LAUSR

O‐Succinyl‐L‐homoserine mercaptotransferase (MetZ) is a key enzyme for the synthesis of L‐methionine. However, the limited tolerance to high concentrations of substrate methyl mercaptan sodium restricts its industrial applications, while the “trade‐off” between activity and stability has always been a challenge for protein engineering. To address this problem, a strategy of dynamic network rigidification‐local flexibility regulation was applied in this study to engineer the enzyme from Chromobacterium violaceum (CvMetZ). Through consensus sequence analysis and structure‐guided design, the hotspot amino acid residues were anchored for mutagenesis. The best mutant T59I/T119Y/A241T was obtained with synergistic enhancement of activity and stability. The half‐life (t1/2) of whole cells harboring the mutant was increased from 3.3 and 1.3 h to 20.6 and 7.2 h, in the presence of 5% and 10% methyl mercaptan sodium, respectively. The engineered enzyme was successfully immobilized with ideal usability, which laid the foundation for efficient and scale‐up bioproduction of L‐methionine.