Zhang_2024_Int.J.Biol.Macromol_269_132082

Feruloyl esterase has a wide range of applications, but there are still problems with low enzyme yield and activity, and complex purification steps. Our previous research found Lactobacillus amylovorus feruloyl esterase could be secreted extracellular in Escherichia coli. In this study, multiple strategies were implemented to maximize the extracellular production of feruloyl esterase with improved activity in E. coli. Firstly, codon-optimized feruloyl esterase was obtained based on the preference of E. coli, resulting in 41.97 % increase in extracellular secretion. Furthermore, by cascading T7 promoters, replacing the 5' UTR, randomly mutating the N-terminal sequence, and co-expressing secretory cofactors, the extracellular secretion was increased by 36.46 %, 31.25 %, 20.66 % and 25.75 %, respectively. Moreover, the feruloyl esterase were mutated to improve the substrate affinity and activity. The catalytic efficiency of Fae-Q134T and Fae-Q198A increased by 4.62-fold and 5.42-fold. Combining above strategies, extracellular feruloyl esterase activity was increased from 2013.70 U/L to 10,349.04 U/L. These results indicated that the activity and yield of feruloyl esterase secreted by E. coli were significantly increased, which laid a foundation for its industrial application.