Abstract: One major problem in the field applications of Bacillus thuringiensis Cry toxins is that their exposure to sunlight can bring about reduced bioactivity, mainly because of the most vulnerable indole ring of tryptophan(W) residue. W544 is the unique tryptophan in the domain Ⅲ of Cry1Ac toxin, which forms part of a buried hydrophobic cluster involving the conserved F578 and F604, and are organized in a “propeller-like” pattern, playing a clear structural role in protein stability. In this study, W544 was conservatively substituted with phenylalanine(F) to determine whether its stability was affected in the resulting mutant. Comparative analysis results by SDS-PAGE show that the protoxin of W544F was more stable than the wild-type Cry1Ac, when treated with ultraviolet irradiation, trypsin and preserved at room temperature. The distance between two vertex of crystals of W544F were 0.6 μm longer and unsmooth when compared with that of the wild-type Cry1Ac under an atomic force microscope. Addionally the mutation W544F had similar insecticidal activity to wild-type Cry1Ac, but when treated with ultraviolet irradiation for 9 h, it still maintained more than 4 times higher toxicity against Helicoverpa armigera than the wild-type Cry1Ac. In conclusion, the W544F mutation can enhance the stability of the Cry1Ac protein and may contribute to solving the major problem of the field applications of Cry1Ac toxin.

Key words:

Cry1Ac protein, Site-directed mutagenesis, Tryptophan, Phenylalanine, Stability