1. Discovery of a Novel Alginate Lyase from Nitratiruptor sp. SB155-2 Thriving at Deep-sea Hydrothermal Vents and Identification of the Residues Responsible for Its Heat Stability
- Author
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Satoshi Nakagawa, Takao Ojima, Akira Inoue, and Moe Anraku
- Subjects
0301 basic medicine ,Hot Temperature ,Oceans and Seas ,030106 microbiology ,Hypothetical protein ,medicine.disease_cause ,Biochemistry ,03 medical and health sciences ,Hydrothermal Vents ,Bacterial Proteins ,Protein Domains ,Enzyme Stability ,medicine ,Extremophile ,Lyase activity ,Molecular Biology ,Escherichia coli ,Peptide sequence ,Polysaccharide-Lyases ,chemistry.chemical_classification ,biology ,Substrate (chemistry) ,Cell Biology ,Hydrogen-Ion Concentration ,biology.organism_classification ,030104 developmental biology ,Enzyme ,chemistry ,Enzymology ,Epsilonproteobacteria ,Bacteria - Abstract
Extremophiles are expected to represent a source of enzymes having unique functional properties. The hypothetical protein NIS_0185, termed NitAly in this study, was identified as an alginate lyase-homolog protein in the genomic database of epsilon-Proteobacteria Nitratiruptor sp.SB155-2, which was isolated from deep-sea hydrothermal vents at a water depth of 1,000 m. Among the characterized alginate lyases in the polysaccharide lyase family 7 (PL-7), the amino acid sequence of NitAly showed the highest identity (39%) with that of red alga Pyropia yezoensis alginate lyase PyAly. Recombinant NitAly (rNitAly) was successfully expressed in Escherichia coli. Purified rNitAly degraded alginate in an endolytic manner. Among alginate block types, polyM was preferable to polyG and polyMG as a substrate, and its end degradation products were mainly tri-, tetra-, and penta-saccharides. The optimum temperature and pH values were 70 degrees C and around 6, respectively. Ahigh concentration of NaCl (0.8-1.4 M) was required for maximum activity. In addition, a 50% loss of activity was observed after incubation at 67 degrees C for 30 min. Heat stability was decreased in the presence of 5 mM DTT, and Cys-80 and Cys-232 were identified as the residues responsible for heat stability but not lyase activity. Introducing two cysteines into PyAly based on homology modeling using Pseudomonas aeruginosa alginate lyase PA1167 as the template enhanced its heat stability. Thus, NitAly is a functional alginate lyase, with its unique optimum conditions adapted to its environment. These insights into the heat stability of NitAly could be applied to improve that of other PL-7 alginate lyases.
- Published
- 2016