鱼腥藻PCC7120中琥珀酸半醛脱氢酶的初步表征及结构分析  

Preliminary Characterization and Structural Analysis of Succinic Semialdehyde Dehydrogenase from Anabaena sp. PCC7120

作　　者：王小琴[1] 李至敏[2] 雷国风 汤亚兰 李志敏[1,3] 

机构地区：[1]江西农业大学生物科学与工程学院,中国江西南昌330045 [2]江西农业大学理学院,中国江西南昌330045 [3]江西省农业微生物资源开发与利用工程实验室,中国江西南昌330045

出　　处：《生命科学研究》2017年第4期318-324,共7页Life Science Research

基　　金：国家自然科学基金资助项目(31400054;31560250);江西省青年科学基金重大项目(20161ACB21012);教育部留学回国人员科研启动基金项目(第46批)

摘　　要：蓝藻琥珀酸半醛脱氢酶催化琥珀酸半醛到琥珀酸的转化,使得蓝藻的三羧酸循环变得完整。BLAST序列分析预测鱼腥藻PCC7120中all3556基因编码琥珀酸半醛脱氢酶。为了证实all3556基因编码蛋白的催化功能,构建了p ET28a-all3556表达质粒,并在大肠杆菌BL21(DE3)菌体中进行重组蛋白诱导表达;利用镍亲和层析方法对all3556蛋白进行了分离纯化。酶动力学测试表明all3556蛋白是一个NADP+-依赖型的琥珀酸半醛脱氢酶。生物信息学分析发现all3556蛋白和其他来源的琥珀酸半醛脱氢酶的氨基酸序列具有一定的同源性,在催化中心的氨基酸残基高度保守。Succinic semialdehyde dehydrogenases （SSADHs） of cyanobacteria catalyze the oxidation of succinic semialdehyde to succinic acid and complete the cyanobacterial TCA cycle. The all 3556 gene of A nabaena sp. PCC7120 was predicted to encode a succinic semialdehyde dehydrogenase by BLAST analysis. To prove the catalytic function of the protein encoded by all 3556 gene, the plasmid pET28a-all 3556 was constructed and transformed into E. coli BL21（DE3） competent cells. Then the all 3556 protein was purified by Ni-NTA affinity chromatography. Steady-state kinetic studies showed that a113556 was a NADP＋-dependent succinic semialdehyde debydrogenase. Bioinformatic analysis indicated that all 3556 protein and other succinic semialdehyde dehydrogenases had identities in amino acid sequences and that the residues in the catalytic center were highly conserved in different succinic semialdehyde dehydrogenases.

关 键 词：鱼腥藻PCC7120 琥珀酸半醛脱氢酶(SSADH) 重组表达 蛋白质纯化及表征 结构分析 

分 类 号：Q936[生物学—微生物学]