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作 者:李剑[1] 唐赟[1] 梁凤来[1] 张心平[1] 刘如林[1]
出 处:《生物工程学报》2004年第5期725-729,共5页Chinese Journal of Biotechnology
摘 要:构建了一株产D ,L_乳酸的乳杆菌 (Lactobacillussp .)MD_1的基因文库。利用乳酸脱氢酶和丙酮酸裂解酶缺陷的EscherichiacoliFMJ14 4作为宿主 ,通过互补筛选分离克隆到乳酸脱氢酶基因 (ldhL)。核酸序列分析表明 ,该基因以ATG为起始密码子编码 316个氨基酸残基组成的蛋白质 ,预测的分子量为 33 84kD ;5′端存在典型的启动子结构 ,3′端的终止子是不依赖于 ρ因子的转录终止子。ldhL编码的蛋白质有 3个保守区域 ,其中Gly13~Asp5 0保守区域是NADH的结合位点 ,Asp73~Ile10 0和Asn12 3~Arg15 4保守区是酶的活性部位。该ldhL和其他乳杆菌的ldhL基因和编码的氨基酸序列相似性较低 ,核苷酸序列相似性最高仅为 6 4 1% ,氨基酸序列相似性最高仅为 6 8 9% 。It was constructed that a genomic DNA library from Lactobacillus sp . MD-1 yielding D,L-lactic acid. The gene encoding L-lactate dehydrogenase (L-LDH) was cloned from the genomic library of strain MD-1 by complementation in E. coli FMJ144 which was lactate dehydrogenase and pyruvate-formate lyase double defective mutant. The nucleotide sequence of the ldhL gene predicted a protein of 316 amino acid starting with ATG. The putative molecular weight of the L-LDH amino acid sequence was 33 84kD. A putative typical promoter (-35 and -10 boxes) had been observed in the 5′ noncoding region. An rho-independent transcriptional terminator has been observed in the 3′ noncoding region. Three highly conserved regions (Gly13~Asp50, Asp73~Ileu100 and Asn123~Arg154) with several conserved residues had been identified. Gly13~Asp50 was NADH-binding site domain. Asp73~Ileu100 and Asn123~Arg154 were reported to be the active site domains. The ldhL and the L-LDH of Lactobacillus sp . MD-1 showed the low identity and similarity with other Lactobacilli , and the highest percentage were 61 9% and 68 9% respectively. All the above indicated this gene is a novel ldhL .
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