Gene Cloning,Expression And Characterization Of A Novel Phosphatase Isolated From A Marine Bacterium Fulvimarina Manganoxydans

The ocean is a huge and complicated environment which contains a rich microbial resource for screening and researching of new functional genes. And the deep-sea hydrothermal vent is a kind of extreme ocean ecosysterm, there are a variety of enzymes which have special enzymatic properties in it. Based on discovering new genes and developing potential application enzymes, we used concentrated in situ seawater which collected in Southwest Indian Ocean from 2800 m below sea level as a sample to screen new enzyme.In this study, we cloned a novel gene fm2382 from a marine bacterium Fulvimarina manganoxydans which isolated from deep-sea hydrothermal plume, and expressed in Escherichia coli. The recombinant protein was purified to investigate enzymatic properties. The results showed that FM2382 is a HAD superfamily phosphatase, and also has ATPase functions, but there are some differences between FM2382 blocks and HAD superfamily motifs. So it will be identified as a novel phosphatase in HAD superfamily, and recommended it in the subfamily designated as "C" subfamily. The recombinant protein is a metal ion-dependent phosphatase with optimum pH at 7.1 and optimum temperature at 45?, and it has a certain temperature tolerance. Structural stability experiment revealed that FM2382 unfolding process is irreversible, it is also means that phosphatase activity is irreversible by heat denaturation. The site-directed mutagenesis assays exhibited several amino acid residues that might be crucial in catalysis for FM2382,such as C15?C33?D34?W36?T67?C171?C187?L220?D232?D237? D247 are the key residues for phosphatase function, and C15?D34?W36?T67?C187? R163?D247 are the critical sites for ATPase.