(442b) Development of a Phosphite Dehydrogenase-Based Nicotinamide Cofactor Regeneration System

NAD(P)H-dependent
oxidoreductases are valuable tools for synthesis of chiral compounds. The
expensive cost of the cofactors, however, requires in situ cofactor
regeneration for preparative applications. We have attempted to develop an
enzymatic system based on phosphite dehydrogenase (PTDH) from Pseudomonas stutzeri
to regenerate the reduced nicotinamide cofactors NADH and NADPH. We used directed
evolution to address one of the main limitations with the wild-type PTDH
enzyme, its low stability. After three rounds of random mutagenesis and high
throughput screening, twelve thermostable amino acid substitutions were
identified. These twelve mutations were combined by site-directed mutagenesis,
resulting in a mutant whose T₅₀ is 20°C higher and half-life
of thermal inactivation at 45°C is >7000-fold greater than that of the
parent PTDH. The engineered PTDH has a half-life at 50°C that is 2.4-fold
greater than the Candida boidinii formate dehydrogenase (FDH), an enzyme
widely used for NADH regeneration.  The improved stability and effectiveness of
the thermostable PTDH mutant was shown using the industrially important
bioconversion of trimethylpyruvate to L-tert-leucine. Site-directed
mutagenesis was also used to incorporate a cofactor specificity mutation (A176R)
identified in previous work into the thermostable PTDH construct to create a
powerful new NADPH regenerating enzyme.  Several regeneration reactions
were selected and conducted in small-scale batch reactions and in an enzyme
membrane reactor to evaluate the capabilities of the PTDH mutants.  The engineered PTDH will be useful in NAD(P)H regeneration
for industrial biocatalysis.