Enhanced thermostability of formate dehydrogenase via semi-rational design

Abstract

An NAD+-dependent formate dehydrogenase from Lodderomyces elongisporus (LeFDH) was expressed in Escherichia coli and then purified by nickel affinity chromatography. The analysis of enzymatic properties showed that the specific activity of LeFDH was much high (16.26 U·mg–1), while the thermal stability may be insufficient for industrial application. To further enhance the thermal stability by semi-rational design, sequences alignment, homologous modeling and molecular dynamic simulations were conducted. As a result, seven non-conserved amino acid residues far from the active center were selected for site-directed mutagenesis. Several mutants as well as a binary mutant displayed improved thermal stability and unreduced specific activity. Then temperature and pH stability, kinetic parameters and specific activity, and structural characteristics of wild-type and mutant LeFDH were compared and evaluated. The variant K21P/K22R exhibited an increased half-life by 211% at 60 °C and a 4.5 °C higher Tm than those of wild type, and good stability in alkaline environment. This binary mutant enhances the operational stability of LeFDH in the catalytic reaction and thus improves its potential for application in coenzyme regenerations.