Laboratory of Protein Engineering and Bioactive Molecules (LIP-MB-LR11ES24), National Institute of Applied Sciences and Technology INSAT-BP 676, University of Carthage, 1080, Tunis Cedex, Tunisia.
Department of Biochemistry and Biotechnology, Institute of Biological Sciences, Maria Curie-Skłodowska University, Akademicka 19, 20-033, Lublin, Poland.
Biotechnol Lett. 2024 Dec;46(6):1199-1218. doi: 10.1007/s10529-024-03532-w. Epub 2024 Oct 28.
This study aimed to produce an engineered recombinant laccase from extremophilic Halalkalibacterium halodurans C-125 (Lac-HhC-125) with higher protein yield, into a more active conformation and with properties that meet the fundamental needs of biotechnological application.
The rLac-HhC125 was partially purified by size exclusion chromatography and concentrated by ultrafiltration (10 kDa) with a yield of 57.6%. Oxidation reactions showed that adding 2 mM CuSO to the assay solution led to activating the laccase. To increase its initial activity, the rLac-HhC125 was treated at 50 °C for 20 min before the assays, improving its performance by fourfold using the syringaldazine as a substrate. When treated with EDTA, methanol, ethanol, and DMSO, the rLac-HhC125 maintained more than 80% of its original activity. Interestingly, the acetonitrile induced a twofold activity of the rLac-HhC125. The putative rLac-HhC125 demonstrated a capability of efficient transformation of different organic compounds at pH 6, known as dye precursors, into coloured molecules.
The rLac-HhC125 was active at high temperatures and alkaline pH, exhibited tolerance to organic solvents, and efficiently transformed different hydroxy derivatives into coloured compounds, which indicates that it can be used in various biotechnological processes.
本研究旨在从嗜盐耐碱极端微生物盐单胞菌（Halalkalibacterium halodurans C-125）中生产一种工程重组漆酶（Lac-HhC-125），使其具有更高的蛋白质产量、更活跃的构象和满足生物技术应用基本需求的特性。
通过大小排阻层析对 rLac-HhC125 进行部分纯化，并通过超滤（10 kDa）浓缩，收率为 57.6%。氧化反应表明，在测定溶液中添加 2 mM CuSO4 可激活漆酶。为了提高其初始活性，在测定前将 rLac-HhC125 在 50°C 下处理 20 分钟，使用愈创木酚作为底物可将其性能提高四倍。当用 EDTA、甲醇、乙醇和 DMSO 处理时，rLac-HhC125 保持了超过 80%的原始活性。有趣的是，乙腈可使 rLac-HhC125 的活性增加两倍。该假定的 rLac-HhC125 在 pH 6 下具有有效转化不同有机化合物（称为染料前体）为有色分子的能力。
rLac-HhC125 在高温和碱性 pH 条件下具有活性，对有机溶剂具有耐受性，并能有效转化不同的羟基衍生物为有色化合物，这表明它可用于各种生物技术过程。
