Transaminase responsible for alienating prochiral ketone compound is applicable to asymmetric synthesis of herbicide L-phosphinothricin (L-PPT). In this work, the covalent immobilization of recombinant transaminase from Citrobacter koseri (CkTA) was investigated on different epoxy resins. Using optimum ES-105 support, a higher immobilized activity was obtained via optimizing immobilization process in terms of enzyme loading, coupling time and initial PLP concentration. Crucially, due to blocking unreacted epoxy groups on support surface with amino acids, the reaction temperature of blocked immobilized biocatalyst was enhanced from 37 to 57 °C. Its thermostability at 57 °C was also found to be superior to that of free CkTA. The Km value was shifted from 36.75 mM of free CkTA to 39.87 mM of blocked immobilized biocatalyst, demonstrating that the affinity of enzyme to the substrate has not been apparently altered. Accordingly, the biocatalyst performed the consecutive synthesis of L-PPT for 11 cycles (yields>91%) with retaining more than 91.13% of the initial activity. The seemingly the highest reusability demonstrates this biocatalyst has prospective for reducing the costs of consecutive synthesis of L-PPT with high conversion.

中文翻译：

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共价固定化重组柠檬酸杆菌转氨酶到环氧树脂上以连续不对称合成L-膦丝菌素。

负责疏离前手性酮化合物的转氨酶适用于除草剂L-膦丝菌素（L-PPT）的不对称合成。在这项工作中，研究了来自科氏柠檬酸杆菌的重组转氨酶（CkTA）在不同环氧树脂上的共价固定。使用最佳的ES-105支持物，可以通过优化固定化过程来优化酶的负载，偶联时间和初始PLP浓度，从而获得更高的固定化活性。至关重要的是，由于用氨基酸封闭了载体表面上未反应的环氧基，封闭的固定化生物催化剂的反应温度从37℃提高到57℃。还发现其在57°C时的热稳定性优于游离CkTA。Km值从36.75 mM的游离CkTA转变为39.87 mM的封闭的固定化生物催化剂，这表明酶与底物的亲和力没有明显改变。因此，生物催化剂进行了11个循环的L-PPT的连续合成（产率＞ 91％），保留了超过91.13％的初始活性。看来最高的可重复使用性表明该生物催化剂具有降低连续合成高转化率L-PPT成本的潜力。