(704a) Improvement of KM of Ammonia and Mechanistic Insight of Amine Dehydrogenases through Rational and Data-Driven Protein Engineering Conference: AIChE Annual MeetingYear: 2015Proceeding: 2015 AIChE Annual MeetingGroup: Food, Pharmaceutical & Bioengineering DivisionSession: Protein Engineering II: Rational and Computational Techniques Time: Thursday, November 12, 2015 - 12:30pm-12:50pm Authors: Au, S., University of Kentucky Bommarius, B., Georgia Institute of Technology Bommarius, A. S., Georgia Institute of Technology Recently, amine dehydrogenase (AmDH) has been developed to catalyze prochiral ketones to chiral primary amines, with the addition of ammonia and the cofactor NADH. Three AmDHs have been developed so far: leucine amine dehydrogenase (L-AmDH) , phenylalanine amine dehydrogenase (F-AmDH)  and a chimeric amine dehydrogenase (cFL1-AmDH) . One of the limitations of the AmDH family is the high KM for its ammonium substrate (KM: 650 mM). This work will investigate the improvements of the AmDH binding to ammonia through 1) protein engineering of the F-AmDH and 2) adapting reaction conditions. The protein engineering work will mostly investigate mutations within the active site of the F-AmDH, with a few variants into the secondary shell. The recently solved crystal structure of the F-AmDH helped to guide the rational protein engineering of the AmDHs. Over 80 active variants were expressed and characterized and the KM of ammonia was improved to two-fold to 350 mM. In addition, specific activity of the F-AmDH was improved 2.5-fold by opening up space within the binding pocket. Various reaction medium conditions were investigated such as pH and ammonium buffers (acetate, bicarbonate, citrate, chloride, formate, phosphate). Changes to KM and specific activity of the F-AmDH were observed in various buffers. Lastly, substituents on the model substrate of the F-AmDH, para-fluoro-phenyl acetone, were investigated and will be presented with this work.  Abrahamson, M. J.; Vazquez-Figueroa, E.; Woodall, N. B.; Moore, J. C.; Bommarius, A. S., Development of an Amine Dehydrogenase for Synthesis of Chiral Amines. Angew Chem Int Edit 2012, 51 (16), 3969-3972.  Abrahamson, M. J.; Wong, J. W.; Bommarius, A. S., The Evolution of an Amine Dehydrogenase Biocatalyst for the Asymmetric Production of Chiral Amines. Adv Synth Catal 2013, 355 (9), 1780-1786.  Bommarius, B. R.; Schurmann, M.; Bommarius, A. S., A novel chimeric amine dehydrogenase shows altered substrate specificity compared to its parent enzymes. Chem Commun 2014, 50 (95), 14953-14955.