[P002] Poster

1:00 PM - 1:50 PM JST(4:00 AM - 4:50 AM UTC)

[P01-014] Semi-rational engineering of Pyrocoelia miyako luciferase

○Phuriporn Suyanon¹, Aisaraphon Phintha¹, Pratchaya Watthaisong¹, Pimchai Chaiyen¹ (1. Vidyasirimedhi Institute of Science and Technology (VISTEC) (Thailand))

Keywords:

Firefly luciferase,Enzyme engineering,Thermostability,ATP detection

Insect luciferases such as firefly luciferase are enzymes that use D-luciferin, ATP, and oxygen as substrates to produce oxyluciferin, AMP, CO₂, pyrophosphate, and light. Luciferase from Photinus pyralis (American firefly, FLuc) is a well-known luciferase that has been extensively studied and widely applied in bioanalytical assays. However, thermostability of FLuc and its light emission wavelengths around 560 nm limit enzyme applications in vitro and in vivo. Pyrocoelia miyako luciferase (PmLuc) shares high sequence homology with FLuc but exhibits distinct properties, including higher catalytic efficiency toward D-luciferin and a longer emission half-life. We performed computational tunnel analysis of PmLuc and found that PmLuc possesses a more hydrophobic substrate access tunnel compared to FLuc. Watthaisong et al. demonstrated that PmLuc can utilize various luciferin analogues as substrates, enabling bioluminescence emission at longer wavelengths compared with the classical FLuc–D-luciferin system. We thus think that PmLuc is an attractive scaffold for further engineering. In this study, we aimed to improve thermostability of PmLuc for applications in ATP detection. Computational tools, including FireProt and PROSS were employed to predict residues likely contributing to thermostability enhancement. Site-saturation mutagenesis was subsequently applied to generate and identify variants with improved properties. Promising variants were further characterized, and mutation combinations were performed to investigate the potential synergistic effects in PmLuc. Several variants exhibited improved thermostability, and some variants showed red-shifted emission while retaining higher bioluminescence activity after heat treatment. In addition, several variants demonstrated decreased Km values along with increased enzyme half-life at various temperatures. Single mutation and combined mutation variants were constructed and characterized to obtain variants with enhanced thermostability and suitability in development of reliable ATP detection and other bioluminescent applications.