259593 A Novel Amine Dehydrogenase for the Selective Production of Chiral Amines
Biocatalysts are increasingly used in industry to create enantiomerically pure compounds; routes employing dehydrogenases and other redox enzymes have been especially successful. Enantiomeric compounds have a broad range of uses including an utmost importance in pharmaceuticals. Contemporary methods of protein engineering, such as applying rational design guided by mechanistic and structural knowledge, have greatly increased the ability to create novel enzyme functionality.[2-3]
Application of degenerate codons and a high-throughput screening assay allowed for simplified, rapid evaluation of enzyme variants. Constraint of mutant library sizes was guided by mechanistic and structural knowledge, ultimately reducing the screening requirements while maintaining an increased chance of generating alternate substrate specificity. Expansion of its substrate specificity was achieved through several rounds of focused mutagenesis, allowing for the creation of chiral amines. Beneficial mutations of each round were carried over into subsquent variants.
Over the course of eleven libraries, we have sucessfully developed a novel amine dehydrogenase (AmDH) from an existing leucine dehydrogenase scaffold with broad substrate specificity. Reductive amination activity was not measureable with the original leucine dehydrogenase scaffold, and was increased to a kcat of 0.46 s-1 with a KM of 15.1 mM. This creates a new green route in the production of chiral amines utilizing ketone and NH3 substrates. Novel activity was achieved toward a number of compounds while maintaining the enzymes‘ native enantioselectivity of 99.8% e.e.
 M. Hall and A.S. Bommarius, Chem. Rev. 2011, 111, 4088-4110.
 A.S. Bommarius, J.K. Blum, M.J. Abrahamson, Curr. Opin. Chem. Biol. 2011, 15, 194-200.
 A.S. Bommarius, Nature Chem. Biol. 2010, 6, 793-794.
 M. Abrahamson, et. al, Angewandte Chemie, 2012, 51, 3969-3972.
See more of this Group/Topical: Food, Pharmaceutical & Bioengineering Division - See also TI: Comprehensive Quality by Design in Pharmaceutical Development and Manufacture