Cloning and Molecular Characterization of an Alpha-Glucosidase (MalH) from the Halophilic Archaeon Haloquadratum walsbyi
AbstractWe report the heterologous expression and molecular characterization of the first extremely halophilic alpha-glucosidase (EC 18.104.22.168) from the archaeon Haloquadratum walsbyi. A 2349 bp region (Hqrw_2071) from the Hqr. walsbyi C23 annotated genome was PCR-amplified and the resulting amplicon ligated into plasmid pET28b(+), expressed in E. coli Rosetta cells, and the resulting protein purified by Ni-NTA affinity chromatography. The recombinant protein showed an estimated molecular mass of 87 kDa, consistent with the expected value of the annotated protein, and an optimal activity for the hydrolysis of α-PNPG was detected at 40 °C, and at pH 6.0. Enzyme activity values were the highest in the presence of 3 M NaCl or 3–4 M KCl. However, specific activity values were two-fold higher in the presence of 3–4 M KCl when compared to NaCl suggesting a cytoplasmic localization. Phylogenetic analyses, with respect to other alpha-glucosidases from members of the class Halobacteria, showed that the Hqr. walsbyi MalH was most similar (up to 41%) to alpha-glucosidases and alpha-xylosidases of Halorubrum. Moreover, computational analyses for the detection of functional domains, active and catalytic sites, as well as 3D structural predictions revealed a close relationship with an E. coli YicI-like alpha-xylosidase of the GH31 family. However, the purified enzyme did not show alpha-xylosidase activity. This narrower substrate range indicates a discrepancy with annotations from different databases and the possibility of specific substrate adaptations of halophilic glucosidases due to high salinity. To our knowledge, this is the first report on the characterization of an alpha-glucosidase from the halophilic Archaea, which could serve as a new model to gain insights into carbon metabolism in this understudied microbial group. View Full-Text
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Cuebas-Irizarry, M.F.; Irizarry-Caro, R.A.; López-Morales, C.; Badillo-Rivera, K.M.; Rodríguez-Minguela, C.M.; Montalvo-Rodríguez, R. Cloning and Molecular Characterization of an Alpha-Glucosidase (MalH) from the Halophilic Archaeon Haloquadratum walsbyi. Life 2017, 7, 46.
Cuebas-Irizarry MF, Irizarry-Caro RA, López-Morales C, Badillo-Rivera KM, Rodríguez-Minguela CM, Montalvo-Rodríguez R. Cloning and Molecular Characterization of an Alpha-Glucosidase (MalH) from the Halophilic Archaeon Haloquadratum walsbyi. Life. 2017; 7(4):46.Chicago/Turabian Style
Cuebas-Irizarry, Mara F.; Irizarry-Caro, Ricardo A.; López-Morales, Carol; Badillo-Rivera, Keyla M.; Rodríguez-Minguela, Carlos M.; Montalvo-Rodríguez, Rafael. 2017. "Cloning and Molecular Characterization of an Alpha-Glucosidase (MalH) from the Halophilic Archaeon Haloquadratum walsbyi." Life 7, no. 4: 46.