WP2: Transaminase screening and optimization

Objectives: Identification of amine transaminases (ATAs) able to catalyze efficiently the amination of a desired set of ketones and aldehydes (expected products of GO oxidation). Optimization of the most potent biocatalysts, in terms of catalytic efficiency, stability, availability of functional groups for covalent immobilization.

Description of Work: ATAs from our construct selection (>30 wild-type and variants of both (R)- and (S)-enantioselectivity) with different substrate selectivity will be recombinantly expressed in Escherichia coli strains and purified via affinity chromatography based on their 6His-tag using standard protocols. The purified ATAs will be screened for the acceptance of a set of selected ketones and aldehydes (expected products of GO oxidation from WP1). For those enzymes that exhibit activity, biochemical characterization will be performed, concerning the amine donor used for the equilibrium displacement (such as isopropylamine or o-xylelene diamine) as well as the thermal and operational stability. A few biocatalysts will be selected based on their promising biochemical profile, and they will be optimized by means of protein engineering for the planned cascade. Protein engineering efforts will have three main targets: (i) improving the acceptance of the substrates, to have higher affinity and utilize even the lower amount of intermediate released from GO (mostly rational design will be used), (ii) improving the thermal and operational stability of the enzyme (mostly directed evolution will be used) and (iii) providing free amine groups for the covalent (and potentially oriented) immobilization of the enzymes (rational design). To guide the rational design, selected biocatalysts for optimization will be investigated structurally. In the case of directed evolution, the structure of the optimized biocatalyst will be also studied, in order to understand the structural features that provide the beneficial effect, to further guide rational engineering efforts.