Improving the Activity of Phi29 DNA Polymerase by SaProt Prediction

ABSTRACT Phi29 DNA polymerase has broad application value due to its excellent functions such as strand displacement. However, the catalytic efficiency of the natural Phi29 DNA polymerase is low, which greatly limits its application scope. This study attempts to modify Phi29 DNA polymerase using the deep prediction model SaProt to improve its catalytic efficiency. First, the 3D structure of Phi29 DNA polymerase was predicted by AlphaFold, and the reliability of the model was verified by tools such as PROCHECK. Subsequently, SaProt was used to perform saturation mutagenesis calculations on the entire sequence of Phi29 DNA polymerase, and 10 potential beneficial mutation sites were selected. Finally, 7 variants were constructed by site‐directed mutagenesis technology, and the activity of these variants were determined using random primers. The experimental results show that compared with the wild type, the activity of the A447R variant is significantly increased, and its relative activity is approximately 33 times that of the wild type. This study not only provides a new strategy for the engineering modification of Phi29 DNA polymerase, but also verifies the strong potential of the SaProt model in enzyme modification, laying a solid foundation for the development of high‐performance nucleic acid amplification tools.