Increase in Enzymatic Activity of Firefly Luciferase by Site-directed Mutagenesis

• 1、School of Food Science and Technology,Jiangnan University,Jiangsu Wuxi 214122

Abstract：In this study, the wild-type North American firefly luciferase Luc encoding gene luc was undertaken site-directed saturation mutagenesis, in an attempt to improve the Luc enyzmatic activity. The mutation point is located at the junction of the N-terminal region and the flexible linker (residue 423), inside the flexible linker (residue 436) and at the C-terminal domain (residue 530), respectively. The mutant luciferase Luc1.1 against these three active sites was constructed, and on the basis of these three sites, saturated mutants were constructed respectively and a total of 57 mutants were obtained. The effects of various mutated amino acid residues on the luciferase activity were investigated. Eight mutants with more than 10-fold higher enzymatic activity to the wild type Luc were screened. The results showed that the activity of Luc could be increased by replacing the amino acid residues 423, 436 and 530 with amino acids with non-polar side chains, amino acids with small steric hindrance of side chains and amino acids with positive charges, respectively. The best mutants obtained in this study had 20-fold higher enzymatic activities than the wild-type.

Keywords： firefly luciferase site-directed saturation mutagenesis aminoacids enzymatic activity