Characterization, Structural Analysis, and Thermal Stability Mutation of a New Zearalenone-Degrading Enzyme Mined from Bacillus subtilis.

Zearalenone (ZEN) is a widespread mycotoxin that causes serious damage to animal husbandry and poses a threat to human health. A screen of ZEN-degrading soil bacteria yielded Bacillus subtilis YT-4, which yielded 80% ZEN degradation after 6 h and 95% after 36 h. The gene sequence encoding the degradative enzyme ZENY was mined from the genome of YT-4 and expressed in yeast. ZENY is an α/ß-hydrolase with an optimal enzyme activity at 37 °C and pH 8. By breaking the lactone ring of ZEN, it produces ZENY-C18H24O5 with a molecular weight of 320.16 g/mol. Sequence comparison and molecular docking analyses identified the catalytic ZENY triad 99S-245H-123E and the primary ZEN-binding mode within the hydrophobic pocket of the enzyme. To improve the thermal stability of the enzyme for industrial applications, we introduced a mutation at the N-terminus, specifically replacing the fifth residue N with V, and achieved a 25% improvement in stability at 45 °C. These findings aim to achieve ZEN biodegradation and provide insight into the structure and function of ZEN hydrolases.

Integrating Benzenesulfonic Acid Pretreatment and Bio-Based Lignin-Shielding Agent for Robust Enzymatic Conversion of Cellulose in Bamboo.

A hydrotrope-based pretreatment, benzenesulfonic acid (BA) pretreatment, was used to fractionate bamboo in this work. With optimized content (80 wt %) of BA in pretreatment liquor, about 90% of lignin and hemicellulose could be removed from bamboo under mild conditions (95 °C, 30 min or 80 °C, 60 min). The potential accessibility of BA pretreated substrate to cellulase was thus significantly improved and was also found to be much higher than those of acidic ethanol and dilute acid pretreatments. But the deposition of lignin on the surface of solid substrates, especially the BA pretreated substrate, was also observed, which showed a negative effect on the enzymatic hydrolysis efficiency. The addition of inexpensive soy protein, a bio-based lignin-shielding agent, could readily overcome this negative effect, leading the increase of enzymatic conversion of cellulose in BA pretreated substrate from 37% to 92% at a low cellulase loading of 4 FPU/g glucan. As compared to acidic ethanol and dilute acid pretreatments, the combination of BA pretreatment and soy protein could not only stably improve the efficiency of non-cellulose components removal, but also could significantly reduce the loading of cellulase.