Busch Florian, Brummund Jan, Calderini Elia, Schürmann Martin, Kourist Robert
InnoSyn B.V., Urmonderbaan 22, NL-6167 RD Geleen The Netherlands.
Junior Research Group for Microbial Biotechnology, Ruhr-Universität Bochum, Universitätsstrasse 150, 44780 Bochum, Germany.
ACS Sustain Chem Eng. 2020 Jun 15;8(23):8604-8612. doi: 10.1021/acssuschemeng.0c01122. Epub 2020 May 13.
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 -succinyl amino acid hydroxylase SadA with an l-glutamate oxidase (LGOX) allowed for coupling 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.
依赖于Fe(II)和α-酮戊二酸的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）的化学计量供应是一个重要的成本因素。将琥珀酰氨基酸羟化酶SadA与L-谷氨酸氧化酶（LGOX）相结合，可在一锅/两步级联反应中将αKG的生成与立体选择性αKG依赖性双加氧酶偶联起来。两种酶均用作固定化酶，并在接近实际生产的条件下进行了制备规模的测试。在放大至1 L搅拌釜反应器之前，先在小规模的受控反应条件下研究了谷氨酸氧化过程中的氧气供应、酶和底物负载情况。LGOX的底物浓度为73.6 g/L（339 mM），时空产率达到14.2 g/L/h。此外，该酶可循环使用多达3次。羟化酶SadA在产物浓度为9.3 g/L（40 mM）时的时空产率为1.2 g/L/h。对于这两个级联反应，氧气供应被确定为关键参数。结果强调了α-酮戊二酸依赖性双加氧酶在活细胞外应用的稳健性和适用性。
