Screening of the candidate inhibitory peptides of subtilisin by in vitro RNA display technique

The application of inhibitors facilitates the stable preservation of enzyme in liquid detergent by mitigating the proteolytic activity of subtilisin. The conventionally used subtilisin inhibitors such as boric acid pose a threat to the environment and human health. Thus, the formulation of novel subtilisin inhibitors demands immediate attention. In the current study, we have screened the peptide inhibitors for subtilisin by employing the in vitro mRNA display technique. It is a sensitive screening technique with a high library capacity. The affinity screening was performed between the biotin-modified subtilisin immobilized on the streptavidin magnetic beads and the cDNA-mRNA-peptide fusion molecular library acquired from the in vitro translation and reverse transcription. The candidate peptides with high affinity were obtained after multiple rounds of screening. Furthermore, the inhibitory effect was evaluated, showing that some candidate peptides had inhibitory effects, but the isothermal titration calorimetry and time dependent experiments ultimately proved that these candidate peptides were not stable inhibitors. However, the in vitro mRNA display method explored in this study can be used as a preliminary screening method to provide candidate peptides for the screening of subtilisin inhibitors.