ObjectivesTo improve the thermostability and organic solvent tolerance of L-phenylserine aldolase, the in vivo SpyTag/SpyCatcher cyclization strategy was applied in this work.ResultsThe in vivo cyclization of L-phenylserine aldolase was achieved by fusing the tags of SpyCatcher and SpyTag to the N- and C-termini of the enzyme, respectively. The k(cat) values and the circular dichroism spectra of the linear and cyclized LPAs are very similar, indicating that the cyclized LPA can be folded appropriately like the wild type. The cyclized enzyme has better thermostability and organic solvent tolerance than does the wild type. The half-life of L-phenylserine aldolase after cyclization was increased by 8.3 times at 70 degrees C, and the T-50 also increased from 56.8 to 67.1 degrees C. The cyclized enzyme showed a remarkably higher tolerance to organic solvents (e.g., methanol, ethanol and acetone).ConclusionsThese results suggest that the in vivo cyclization using SpyTag/SpyCatcher is an effective strategy to improve the stability of enzymes, which potentially could be applied in industrial bioconversion.