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Expression, Folding, and Activation of Halophilic Alkaline Phosphatase in Non-Halophilic Brevibacillus choshinensis.
The Protein Journal ( IF 3 ) Pub Date : 2019-11-16 , DOI: 10.1007/s10930-019-09874-z
Fina Amreta Laksmi 1 , Hikari Imamura 2 , Hirohito Tsurumaru 1, 2 , Yoshitaka Nakamura 1 , Hiroshi Hanagata 3 , Shigeki Arai 4 , Masao Tokunaga 1, 2 , Matsujiro Ishibashi 1, 2
Affiliation  

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.

中文翻译:

嗜盐碱性磷酸酶在嗜盐性短杆菌中的表达,折叠和活化

嗜盐酶包含大量的酸性氨基酸和少量的大疏水性氨基酸,这使其即使在强疏水性条件下也高度可溶。嗜盐酶的这一特性为其工业应用提供了潜力。但是,由于其在低盐环境中的不稳定性,与其他极端嗜热菌的酶相比,用于工业应用的嗜盐酶容易降解。我们旨在阐明嗜盐酶的稳定机制。我们之前曾尝试在非嗜盐性大肠杆菌中表达来自Halomonas(HaALP)的嗜盐碱性磷酸酶。然而,表达的HaALP显示很少活性。因此,我们在革兰氏阳性非嗜盐菌中过表达HaALP在这项研究中,成功​​地表达和纯化了Choshinensis短杆菌。HaALP在6 M尿素中变性,使用各种盐和非离子渗透压三甲胺N-氧化物(TMAO)重折叠,并通过天然聚丙烯酰胺凝胶电泳进行评估。HaALP在含有Na +离子的3 M NaCl或3 M TMAO中重新折叠。高盐浓度或TMAO引起的疏水相互作用增强了折叠中间体(单体前体)的形成,只有Na +离子激活了二聚体形式。对HaALP稳定机制的这种洞察可能会导致在低盐条件下开发嗜盐酶的工业应用。
更新日期:2019-11-16
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