Developing of a novel enantioselective biocatalyst acting on L-amino acids of biotechnological interest through a "semi-rational design" approach supported by computational analyses.

The synthesis of optically pure enantiomers is a challenge for traditional chemistry. Enzymes often perform better than inorganic catalysts because of their enantioselectivity, effectiveness and ecological sustainability. However, natural enzymes that evolved under the drive of natural selective pressure for their biological function, are not suitable for most industrial applications 1. For this reason, enzymes must be improved by protein engineering. L-amino acid deaminase from Proteus myxofaciens (PmaLAAD) catalyzes the oxidative deamination of L-amino acids (AAs) to the corresponding -keto acids (KA). This biocatalyst can be used for the setup “green processes” to produce pure KA and D-AAs, which are used as raw materials for the synthesis of drugs and agrochemicals. In this project, several biocatalytic processes based on PmaLAAD have been optimized for the bioconversion, deracemization and stereoinversion of (un)natural L-AAs 2. Several natural or synthetic L-AAs have been used as substratet obtaining conversion yields close to 100%. The knowledge of the 3D structure of PmaLAAD 3 was exploited to produce several enzyme variants possessing animproved activity on L-1-naphthylalanine (L-1-Nal) through a semi-rational design approach supported by computational analysis. The most interesting variant (F318A/V412A/V438P-PmaLAAD) was able to convert D,L-1-Nal with a rate 7.5 fold higher than the wild type.