OBJECTIVES
Objective 1.
Creation of MIO-enzyme toolbox, containing known and newly reported MIO-enzymes:
the enzyme library will consist of various wt-ALs and AMs from various mesophiles and termophiles as well as mutant variants reported to have promising catalytic properties. Furthermore the library will contain several chimeras, with increased heat stability developed from the eukaryotic PcPAL, RtPAL, TcPAM and bacterial AvPAL.
Related work-packages:
- WP1. molecular cloning steps;
- WP2. fermentation optimization;
- WP3. production and purification of enzymes.
Objective 2.
Development of high throughput activity screening assay:
the method proposed by us for the development of the high-throughput activity assay, is based on the enzymatic decarboxylation of the cinnamic acid derivatives, providing styrene like derivatives, which in the presence of diaryltetrazole based probes can be photoinduced, generating fluorescent pyrazoline products.
Related work-packages:
- WP1. molecular cloning, production and purification of phenylacrylic acid decarboxilase system;
- WP2. synthesis of diaryltetrazole based probes;
- WP3. activity assay set-up for the whole cell and purified protein MIO-enzyme kit.
Objective 3.
Exploring and broadening the substrate scope of the MIO-enzyme toolbox:
we will explore the subsrate scope of the MIO-enzyme library towards non-natural substrates with the aim to develop highly efficient biocatalytic synthetic procedures for the non-natural aminoacids.
Related work-packages:
- WP1. chemical synthesis of known or/and novel unnatural a- and b-arylalanines as well as their acrylic counterparts;
- WP2. determination of activity of the MIO-enzyme (whole cell and purified enzyme) library towards the substrate library;
- WP3. enzyme kinetic measurements and for the preparative scale biotransformations with optimal purified enzymes;
- WP4. docking studies based on the obtained activity spectra.