Institut für Pharmazeutische und Medizinische Chemie, PharmaCampus, Westfälische Wilhelms-Universität Münster, Corrensstr. 48, Münster, 48149, Germany.
Institute of Biochemistry, Department of Biotechnology and Enzyme Catalysis, Greifswald University, Felix-Hausdorff-Str.17489, Greifswald, Germany.
Microb Biotechnol. 2022 Aug;15(8):2235-2249. doi: 10.1111/1751-7915.14062. Epub 2022 Apr 27.
The application of enzymes as biocatalysts in industrial processes has great potential due to their outstanding stereo-, regio- and chemoselectivity. Using autodisplay, enzymes can be immobilized on the cell surface of Gram-negative bacteria such as Escherichia coli. In the present study, the surface display of an alcohol dehydrogenase (ADH) and a cyclohexanone monooxygenase (CHMO) on E. coli was investigated. Displaying these enzymes on the surface of E. coli resulted in whole-cell biocatalysts accessible for substrates without further purification. An apparent maximal reaction velocity V for the oxidation of cyclohexanol with the ADH whole-cell biocatalysts was determined as 59.9 mU ml . For the oxidation of cyclohexanone with the CHMO whole-cell biocatalysts a V of 491 mU ml was obtained. A direct conversion of cyclohexanol to ε-caprolactone, which is a known building block for the valuable biodegradable polymer polycaprolactone, was possible by combining the two whole-cell biocatalysts. Gas chromatography was applied to quantify the yield of ε-caprolactone. 1.12 mM ε-caprolactone was produced using ADH and CHMO displaying whole-cell biocatalysts in a ratio of 1:5 after 4 h in a cell suspension of OD 10. Furthermore, the reaction cascade as applied provided a self-sufficient regeneration of NADPH for CHMO by the ADH whole-cell biocatalyst.
由于其出色的立体、区域和化学选择性，酶作为生物催化剂在工业过程中的应用具有巨大的潜力。通过自展示，酶可以固定在革兰氏阴性细菌如大肠杆菌的细胞表面。在本研究中，研究了醇脱氢酶 (ADH) 和环己酮单加氧酶 (CHMO) 在大肠杆菌上的表面展示。将这些酶展示在大肠杆菌表面上，可得到无需进一步纯化即可接触底物的全细胞生物催化剂。用 ADH 全细胞生物催化剂氧化环己醇的表观最大反应速度 V 确定为 59.9 mU/ml。用 CHMO 全细胞生物催化剂氧化环己酮的 V 为 491 mU/ml。通过组合两种全细胞生物催化剂，可将环己醇直接转化为 ε-己内酯，ε-己内酯是有价值的可生物降解聚合物聚己内酯的已知结构单元。气相色谱法用于定量 ε-己内酯的产率。在 OD10 的细胞悬浮液中反应 4 小时后，使用 ADH 和 CHMO 展示全细胞生物催化剂的比例为 1:5，可生产出 1.12 mM 的 ε-己内酯。此外，所应用的反应级联为 CHMO 提供了由 ADH 全细胞生物催化剂自足再生 NADPH。
