title: Expression of a metagenome-derived fumarate reductase from marine microorganisms and its characterization.
abstract: A potential novel fumarate reductase gene designated frd1A was isolated by screening a marine metagenomic library through a sequence-based strategy. Sequence analyses indicated that Frd1A and other putative fumarate reductases were closely related. The putative fumarate reductase gene was subcloned into a pETBlue-2 vector and expressed in Escherichia coli Tuner(DE3)pLacІ cells. The recombinant protein was purified to homogeneity. Functional characterization by high-performance liquid chromatography demonstrated that the recombinant Frd1A protein could catalyze the hydrogenation of fumarate to succinate acid. The Frd1A protein displayed an optimal activity at pH 7.0 and 28 °C, which could be stimulated by adding metal ions such as Zn(2+) and Mg(2+). The Frd1A enzyme showed a comparable affinity and catalytic efficiency under optimal reaction conditions: k m =0.227 mmol/L, v max= 29.9 U/mg, and k cat/k m=5.44 × 10(4) per mol/s. The identification of Frd1A protein underscores the potential of marine metagenome screening for novel biomolecules.
authors.affiliation: The Key Laboratory of Ministry of Education for Microbial and Plant Genetic Engineering; and State Key Laboratory for Conservation and Utilization of Subtropical Agro-bioresources, College of Life Science and Technology, Guangxi University, 100 Daxue East Road, Nanning, Guangxi, 530004, China.
authors.forename: Chengjian
authors.initials: C
authors.lastname: Jiang
authors.forename: Yu
authors.initials: Y
authors.lastname: Liu
authors.forename: Can
authors.initials: C
authors.lastname: Meng
authors.forename: Lanlan
authors.initials: L
authors.lastname: Wu
authors.forename: Jie
authors.initials: J
authors.lastname: Huang
authors.forename: Jie
authors.initials: J
authors.lastname: Deng
authors.forename: Jinyi
authors.initials: J
authors.lastname: Wang
authors.forename: Peihong
authors.initials: P
authors.lastname: Shen
authors.forename: Bo
authors.initials: B
authors.lastname: Wu
mesh_terms: D059001:Aquatic Organisms; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D020536:Enzyme Activators / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics; D005650:Fumarates / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism; D054892:Metagenome; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013385:Succinate Dehydrogenase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019802:Succinic Acid / Q000378:metabolism; D013696:Temperature; D015032:Zinc / Q000378:metabolism
chemical_list: D020536:Enzyme Activators; D005650:Fumarates; D011994:Recombinant Proteins; D019802:Succinic Acid; D013385:Succinate Dehydrogenase; D008274:Magnesium; D015032:Zinc