NAD(P)H-dependent oxidoreductases are valuable tools for synthesis of chiral compounds. The expense 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. Here we report the use of 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, 12 thermostabilizing amino acid substitutions were identified. These 12 mutations were combined by site-directed mutagenesis, resulting in a mutant whose T 50 is 20°C higher and half-life of thermal inactivation at 45°C is >7,000-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, an enzyme widely used for NADH regeneration. In addition, its catalytic efficiency is slightly higher than that of the parent PTDH. Various mechanisms of thermostabilization were identified using molecular modeling. The improved stability and effectiveness of the final mutant were shown using the industrially important bioconversion of trimethylpyruvate to L-tert-leucine. The engineered PTDH will be useful in NAD(P)H regeneration for industrial biocatalysis.The potential of enzyme-catalyzed transformations in the pharmaceutical and fine-chemical industries has long been considered to have great promise (7,10,23). Despite the development of rapid and efficient methods for creating designer biocatalysts, inherent limitations in the chemistry that these enzymes perform still persist. The majority of biocatalysts used in industry are hydrolytic in action (ϳ65%) and perform rather simple chemistry (4). More complicated enzymatic reactions often require one or more costly cofactors, which, when added in stoichiometric quantities, make the process not economically feasible. Oxidoreductases, for example, catalyze a myriad of regio-, chemo-, and stereospecific reactions on a variety of functional groups, but often require either NADH or NADPH as a cofactor (8, 11).Various in situ regeneration methods have been developed to allow the use of catalytic quantities of NAD(P) ϩ and NAD(P)H (20). The most successful and widely used enzymatic NADH regeneration system is based on the Candida boidinii formate dehydrogenase (FDH) (1). We have attempted to develop an NADH regeneration process using the enzyme phosphite dehydrogenase (PTDH) from Pseudomonas stutzeri (2). This enzyme catalyzes the nearly irreversible oxidation of phosphite to phosphate with the concomitant reduction of NAD ϩ to NADH. The kinetic and practical advantages of using PTDH for NADH regeneration have been recently reported (22).In addition to NADH regeneration, the enzyme PTDH also has considerable potential in regenerating NADPH. The cost of NADPH is significantly higher than that of NADH, and no widel...