DEVELOPMENT AND APPLICATION OF NOVEL ENGINEERED TRANSAMINASE PANELS ASSISTED BY IN-SILICO RATIONAL DESIGN FOR THE PRODUCTION OF CHIRAL AMINES

摘要

There is a high demand for the synthesis of chiral amines as building blocks for a large number of industrially valuable compounds. Transaminases (TAm) offer an enzymatic route for the synthesis of chiral amines that avoids complex chemical synthesis . However, their catalytic efficiency towards bulky ketone substrates is greatly limited by steric hinderance . This poster highlights a rational design strategy of combining in silico and in vitro methods to engineer the transaminase enzyme with a minimal number of mutations, achieving high catalytic activity and high enantioselectivity. The wildtype TAm showed no detectable activity towards the ketone 2-acetylbiphenyl but upon introduction of two mutations detectable enzyme activity was observed. The reaction rate was improved a further 1716-fold with the rationally designed variant, that contained a further 5 mutations, producing the corresponding enantiomeric pure (S)-amine (enantiomeric excess (ee) value of >99%). In addition, screening of in silico designed (R)-TAm mutant panels in resolution mode offered an attractive and efficient route for the preparation of problematic (S)-amines. A mutant was identified from the panels that gave complete resolution of the racemic amine (high substrate loading) to leave the desired enantiomer at a low enzyme loading fit for process development towards an economically viable scale up process.