Improving the Stability of Formate Dehydrogenase（LeFDH）by Site-Directed Mutagenesis

• 1、School of Bioengineering, Jiangnan University, Wuxi,Jiangsu 214000

Abstract：Formate dehydrogenase (FDH), which can reduce NAD+ to NADH using low cost substrate, is one of the most promising cofactor regenerating enzymes for industrial applications. However, the popularity in commercial processes of native FDHs are limited by its weaknesses in catalytic efficiency and operational stability. In this work, the three-dimensional model of Lodderomyces elongisporus FDH(LeFDH) was created by homology modeling, and the active center as well as the binding sites of cofactor and substrate were predicted. In order to improve the thermal stability, four amino acid residues on the loops which are far away from the active center, were mutated by a comprehensive strategy of molecular dynamics simulations and multiple sequence alignments. The results of enzymatic property analyses showed that the binary mutant K21PK22R obtained the greatest improvement in thermal stability, which can retain more than 80% of enzyme activity after 60 minites incubation at 65 C, while wild-type LeFDH was completely inactivated by incubation up to 30 minites. This study lays the foundation for further protein engineering of LeFDH, and the mutantsobtained in this work increase the potential of LeFDH for cofactor regeneration.

Keywords： Molecular Biology Formate Dehydrogenase Site-Directed Mutagenesis Stability