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.
Reconstitution properties of biologically active polymersomes after cryogenic freezing and a freeze-drying process