Design of GH16 XET/XEH chimeric enzymes with SCHEMA: Manuscript
(English)Manuscript (preprint) (Other academic)
This manuscript contains experimental data obtained during a pilot study for the application of the SCHEMA method for structure-guided recombination on PttXET16-34 and TmNXG1, a model system for the evolution of different catalytic routes of GH16 XETs and XEHs.
A restricted library of PttXET16-34/TmNXG1 chimeras with high diversity and low calculated SCHEMA disruption was generated based on crossover points identified by the RASPP algorithm. Analysis of the library revealed a bias among certain regions to remain intact and recalcitrant to recombination, in particular the upper and lower β-sheet structures forming the part of the protein that binds the donor substrate. In contrast, sequence diversity was preferentially introduced at the N-terminus, the major part of the acceptor side of the protein, and most of the C-terminal extension characteristic to XET/XEH in the GH16 family. Finally, in order to test the predictive capacity of SCHEMA, six chimeras with low calculated disruption were chosen for subsequent cloning and expression in Pichia pastoris.
IdentifiersURN: urn:nbn:se:kth:diva-24298OAI: oai:DiVA.org:kth-24298DiVA: diva2:346379
QC 201009022010-08-312010-08-312010-09-02Bibliographically approved