Objectives

Objective 1. Site-specific covalent immobilization of PcPAL by incorporation of unnatural amino acids

The site-specific incorporation of the unnatural amino acid, p-azido-phenylalanine (p-azido-Phe), will allow site-specific immobilization of PcPAL (PAL from Petroselinum crispum) via CuAAC click reaction (copper-catalyzed azide-alkyne cycloaddition). Novel immobilization supports functionalized with an alkynyl group will be designed. The activity and stability of the novel biocatalysts will be investigated.

Objective 2. Site-specific covalent immobilization of PcPAL through maleimide/thiol coupling

Immobilization of PcPAL will be accomplished through covalent binding of unique surface accessible cysteine residues (introduced by site-direct mutagenesis) to maleimide-functionalized supports. Novel immobilization supports bearing a maleimide group will be developed. The activity and stability of the novel biocatalysts will be investigated.

Objective 3. Biocatalytic procedures with novel immobilized PcPALs in batch and continuous-flow systems

The efficient developed immobilization techniques will be applied to the novel PAL variants evolved by the host group and tested in ammonia elimination and addition reactions of various unnatural substrates in batch and continuous-flow packed-bed microreactors, with the aim of obtaining l– and d–unnatural amino acids of industrial interest.