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

Abstract

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/β-hydrolase with an optimal enzyme activity at 37 °C and pH 8. By breaking the lactone ring of ZEN, it produces ZENY-C 18 H 24 O 5 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.

Biodegradation Characteristics and Mechanism of Aflatoxin B1 by Bacillus amyloliquefaciens from Enzymatic and Multiomics Perspectives

“…B. amyloliquefaciens ZG08 was cultured in LB broth at 37 °C for 24 h. Whole-genome sequencing was subsequently performed by Novogene Co., Ltd. (Beijing, China), providing a reference genome for transcriptomics. The extraction, sequencing, and assembly of the genome were performed according to the methods described by Shi et al Following another 24 h of cultivation in LB broth containing 50 ng/mL AFB 1 , total RNA extraction followed by mRNA purification, quality assessment, cDNA library construction, and mRNA sequencing were carried out using the methods described by Xu et al Gene read counts were determined using HTSeq v0.6.1, and expression normalization was achieved by applying fragments per kilobase of transcript per million mapped reads (FPKM) based on gene length and read count. Differential expression analysis was then performed using the R package DESeq2, comparing the AFB 1 -exposed group to the control group.…”

A novel glycosyltransferase from Bacillus subtilis achieves zearalenone detoxification by diglycosylation modification

“…26 More and more work is focusing on the degradation of ZEN by B. subtilis , where the presence of the phosphatase ZPH in B. subtilis Y816 allows for the phosphorylation of ZEN, 27 and the degradation of ZEN by B. subtilis Y-4 occurs through the action of the enzyme ZENY, which is a member of the lactone hydrolase family. 28…”