Engineering Thermostability Of Luciferase By Rational Design

Compared to traditional chemical catalysts, biocatalysts have a great number ofadvantages, which are becoming the main tools in industry. However, most of theprotein enzymes cannot maintain activity under harsh industrial conditions. There is aneed to engineering thermostatiblity of biocatalysts using protein engineeringstrategies. Photinus pyralis luciferase can catalyze a bioluminescence reaction makingitself a useful enzyme. However, its limited stability prohibits its broad applications inindustry.In this research, we attempted to engineering thermostability of luciferase througha new rational design method named RFSP (rigidify flexible sites through prolinesubstitutions). At first, molecular dynamics software Gromacs v4.5.5, geometricsimulation software FRODA and B-FITTER were used to predict the flexible regionsof luciferase, Fragment471-481and Fragment487-495. Then β-turn sequencestatistics and stereoscopic criteria of introducing proline were combined to pinpointappropriate substitution sites by prolines, D476P and H489P. S307P from a rigidregion was chosen as control. Quikchange directed mutagenesis method was appliedto mutate the WT recombinant plasmid (pET28a-luc). After inducted under lowtemperature and purified using Ni-IMAC, the WT luciferase and its mutants weresuccessfully achieved. We investigated the thermostability, kinetic parameters,bioluminescence emission spectra of luciferases. In the results, the thermostability ofH489P mutant was enhanced by about1.5fold. Its activity and emission spectra didnot change too much.In this study, a strategy named RFS to enhance thermostability of enzymes wasfirstly proposed. Using this strategy, we successfully enhanced the thermostability ofluciferase without changing its activity. RFS strategy has potential to be applied toengineer thermostability of other enzymes, which is greatly beneficial for the widerapplication of biocatalysts in industry.