The aim of this work was to develop a stable immobilized enzyme biocatalyst for the isomerization of d‐galactose to d‐tagatose at high temperature. l‐Arabinose isomerase from the hyperthermophilic bacterium Thermotoga maritima (TMAI) was produced as a (His)₆‐tagged protein, immobilized on a copper–chelate epoxy support and subjected to several postimmobilization treatments aimed at increasing its operational and structural stability. Treatment with glutaraldehyde and ethylenediamine resulted in a more than twofold increase in the operational stability and in all enzyme subunits linked, directly or indirectly, to the support via covalent bonds. A postimmobilization treatment of the immobilized derivatives with mercaptoethanol for the removal of any remaining copper ions, determined a further increase of the operational biocatalytic activity. Immobilized derivatives subjected to both treatments were used for the bioconversion of 18 g/L d‐galactose to d‐tagatose at 80°C in a packed bed reactor in three repeated cycles and showed a better operational stability compared with the literature data. This study shows that a postimmobilization stabilization treatment with glutaraldehyde and ethylenediamine can stabilize the multi‐subunit structure of an enzyme immobilized on a metal‐chelate epoxy support with an increase of its operational stability, results that are not easily achievable with the sole immobilization on epoxy or metal chelate‐epoxy supports in the case of complex multimeric enzymes with geometric incongruence with the support.

中文翻译：

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用于从 d-半乳糖生产 d-塔格糖的固定化 l-阿拉伯糖异构酶的稳定性。

这项工作的目的是开发一种稳定的固定化酶生物催化剂，用于在高温下将d-半乳糖异构化为d-塔格糖。来自超嗜热细菌海栖热袍菌(TMAI) 的l-阿拉伯糖异构酶作为 (His) ₆标记的蛋白质，固定在铜螯合环氧树脂载体上，并经过多次固定化后处理，以提高其操作和结构稳定性。用戊二醛和乙二胺处理导致操作稳定性和通过共价键直接或间接连接到支持物的所有酶亚基增加两倍以上。用巯基乙醇对固定化衍生物进行后固定化处理以去除任何残留的铜离子，进一步提高了生物催化活性。经过两种处理的固定化衍生物用于将 18 g/L d-半乳糖生物转化为d-塔格糖在 80°C 的填充床反应器中重复三个循环，与文献数据相比显示出更好的操作稳定性。该研究表明，用戊二醛和乙二胺进行后固定化处理可以稳定固定在金属螯合环氧树脂载体上的酶的多亚基结构，并增加其操作稳定性，这是单独固定在环氧树脂上不容易实现的结果。在复杂的多聚酶与载体几何不一致的情况下，金属螯合物-环氧树脂载体。