A secretion‐based dual fluorescence assay for high‐throughput screening of alcohol dehydrogenases. Issue 4 (25th January 2021)

• Record Type: Journal Article
• Authors: Lu, Hongyuan; Yu, Shiqin; Qin, Fengyu; Ning, Wenbo; Ma, Xiaoqiang; Tian, Kaiyuan; Li, Zhi; Zhou, Kang
• Is Part Of: Biotechnology and bioengineering. Volume 118:Issue 4(2021)
• Page Start: 1605
• Page End: 1616
• Publication Date: 2021-01-25
• Subjects: alcohol dehydrogenase -- cascade reaction -- directed protein evolution -- fluorescence assay -- protein secretion; Biotechnology -- Periodicals; Bioengineering -- Periodicals; 660.6
• DOI: 10.1002/bit.27677
• Languages: English
• ISSNs: 0006-3592

Abstract

Abstract: Alcohol dehydrogenases (ADHs) play key roles in the production of various chemical precursors that are essential in pharmaceutical and fine chemical 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 k cat / k m value toward ( S )‐2‐octanol compared to its wild type. Abstract : A secretion‐based dual fluorescence assay for high‐throughput screening of alcohol dehydrogenases was developed in this study, which offers an easy and efficient way to normalize the catalytic activity of different enzyme variants. By using this assay, the authors screened a combinatorial library of an alcohol dehydrogenase from Pichia finlandica and identified a variant with a 197‐fold higher kcat /km value toward (S)‐2‐octanol compared to its wild‐type.