Sustainable Production of Ferulic Acid and Prebiotic Sugars via Spore-Displayed Bifunctional Enzymes Purified through Integrated ELP-Intein Strategies.

The His-ELP-intein tag, containing His-tag, intein, and elastin-like polypeptide (ELP), and the ELP-intein tag, containing intein and ELP, were designed to fuse with bifunctional xylanase/feruloyl esterase XynII-Fae to generate recombinant proteins His-ELP-intein-XynII-Fae and ELP-intein-XynII-Fae, respectively. XynII-Fae was purified via nonchromatographic strategies and covalently cross-linked onto the surface of Bacillus subtilis 168 trpC2 spores, yielding Sp-G-XynII-Fae. Compared with the free enzyme, Sp-G-XynII-Fae exhibited enhanced stability and catalytic performance. Additionally, Sp-G-XynII-Fae showed remarkable reusability and storage stability. It maintained 67%-73% of its initial activity after 10 reuse cycles and retained approximately 60% residual activity after storage at 4 °C for 35 days. Furthermore, Sp-G-XynII-Fae showed outstanding catalytic activity in the hydrolysis of delignified corn stover, wheat straw, and sugarcane bagasse, effectively releasing ferulic acid and reducing sugars. These results highlight the great potential of Sp-G-XynII-Fae for applications in agriculture, food technology, biosciences, and related industries.