(26f) 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