海洋未培养微生物延胡索酸水合酶的基因克隆和功能鉴定
Identification of a novel fumarase gene by metagenome expression cloning from marine microorganisms
延胡索酸水合酶催化延胡索酸的水合然后生成L-苹果酸，它也是TCA循环和氨基酸代谢中一种关键的酶。来自于特殊生境的高活性和耐热性良好的延胡索酸水合酶在工业生产L-苹果酸中具有广泛的用途。本研究基于序列特异性筛选的策略从一个海洋宏基因组文库中分离得到了一个新型延胡索酸水合酶基因（fumF）。氨基酸序列分析揭示FumF蛋白和来自于Bacteroides sp. 2_1_33B 和 Parabacteroides distasonis ATCC 8503的延胡索酸水合酶具有26%的一致性和43%的相似性。fumF基因被克隆到pETBlue-2 载体上，在大肠杆菌BL21(DE3)pLysS中实现了高效表达。HPLC实验结果证实了纯化的重组FumF 蛋白可以催化延胡索酸与水的水合生成L-苹果酸。fumF基因的克隆和生化性质鉴定研究显示了FumF蛋白在工业上生产L-苹果酸具有良好的改造性，也显示了利用宏基因组学技术从海洋微生物中分离筛选新型生物活性物质的巨大潜力。
Fumarase catalyzes the reversible hydration of fumarate to L-malate and is a key enzyme in the tricarboxylic acid (TCA) cycle and in amino acid metabolism. Fumarase is also used for the industrial production of L-malate from the substrate fumarate. A plasmid metagenomic library was constructed from uncultivated marine microorganisms within marine water samples. Through sequence-based screening of the DNA library, a gene encoding a novel fumarase (named FumF) was isolated.The putative fumarase gene was subcloned into pETBlue-2 vector and expressed in E. coli BL21(DE3)pLysS. The recombinant protein was purified to homogeneity. Functional characterization by high performance liquid chromatography confirmed that the recombinant FumF protein catalyzed the hydration of fumarate to form L-malate. The properties of FumF protein may be ideal for the industrial production of L-malate under higher temperature conditions. The identification of FumF underscores the potential of marine metagenome screening for novel biomolecules.
武波、申佩弘、赵高超、吴兰兰、蒋承建
生物化学分子生物学微生物学
延胡索酸水合酶L-苹果酸宏基因组文库海洋微生物
FumaraseL-malatemetagenomic librarymarine microorganisms
武波,申佩弘,赵高超,吴兰兰,蒋承建.海洋未培养微生物延胡索酸水合酶的基因克隆和功能鉴定[EB/OL].(2012-11-28)[2025-07-22].http://www.paper.edu.cn/releasepaper/content/201211-510.点此复制
评论