Expression, Folding, and Activation of Halophilic Alkaline Phosphatase in Non-Halophilic Brevibacillus choshinensis
| Autor: | Yoshitaka Nakamura, Hiroshi Hanagata, Hirohito Tsurumaru, Shigeki Arai, Matsujiro Ishibashi, Fina Amreta Laksmi, Hikari Imamura, Masao Tokunaga |
|---|---|
| Rok vydání: | 2019 |
| Předmět: |
Protein Folding
Bioengineering Sodium Chloride Biochemistry Analytical Chemistry Hydrophobic effect 03 medical and health sciences Methylamines Bioorganic chemistry Cloning Molecular 030304 developmental biology chemistry.chemical_classification 0303 health sciences Halomonas biology Brevibacillus Chemistry 030302 biochemistry & molecular biology Organic Chemistry biology.organism_classification Alkaline Phosphatase Halophile Amino acid Enzyme Osmolyte Alkaline phosphatase |
| Zdroj: | The protein journal. 39(1) |
| ISSN: | 1875-8355 |
| Popis: | Halophilic enzymes contain a large number of acidic amino acids and marginal large hydrophobic amino acids, which make them highly soluble even under strongly hydrophobic conditions. This characteristic of halophilic enzymes provides potential for their industrial application. However, halophilic enzymes easily degrade when used for industrial applications compared with enzymes from other extremophiles because of their instability in low-salt environments. We aimed to clarify the stabilization mechanism of halophilic enzymes. We previously attempted to express halophilic alkaline phosphatase from Halomonas (HaALP) in non-halophilic E. coli. However, the expressed HaALP showed little activity. Therefore, we overexpressed HaALP in Gram-positive non-halophilic Brevibacillus choshinensis in this study, which was successfully expressed and purified in its active form. HaALP was denatured in 6 M urea, refolded using various salts and the non-ionic osmolyte trimethylamine N-oxide (TMAO), and assessed by native polyacrylamide gel electrophoresis. HaALP refolded in 3 M NaCl or 3 M TMAO containing Na+ ions. Hydrophobic interactions due to a high salt concentration or TMAO enhanced the formation of the folding intermediate (the monomer precursor), and only Na+ ions activated the dimer form. This insight into the stabilization mechanism of HaALP may lead to the development of industrial applications of halophilic enzymes under low-salt conditions. |
| Databáze: | OpenAIRE |
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