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Development of 96 Multiple Injection-GC-MS Technique and Its Application in Protein Engineering of Natural and Non-Natural Enzymatic Reactions

preprint
submitted on 15.11.2019 and posted on 03.12.2019 by Anja Knorrscheidt, Pascal Püllmann, Eugen Schell, Dominik Homann, Erik Freier, Martin Weissenborn
Directed evolution requires the screening of enzyme libraries in biological matrices. Available assays are mostly substrate or enzyme specific. Chromatographic techniques like LC and GC overcome this limitation, but require long analysis times. The herein developed multiple injections in a single experimental run (MISER) using GC coupled to MS allows the injection of samples every 33 s resulting in 96-well microtiter plate analysis within 50 min. This technique is implementable in any GC-MS system with autosampling. Since the GC-MS is far less prone to ion suppression than LCMS, no chromatographic separation is required. This allows the utilisation of an internal standards and the detection of main and side-product. To prove the feasibility of the system in enzyme screening, two libraries were assessed: i) YfeX library in an E. coli whole cell system for the carbene-transfer reaction on indole revealing the novel axial ligand tryptophan, ii) a library of 616 chimeras of fungal unspecific peroxygenase (UPO) in S. cerevisiae supernatant for hydroxylation of tetralin resulting in novel constructs. The data quality and representation are automatically assessed by a new R-script.

Funding

Biotechnology2020 + Structural Projects: Leibniz Research Cluster (LRC) - Bio / Synthetic Multifunctional Micro-Production Units - Novel Pathways to Drug Development, Subproject B

Federal Ministry of Education and Research

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Biotechnology2020 + Structural Projects: Leibniz Research Cluster (LRC) - Bio / Synthetic Multifunctional Micro-Production Units - Novel Pathways to Drug Development, Subproject E

Federal Ministry of Education and Research

Find out more...

Landesgraduiertenförderung Sachsen-Anhalt

History

Email Address of Submitting Author

martin.weissenborn@ipb-halle.de

Institution

Leibniz-Institut für Pflanzenbiochemie & MLU Halle Wittenberg

Country

Germany

ORCID For Submitting Author

0000-0002-1200-4485

Declaration of Conflict of Interest

No conflict of interest

Licence

Exports