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dc.contributor.advisorBommarius, Andreas
dc.contributor.authorAbrahamson, Michael J.
dc.date.accessioned2014-01-10T19:36:40Z
dc.date.available2014-01-10T19:36:40Z
dc.date.issued2012-08-13
dc.identifier.urihttp://hdl.handle.net/1853/50138
dc.description.abstractBiocatalysts are increasingly prevalent in the large-scale synthesis of enantiomerically pure compounds. However, many sought-after reactions lack a suitable enzymatic production route. This work describes the development of a novel amine dehydrogenase through the application of directed evolution altering the substrate specificity of an existing leucine dehydrogenase scaffold. Eleven rounds of directed evolution completely altered the enzyme’s specificity and successfully created amination activity. The resulting amine dehydrogenase asymmetrically catalyzes methyl isobutyl ketone and free ammonia to 1, 3-dimethyl butyl amine. The enantioselectivity of the wild-type enzyme was maintained despite the drastic changes to the binding pocket and yielded (R)-1,3-DMBA with nearly complete conversion making it an attractive catalyst in the synthesis of chiral amines. This was the first example of a cofactor-dependent amine dehydrogenase capable of selectively synthesizing chiral amines from a prochiral ketone and free ammonia. Additionally, knowledge gained altering the specificity of the leucine dehydrogenase scaffold was applied to an analogous phenylalanine dehydrogenase scaffold allowing for rapid evolution of novel activity. A single mutational library resulted in a second amine dehydrogenase with enhanced activity toward significantly different substrates, while maintaining comparable conversion and enantioselectivity. These two scaffolds provide examples of the broad applicability of the identified mutations in creating amine dehydrogenase activity.
dc.language.isoen_US
dc.publisherGeorgia Institute of Technology
dc.subjectChiral amines
dc.subjectDirected evolution
dc.subjectAmine dehydrogenase
dc.subjectBiocatalysis
dc.subject.lcshDehydrogenases
dc.subject.lcshAmines
dc.titleDevelopment of an amine dehydrogenase
dc.typeDissertation
dc.description.degreePh.D.
dc.contributor.departmentChemical Engineering
dc.contributor.committeeMemberJones, Christopher
dc.contributor.committeeMemberSkolnick, Jeffrey
dc.contributor.committeeMemberWong, John
dc.contributor.committeeMemberKawajiri,Yoshiaki


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