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

03 December 2019, Version 1
This content is a preprint and has not undergone peer review at the time of posting.

Abstract

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.

Keywords

Directed Evolution
High throughput
protein engineering
low matrix effect
carbene transfer

Supplementary materials

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Supporting
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R-Script template
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R-scriptedEvaluation
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Comment number 1, Barbara: Aug 14, 2024, 13:41

The innovative 96-well plate template (https://96wellplatetemplate.com/) approach presented in this study enhances enzyme screening efficiency with MISER-GC-MS, achieving rapid analysis and improved detection of enzymatic reactions.