cDNA cloning, heterologous expression and characterization of a cell wall invertase from copper tolerant population of Elsholtzia haichowensis
The main objective of the present study was to clone, heterologously express and characterize a novel cell wall invertase (FCWI) from a Cu tolerant population of Elsholtzia haichowensis. The full-length FCWI cDNA contained an open reading frame (ORF) of 1671 bp which encoded a 556-amino-acid protein. The theoretical molecular mass and pI of the deduced protein were 62.5 kDa and 9.29, respectively. Phylogenetic analysis showed that FCWI had a closer evolutionary relationship to cell wall invertase of dicot. FCWI was expressed in methylotrophic yeast Pichia pastoris and purified to near homogeneity. Recombinant FCWI enzyme had pH optima of 4.0 and temperature optima of 50 °C. Activity analyses in the presence of various metal cations indicated that FCWI was completely inhibited by Hg2+ (0%), while retained 77.4% activity when exposure to Cu2+. The Km and Kmax values of FCWI for hydrolyzing sucrose were 0.282 mM and 1.576 μkat/mg, respectively. This is the first report that the heterologous expression and characterization of a cell wall invertase from a Cu tolerant population of E. haichowensis. These results helped to understanding the characteristics of FCWI and its physiological role in the resistance mechanisms of Cu tolerant plants.
Key wordscell wall invertase copper enzyme kinetics Elsholtzia haichowensis heterologous expression Pichia pastoris
Unable to display preview. Download preview PDF.
The work was supported by the National Nature Science Foundation of China (Projects 20677046, 30870365, 31270432, 21477093). The authors declare that there is no conflict of interest.
- Albacete A., Grosskinsky D.K. & Roitsch T. 2011. Trick and treat: a review on the function and regulation of plant invertases in the abiotic stress response. Phyton 50: 181–204.Google Scholar
- Cai S., Xiong Z., Li L., Li M., Zhang L., Liu C. & Xu Z. 2014. Differential responses of root growth, acid invertase activity and transeript level to copper stress in two contrasting populations of Elsholtzia haichowensis. Ecotoxicology 23: 76–91.Google Scholar
- Charng Y., Juang R., Su J. & Sung H. 1994. Partial puri fication and characterization of invertase isozymes from rice grains (Oryza sativa). Biochem. Mol. Biol. Int. 33: 607–615.Google Scholar
- CortésRomero C., MartínezHernández A., MelladoMojica E., López M.G. & Simpson J. 2012. Molecular and functional characterization of novel fructosyltransferases and invertases from Agave tequilana. PLOS ONE 7: e35878.Google Scholar
- Le Roy K., Lammens W., Verhaest M., De Coninck B., Rabijns A., Van Laere A. & Van den Ende W. 2007. Unraveling the difference between invertases and fructan exohydrolases: a single amino acid (Asp-239) substitution transforms Arabidopsis cell wall invertasel into a fructan 1-exohydrolase. Plant Physiol. 145: 616–625.CrossRefGoogle Scholar
- Somogyi M. 1952. Notes on sugar determination. J. Biol. Chem. 195: 19–23.Google Scholar