ИСТИНА |
Войти в систему Регистрация |
|
ИСТИНА ИНХС РАН |
||
Formate dehydrogenase (FDH, EC 1.2.1.2.) plays a very important role in different kinds of organisms. In plants it is an enzyme of stress response. In bacteria and yeasts it is involved in energy production. FDH is widely used for cofactor regeneration in different types of biotechnology processes and in enzyme systems such as whole cell biocatalysts, fusions etc. Many genes of FDHs from different sources have been already cloned and expressed. All of them are different in kinetic properties and thermal stability. That’s why now the search of new FDH genes is also under the process. One of the most interesting sources of FDH is eukaryote. It is well known from the literature, that plant FDH genes have a special signal peptide on its N-terminus, which is responsible for enzyme transportation from cytoplasm to mitochondria. Normally the signal peptide is eliminated and the active form of the enzyme is transferred to the mitochondria. In this work we provide the cloning, expression and properties of the new FDH from moss Physcomitrella patens. The gene of the enzyme was cloned in E.coli and for the first time a new FDH was expressed in soluble and active form as a full-size protein including signal peptide. Before the plant FDHs with signal peptide were obtained only in the form of inclusion body. Structure of signal peptide was analyzed and several new forms of FDH with partially deleted N-terminus were obtained by site-directed mutagenesis. All variants were purified and their kinetic parameters and thermal stability were studied. The comparison of properties showed small difference between FDH variants, but at the same time the activity yield for one of the mutants was much higher. One can suggest, that this FDH variant is natural form of the enzyme in the moss in nature. Comparison of properties showed, that new PpaFDH is similar to other plant FDHs and can be used as a biocatalyst for cofactor regeneration. This work was supported by grant of Russian Foundation for Basic Research (grant 14-04-01665-a)