title: Isolation and molecular characterization of thermostable phytase from Bacillus subtilis (BSPhyARRMK33).
abstract: The thermostable phytase gene was isolated from Bacillus subtilis ARRMK33 (BsPhyARRMK33). The gene has an ORF of 1152 bp and that encodes a protein of 383 amino acids. Sequence analysis showed high homology with Bacillus sp. phytase proteins, but no similarity was found with other phytases. SDS-PAGE analysis exhibited a predicted molecular mass of 42 kDa. Homology modeling of BsPhyARRMK33 protein based on Bacillus amyloliquefaciens crystal structure disclosed its β-propeller structure. BsPhyARRMK33 recombinant plasmid in pET-28a(+) was expressed in Rosetta gami B DE3 cells and the maximum phytase activity 15.3 U mg(-1) obtained. The enzyme exhibits high thermostability at various temperatures and broad pH ranges. The recombinant protein retained 74% of its original activity after incubation at 95 °C for 10 min. In the presence of Ca(2+), the recombinant phytase activity was maximal where as it was inhibited by EDTA. The optimal pH and temperature for the recombinant phytase activity is achieved at 7.0 and 55 °C, respectively. Thermostable nature and wide range of pH are promising features of recombinant BsPhyARRMK33 protein that may be employed as an efficient alternative to commercially known phytases and thereby alleviate environmental eutrophication.
authors.affiliation: Plant Molecular Biology Group, International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, New Delhi, 110 067, India, suumani@gmail.com.
authors.forename: Chinreddy Subramanyam
authors.initials: CS
authors.lastname: Reddy
authors.forename: V Mohan Murali
authors.initials: VM
authors.lastname: Achary
authors.forename: Mrinalini
authors.initials: M
authors.lastname: Manna
authors.forename: Jitender
authors.initials: J
authors.lastname: Singh
authors.forename: Tanushri
authors.initials: T
authors.lastname: Kaul
authors.forename: Malireddy K
authors.initials: MK
authors.lastname: Reddy
mesh_terms: D010832:6-Phytase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D000595:Amino Acid Sequence; D001412:Bacillus subtilis / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016366:Open Reading Frames; D013696:Temperature
chemical_list: D001426:Bacterial Proteins; D010832:6-Phytase