Lu, Hongyuan; Yu, Shiqin; Qin, Fengyu; Ning, Wenbo; Ma, Xiaoqiang; Tian, Kaiyuan; Li, Zhi; Zhou, Kang published the artcile< A secretion-based dual fluorescence assay for high-throughput screening of alcohol dehydrogenases>, Related Products of 403-41-8, the main research area is alc dehydrogenase secretion dual fluorescence assay high throughput screening; alcohol dehydrogenase; cascade reaction; directed protein evolution; fluorescence assay; protein secretion.
Alc. dehydrogenases (ADHs) play key roles in the production of various chem. precursors that are essential in pharmaceutical and fine chem. industries. To achieve a practical application of ADHs in industrial processes, tailoring enzyme properties through rational design or directed evolution is often required. Here, we developed a secretion-based dual fluorescence assay (SDFA) for high-throughput screening of ADHs. In SDFA, an ADH of interest is fused to a mutated superfolder green fluorescent protein (MsfGFP), which could result in the secretion of the fusion protein to culture broth. After a simple centrifugation step to remove the cells, the supernatant can be directly used to measure the activity of ADH based on a red fluorescence signal, whose increase is coupled to the formation of NADH (a redox cofactor of ADHs) in the reaction. SDFA allows easy quantification of ADH concentration based on the green fluorescence signal of MsfGFP. This feature is useful in determining specific activity and may improve screening accuracy. Out of five ADHs we have tested with SDFA, four ADHs can be secreted and characterized. We successfully screened a combinatorial library of an ADH from Pichia finlandica and identified a variant with a 197-fold higher kcat/km value toward (S)-2-octanol compared to its wild type.
Biotechnology and Bioengineering published new progress about Catalysis. 403-41-8 belongs to class alcohols-buliding-blocks, and the molecular formula is C8H9FO, Related Products of 403-41-8.
Referemce:
Alcohol – Wikipedia,
Alcohols – Chemistry LibreTexts