。以此引物从地中海拟无枝菌酸菌U32中扩增到一 5 5 5bp的片段 ,并以此片段为探针从地中海拟无枝菌酸菌U32基因组cosmid文库中成功的克隆到了丙氨酸脱氢酶结构基因 (ald)。它编码了一个 371个氨基酸的蛋白质 ,基因的GC含量为 72 5 % ,符合链霉菌的基因结构特征。在起始密码子的上游 6个碱基处 ,有一典型的链霉菌核糖体结合位点(RBS) :AGGAGG ,第 75位的氨基酸为赖氨酸 ,是丙酮酸结合位点。以pET2 8b为载体 ,在E .coliBL2 1 (DE3)中高效表达了ald基因。用IPTG在 2 2℃时诱导得到的丙氨酸脱氢酶活力最高。用His Tag柱纯化了表达的丙氨酸脱氢酶。酶学性质研究表明该酶专一性以L Ala和NAD(H)Alanine dehydrogenase (L Alanine: NAD + oxidoreductase, deaminating, EC 1 4 1 1) catalyzes a reversible oxidative deamination of L Alanine to pyruvate and ammonia, with NAD + as cofactor. It is a key enzyme in alanine catabolism and ammonia assimilation. According to the biased usage in the third codon of G or C, and conservative regions in amino acid sequence of Alanine dehydrogenase from S. coelicolor and M. tuboculosis, a pair of degenerate primers were designed. The product of the degenerate primers PCR, 555bp , was amplified from U32 genomic DNA. The ald gene was cloned and sequenced after screening the cosmid library of U32 strain, with the PCR product as a probe. The open reading frame of ald encodes a protein of 371 amino acids with a calculated molecular mass of around 43kD. There is a typical strepomyces RBS: AGGAGG at the -6 position upstream of start codon GTG. Lys75 is a pyruvate binding site. The ald gene was introduced into E.coli BL21(DE3) by pET28b vector. The ald was induced by IPTG at 22℃, 28℃, 37℃. The activity of alanine dehydrogenase was highest when it was induced at 22℃. It is seen the enzyme is NADH/NAD + and L Ala specific.

关 键 词：地中海拟无枝菌酸菌 丙氨酸脱氢酶 克隆 表达 基因 

分 类 号：Q78[生物学—分子生物学]