Directed evolution of a thermostable phosphite dehydrogenase for NAD(P)H regeneration.
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 T50 is 20 degrees C higher and half-life of thermal inactivation at 45 degrees C is >7,000-fold greater than that of the parent PTDH. The engineered PTDH has a half-life at 50 degrees 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.
Johannes
Tyler W
TW
Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, 600 S. Mathews Ave., Urbana, IL 61801, USA. zhao5@uiuc.edu
Woodyer
Ryan D
RD
Zhao
Huimin
H
D019943:Amino Acid Substitution; D019020:Directed Molecular Evolution; D004795:Enzyme Stability; D006358:Hot Temperature; D007218:Industrial Microbiology / Q000379:methods; D008958:Models, Molecular; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D009249:NADP / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D015202:Protein Engineering / Q000379:methods; D044223:Pseudomonas stutzeri / Q000201:enzymology
D009249:NADP; D009247:NADH, NADPH Oxidoreductases; C429539:NAD phosphite oxidoreductase