The BioCaPS seed fund project aimed to gain deep structural insight into promising biohybrid catalyst for the stereoselective production of polylactic acid (PLA). Biohybrid catalysts or so called artificial metalloenzymes consist of a protein that harbors an (artificial) metal catalyst. The surrounding protein shell controls the chemo- and regioselectivity through space like observed for natural enzymes (specifically orienting substrate to the catalytic metal center). PLA is produced from sustainable resources and represents a promising and biodegradable alternative to petroleum-based polymers.
In working program (WP) 1 (Biotech), the production and reengineering of the b-barrel transmembrane protein Ferric hydroxamate uptake protein component: A to harbor the metal catalyst was performed. An improved purification protocol to achieve high purity for crystallization was established and the protein production was increased to gram-scale amounts in a 80 L fermenter.
Within WP 2 (IAC), a ligand platform based on the 2,2’:6’,2’’-terpyridnie ligand framework bearing a maleimide function for covalent anchoring to the protein was developed. Into this framework various lewis acidic metals (namely: cobalt, zinc, indium, scandium, copper, iron) were incorporated. After purification of the metal-conjugates, conjugation to the protein FhuA was performed and the b-barrel structure renatured. The Biohybrid conjugates were analyzed by CD- and UV/Vis spectroscopy, ThioGlo fluorescence titration, MALDI-TOF MS and BCA assay, indicating successful anchoring and refolding.
In WP 3 (BPP), the crystallization of the FhuA was attempted. The improved purification protocol developed within WP 1 provided the protein in a sufficient purity. Initial trials (576 conditions) lead to precipitates in 90% of the tried conditions. For further experiments, the addition of various additives was tested to improve the protein stability.
WP 4 (ICS-6) established the use of viable isotope labelling protocols which are suitable for extensive and sparse isotope labeling.
Participating Core Groups
Prof. Dr. Ulrich Schwaneberg, Dr. Leilei Zhu, Institute of Biotechnology, RWTH Aachen University
Prof. Dr. Georg Groth, Institut of Biochemical Plant Physiology, Heinrich Heine University Düsseldorf
Prof. Dr. Henrike Heise, Institut of Physical Biology, Heinrich Heine University Düsseldorf / Institute of Complex Systems, ICS-6: Structural Biochemistry, Forschungszentrum Jülich GmbH
01.01.2016 – 31.12.2016