Gene Cioning, Expression And Enzymatic Properties Of A Novel Fumarate Reductase From Uncultured Mairne Microorganisms

Fumarate reductase （FRD, EC1.3.1.6） catalyses the reduction of fumarate to succinate and plays a key role for the anaerobic respiration system of many organisms with fumarate as terminal electron acceptor. It also exits widely in the Gram-negative bacteria, facultative anaerobic Gram-positive bacteria and some algae, protozoa, parasitic worms and other lower marine eukaryotes. Not only can be applied to a variety of human diseases, its products, succinate also has a wide range of applications in industrial production. This research foucs on a positive clones （pGXAR313G） convea a novel fumarate reductase from uncultured marine microorganisms in Guangxi Province, and the possible novel gene was named frdlA.Bioinformatics analysis showed that the frdlA gene had444base pairs, encoded147amino acids, and its correspongding protein had a theoretical isoelectric point of5.59, a relative molecular mass of16.11 kDa. Through analysis with the others known genes in the GenBank database, the reusults indicated that it had low similarity at the nucleic acid level, and had a comparable similarity （82%） and positive （69%） to a b subunit of a fumarate reductase（Runella slithyformis YP₀04658175） at the amino acid level. The possible catalytic domain （56-64amino acids） contained a2Fe-2S iron oxide protein binding sites.Through the recombinant DNA technology, ligated the expression plasmid pETBlue-2and frdl A gene, then transfered into Escherichia Coli Tuner （DE3）pLacI competent cells, and constructed the recombinant strains E.coli Tuner（DE3）pLacI/pGXAR313G Cultured under the optimal conditions, the recombinant Frd1A protein had been expression, and further purified with the Ni2+column technology. The physiological and biochemical properties had been characterized. The optimum reaction temperature was28℃, and the optimum pH was7.0; the enzymatic activity was9.59U/mL. Under the above optimal reaction, specific activity was10.644U/mg. By Lineweaver-Burk drawing, measured Km value was0.227mM, Vmax was29.94mM/min, kcat was12.35s-1; Zn2+and Mg2+may has a promoting role in enzyme reaction, but Cu2+, Fe2+, Fe3+, Ca2+, SDS and EDTA may inhibitory.Successful implementation of this research provides a technical reference and new study mode for new type fumarate reductase, has an important theoretical guiding significance for the next step on constructing efficient engineered bacteria.