Fe(II)- and α-ketoglutarate dependent dioxygenases have emerged as important catalysts for the preparation of non-natural amino acids. The stoichiometric supply of the cosubstrate α-ketoglutarate (αKG) is an important cost factor. A combination of the N-succinyl amino acid hydroxylase SadA with an l-glutamate oxidase (LGOX) allowed for coupling in situ production of αKG to stereoselective αKG-dependent dioxygenases in a one-pot/two-step cascade reaction. Both enzymes were used as immobilized enzymes and tested in a preparative scale setup under process-near conditions. Oxygen supply, enzyme, and substrate loading of the oxidation of glutamate were investigated under controlled reaction conditions on a small scale before upscaling to a 1 L stirred tank reactor. LGOX was applied with a substrate concentration of 73.6 g/L (339 mM) and reached a space-time yield of 14.2 g/L/h. Additionally, the enzyme was recycled up to 3 times. The hydroxylase SadA reached a space-time yield of 1.2 g/L/h at a product concentration of 9.3 g/L (40 mM). For both cascade reactions, the supply with oxygen was identified as a critical parameter. The results underline the robustness and suitability of α-ketoglutarate dependent dioxygenases for application outside of living cells.

中文翻译：

---

α-酮戊二酸和Fe（II）依赖双加氧酶的辅因子生成级联。

Fe（II）-和α-酮戊二酸依赖性双加氧酶已成为制备非天然氨基酸的重要催化剂。共底物α-酮戊二酸（αKG）的化学计量供应是重要的成本因素。所述的组合Ñ与丁二酰氨基酸羟化酶SADA升谷氨酸盐氧化酶（LGOX）允许用于耦合原位一锅/两步级联反应可将αKG生成为立体选择性αKG依赖性双加氧酶。两种酶均用作固定化酶，并在接近过程的条件下以制备规模设置进行测试。在规模扩大到1 L搅拌釜反应器之前，在受控反应条件下以小规模研究了谷氨酸氧化的供氧，酶和底物负载。LGOX的底物浓度为73.6 g / L（339 mM），时空产量为14.2 g / L / h。此外，该酶最多可循环3次。在产物浓度为9.3 g / L（40 mM）时，羟化酶SadA的时空产量达到1.2 g / L / h。对于两个级联反应，氧气的供应被确定为关键参数。