Expanding the Utility of Amine Dehydrogenases Through Protein and Reaction Engineering for the Biocatalytic Production of Chiral Amines

The purpose of this project was to expand the understanding and utility of a pre-existing family of engineered amine dehydrogenases (AmDHs) which catalyze the reductive amination of prochiral ketones to chiral amines. Work toward this goal was focused in three generally independent directions, enzyme improvement through site-directed mutagenesis, understanding of the kinetic mechanism, and reaction engineering toward scalable continuous amine production. Through a combination of mutations shown to have positive effects in other related amine and amino acid dehydrogenases, new AmDH variants were produced with multiple-fold higher activity, 8 °C higher melting temperature, and new activity for large substrates not found for the base case. A detailed series of initial rate experiments allowed us to propose a kinetic mechanism for L-AmDH and showed that the mutations which produced the amine dehydrogenase from the parent amino acid dehydrogenase also caused changes in the kinetic mechanism. After investigations of multiple reactor platforms, enzyme immobilization strategies, and analytical methods, a robust packed bed reactor platform was characterized. The accessible design space of the reactor was explored. Impacts of flow rate, temperature, substrate concentration, rapid recycle, and bed height on conversion, productivity, and enzyme stability were examined. The reported work, especially aim 3, fills an important gap in the field. Detailed discussions of the impact of physical process parameters on productivity allow for concrete engineering decisions. The growing importance of enzymes in pharmaceutical and fine chemical production requires an increased focus on biocatalysis from an engineering prospective.

Date

2020-05-19

Resource Type

Text

Resource Subtype

Dissertation

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