Rational Design of Spontaneous Self-Cyclization Enzymes In Vivo and In Vitro with Improved Thermal Tolerance and Activity

Enzymes have selectivity, require mild catalytic conditions, and are important cornerstones in many industrial catalytic processes. Protein self-cyclization has opened up the possibility of preserving fragile enzymes during long-term high-temperature catalysis. However, the mechanism for self-cyclization and improvement of thermal tolerance have not been elucidated, severely limiting their industrial applications. Herein, we provide a strategy for the rational design of fusion proteins based on structural analysis to obtain cyclized enzymes with improved properties. First, we constructed fusion proteins that preferentially translated SpyCatcher (CBT) or SpyTag (TBC), both of which could form stable single self-cyclization with significantly improved thermal tolerance. Especially, the thermal half-life of TBC obtained by modifying the N-terminal SpyTag at 40 °C was 10.83 times that of wild enzymes. Structural analysis revealed that the terminus of the protein, which preferentially translated to SpyCatcher, folded into a conformation that adversely affected stability. In addition, the structure of the catalytic pocket and the orientation of the catalytic residues of CBT were significantly different from those of the wild-type enzymes, which led to a decrease in the catalytic activity. These conclusions were confirmed when another industrial enzyme, sucrose phosphorylase, was cyclized. Finally, the cyclized glucosidase was also superior to the wild-type strain for the preparation of ginsenoside F1 at high titers and as a whole-cell catalyst for extended use. In conclusion, we demonstrated for the first time that conjugated long oligopeptide SpyCatcher significantly affected the catalytic activity and stability of cyclized enzymes. It was necessary to preferentially translate units with less steric hindrance to reduce their impact on the protein structure. The rational design of cyclized enzymes based on structural analysis provides a simple and effective strategy for the modification of industrial enzymes with poor thermal tolerance, providing considerable prospects for biosynthesis in vivo and in vitro .