Development of a Biphasic Organic Solvent System to Broaden the Substrate Range of the Phenylalanine Amine Dehydrogenase

Title: Development of a Biphasic Organic Solvent System to Broaden the Substrate Range of the Phenylalanine Amine Dehydrogenase

Authors: Samantha K. Au, Bettina Bommarius, Andreas Bommarius

The novel enzyme amine dehydrogenase (AmDH) derived through introduction of mutations to the phenylalanine dehydrogenase gene and thus termed F-AmDH, catalyzes the reduction of prochiral ketones to chiral amines [1]. Many desired substrates of F-AmDH show little to no solubility in aqueous media. Organic solvents can be incorporated into the reaction system to improve solubility of the hydrophobic substrates. Dehydrogenases have been scarcely employed in organic solvents because of limited stability of the enzyme in organic media and low solubility of the cofactor. The introduction of water-soluble organic solvents was unsuccessful because of enzyme deactivation in the presence of organic solvents, which led us to transition into biphasic organic solvent reaction systems. In biphasic media, the enzyme and hydrophilic cofactors are envisioned to stay in the aqueous phase while the hydrophobic substrate partitions between the phases but mostly remain in the organic phase. The advantages include a larger amount of total substrate present in the system and absence or reduced substrate and product inhibition.

Hydrophobic substrates were investigated in a variety of organic solvents. We found that for a reaction to occur, a significant amount of the hydrophobic substrate must be present in the aqueous phase. Thus, the best results are obtained with heptane as a second phase in the biphasic reaction system.

In our biphasic reaction system coupled with cofactor regeneration enzyme formate dehydrogenase, the double variant F-AmDH from Bacillus badius PheDH creates the product amine of several hydrophobic substrates that were previously unattainable due to low solubility of the ketone substrate in aqueous buffers.  An example is adamantylethyl-1-amine, obtained from adamantylmethylketone, which reacts with significant rate in biphasic but not at all in monophasic systems.

[1] Michael J. Abrahamson, John W. Wong, Andreas S. Bommarius. The Evolution of an Amine Dehydrogenase Biocatalyst for the Asymmetric Production of Chiral Amines. Adv. Synth. Catal., 2013. 355: p. 1-8.

[2] Michael J. Abrahamson, Eduardo Vazquez-Figueroa, Nicholas B. Woodall, Jeffrey C. Moore, Andreas S. Bommarius. Development of an Amine Dehydrogenase for Synthesis of Chiral Amines. Angewante Chemie Int. Ed., 2012. 51: p. 3696-3972.

[3] Mélanie Hall and Andreas S. Bommarius. Enantioenriched compounds via enzyme-catalyzed redox reactions. Chem. Rev. 2011, 111, 4088-4110