WP1: Enzyme production
Recombinant production of relevant enzymes for use in characterization and application testing. The expression of enzymes will be optimized and a cost-analysis for pilot scale-up of enzyme production will be performed.
WP2: Application testing/Proof of Concept studies
We aim to optimize the use of aldolases for production of sialic acid. NALs will initially be tested for synthesis of sialic acid from both N-acetylglucosamine and rest raw materials (chitin hydrolysates). We will verify the use of NALs both in a one-step synthesis of sialic acid at elevated pH (chemical epimerization) and in a two-step combination with AGEs. Optimization of the enzymes in these applications will be part of this workpackage. The most promising enzymes evolved by nature itself will be selected, and if needed through rational design and redesign (WP4), further develop the biocatalyst properties to meet various demands. Newly selected NALs will in particular be relevant for production of sialic acid derivatives.
WP3: Enzyme characterization
Functionality and biocatalytic properties of newly selected aldolases will be investigated. Crystallographic studies will be performed. Knowledge of the structure of natural enzymes, and their binding properties for ligands as well as structural modifications induced by these ligands are obligate tools to predict mutation positions within the enzymes that will increase their efficiency or alter their specificity. The X-ray structures will provide crucial information regarding construction of such rationally designed mutants (WP4), as will structures with substrate, substrate analogues and other known ligands bound.
WP4: Rational design of enzymes
This workpackage include structure based rational design of mutants for production of the desirable sialic acid derivatives or mutants with improved biocatalytic properties (if needed).
WP5: Organic synthesis of substrates
Relevant substrates for production of the sialic acid derivatives will be synthesized.
Securing of IPR and work with commercialization will be performed in this workpackage headed by Norinnova Technology Transfer.