Enhanced trypsin thermostability in Pichia pastoris through truncating the flexible region

High thermostability is required for trypsin to have wider industrial applications. Target mutagenesis at flexible regions has been proved to be an efficient protein engineering method to enhance the protein thermostability. The flexible regions in porcine trypsin were predicted using the methods including molecular dynamic simulation, FlexPred, and FoldUnfold. The amino acids 78–90 was predicted to be the highly flexible region simultaneously by the three methods and hence selected to be the mutation target. We constructed five variants (D3, D5, D7, D9, and D11) by truncating the region. And the variant D9 showed higher thermostability, with a 5 °C increase in Topt, 5.8 °C rise in $$T_{50}^{10}$$ , and a 4.5 °C rise in Tm, compared to the wild-type. Moreover, the half-life value of the variant D9 was also found to be dramatically improved by 46 min. Circular dichroism and intrinsic fluorescence indicated that the structures had no significant change between the variant D9 and the wild-type. The surface hydrophobicity of D9 was measured to be lower than that of wild-type, indicating the increased hydrophobic interaction, which could have contributed to the improved thermostability of D9. These results showed that the thermostability of variant D9 was increased. The variant D9 could be expected to be a promising tool enzyme for its wider industrial applications. The method of truncating the flexible region used in our study has the potential to be used for enhancing the thermostability of other proteins.