Rational Substitution of Firefly Luciferase from Photinus Pyralis for Improvement of Thermostability

Firefly luciferase is widely used in many areas of biotechnology and molecular biology, but the application is restricted due to its poor thermal stability. Random mutagenesis and directed evolution paly an important role in the thermal stability of protein; nevertheless, large number of experimental attempts appear to be time- and labor-consuming. In this paper, to enhance the thermal stability of firefly luciferse, we got two mutants (E325K, E354K) by reversing the charges of individual residues at active sites with opposite interaction energies on the basis of molecular dynamic (MD) simulation. Root-mean-square-deviation(RMSD) and radius of gyration were used to evaluate the thermostability of the mutants. The residual activities of wide-type luciferase, E325K, E354K were 4.52%,7.22% and 30%, respectively, after being heated at 37 ℃ for 5 h, which showed the same trends with the computational predictions (E354K > E325K > WT). It was worth nothing that the T₅₀ and T_m of E354K were 4.45 and 3.2 ℃ higher, respectively, than that of the native luciferase. Rational substitution of residue in regulation of thermostability using MD simulation would be a faster, less resource-intensive and more effective than random mutagenesis and directed evolution for instructional design to enhance thethermostability.