Cloning and Characterization of GDSL Esterases from Bacillus paralicheniformis T7

Esterases catalyze the hydrolysis and transesterification of short-chain fatty acid esters, and microbial esterases are used in the production of biofuels, cosmetics, food, and pharmaceuticals. The soil strain Bacillus paralicheniformis T7 secretes enzymes with esterase activity; however, many bacterial enzymes remain insufficiently studied. Therefore, this study aimed to identify and characterize novel GDSL esterases produced by B. paralicheniformis. Protein mass spectrometry, combined with proteomics and genomics, identified genes encoding two GDSL esterases, which were cloned into the pET-28c(+) vector. The resulting proteins were obtained in Escherichia coli BL21(DE3) as the recombinant esterases rEST-24 and rEST-28. These recombinant GDSL esterases showed maximum activity at 40 °C and pH 7.0. Moreover, Ca²⁺, Zn²⁺, Cu²⁺, and Fe²⁺ ions inhibited their activity, and rEST-28 was resistant to the detergents Tween-20, Tween-80, and Triton X-100. High-yield esterase activity was detected in bacteria cultured on feather medium and nutrient broth, and submerged fermentation of the B. paralicheniformis T7 strain on feather medium enabled the production of an esterase extract exhibiting activity of 17,618 ± 610 U/g. These results suggest that the B. paralicheniformis T7 strain can produce esterases and shows promising potential for application in technologies that degrade fatty acid esters using hydrolytic enzymes.