Engineering a thermophilic luciferase variant from Photuris pennsylvanica into a mesophilic-like enzyme for expanded applications potential

Luciferase, known for its exceptional catalytic bioluminescent properties, has been widely utilized in diverse applications within biotechnology and medical research. Currently, enhancing thermostability and catalytic activity is a primary focus for optimizing luciferase modifications to further expand its detection range and accuracy. This study revealed a highly thermostable luciferase variant from Photuris pennsylvanica, Ppe146-1H2, which inherently exhibits thermophilic enzyme characteristics that are not conducive for optimal catalytic performance in practical applications. Building upon structural analysis, this research engineered Ppe146-1H2 into Ppe146-LGR via the residue substitutions I422L, D435G, and I519R. Ppe146-LGR retained notably thermostability, exhibiting a melting temperature (Tm value) of 75.3 ± 0.3 °C. Additionally, the variant demonstrated efficient catalytic activity at moderate temperatures, exhibiting 3.8 and 3.7-fold higher catalytic efficiencies towards D-luciferin and ATP at 37 °C compared to Ppe146-1H2. Overall, Ppe146-LGR displayed mesophilic-like catalytic activity and thermophilic-like thermostability simultaneously. In addition to enhanced catalytic properties, Ppe146-LGR emitted longer-wavelength light (580 nm) and operated optimally at near-neutral pH, coordinating with the current demands of luciferase applications. Through validation via rapid bacterial detection and reporter gene assays, it has been demonstrated that Ppe146-LGR holds promise as a valuable tool in the field of bioluminescence technology.