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  • MK1775 The results described in the present study


    The results described in the present study confirm key restrictions on reaction conditions which must considered to maximize reactor productivity. Increased viscosity should be avoided for the amination of ketones by L-AmDH, as a 4-fold increase in viscosity can lower the reaction rate by as much as 50% at saturating ketone concentration (Fig. 2C). In a reactor setting, detrimental and beneficial KSVEs may be realized through the deliberate addition of glycerol to stabilize the biocatalyst [45] and unintentionally through the introduction of kosmotropic anions such as formate or sulfate [46], [47]. As a result, engineers must optimize processes to balance MK1775 and catalytic efficiently based on relative costs of catalyst and products. Additionally, it is important to avoid the strong product inhibition seen for L-AmDH to optimize amine production, especially, as competitive inhibition by 2-aminopentane on the already slow-binding 2-pentanone increases with conversion. Inhibition by NAD+ can be avoided by employing a large amount of the cofactor regeneration enzyme, which lowers [NAD+].
    Conclusion Through initial rate, product inhibition, and KSVE experiments, we have demonstrated key differences between the kinetic mechanisms of L-AmDH and its parent enzyme, LeuDH. The 40-fold decrease in the appkcat value between the two enzymes can largely be explained by the much lower affinity for the ketone or amine substrate rather than affinity for ammonia. Kinetic viscosity effects elucidated the importance of an isomerization of the Michaelis complex for determining the rate of LeuDH catalysis, but the rate-limiting step is shifted for L-AmDH. A change in substrate binding order between the two enzymes was indicated by the differences in the fitted rate laws and inhibition patterns. An understanding of the kinetic properties of L-AmDH will enable its use in the biocatalytic production of chiral amines and is a required input for reactor design and scale-up.
    Acknowledgments Funding for the reported work by the National Science Foundation through NSF I/UCRC grant IIP-1540017 to the Center for Pharmaceutical Development and through MK1775 CBET grant 1512848 is gratefully acknowledged. The authors thank Adam Caparco for helpful insights and support. We also wish to acknowledge the Biopolymer Characterization (BPC) core facility at the Parker H. Petit Institute for Bioengineering and Bioscience at the Georgia Institute of Technology for the use of its shared equipment, services and expertise.