Zearalenone Decreases Food Intake by Disrupting the Gut-Liver-Hypothalamus Axis Signaling via Bile Acids.

Zearalenone (ZEN) is a mycotoxin that is harmful to humans and animals. In this study, female and male rats were exposed to ZEN, and the results showed that ZEN reduced the farnesoid X receptor (FXR) expression levels in the liver and disrupted the enterohepatic circulation of bile acids (BAs). A decrease in food intake induced by ZEN was negatively correlated with an increase in the level of total BAs. BA-targeted metabolomics revealed that ZEN increased glycochenodeoxycholic acid levels and decreased the ratio of conjugated BAs to unconjugated BAs, which further increased the hypothalamic FXR expression levels. Preventing the increase in total BA levels induced by ZEN via Lactobacillus rhamnosus GG intervention restored the appetite. In conclusion, ZEN disrupted the enterohepatic circulation of BAs to decrease the level of food intake. This study reveals a possible mechanism by which ZEN affects food intake and provides a new approach to decrease the toxic effects of ZEN.

Heme Oxygenase-1 Regulates Zearalenone-Induced Oxidative Stress and Apoptosis in Sheep Follicular Granulosa Cells.

Zearalenone (ZEA) is a common non-steroidal estrogenic mycotoxin found in a range of animal feeds and poses a serious threat to the reproductive health of farm animals and humans. However, the mechanism underlying ZEA-induced reproductive toxicity in sheep remains unknown. Granulosa cells are crucial for egg maturation and the fertility of female sheep. In this study, we aimed to examine the impact of different ZEA concentrations on sheep follicular granulosa cells and to elucidate the potential molecular mechanism underlying ZEA-induced toxicity using transcriptome sequencing and molecular biological approaches. Treating primary sheep follicular granulosa cells with different concentrations of ZEA promoted the overproduction of reactive oxygen species (ROS), increased lipid peroxidation products, led to cellular oxidative stress, decreased antioxidant enzyme activities, and induced cell apoptosis. Using transcriptome approaches, 1395 differentially expressed genes were obtained from sheep follicular granulosa cells cultured in vitro after ZEA treatment. Among them, heme oxygenase-1 (HMOX1) was involved in 11 biological processes. The protein interaction network indicated interactions between HMOX1 and oxidative and apoptotic proteins. In addition, N-acetylcysteine pretreatment effectively reduced the ZEA-induced increase in the expression of HMOX1 and Caspase3 by eliminating ROS. Hence, we suggest that HMOX1 is a key differential gene involved in the regulation of ZEA-induced oxidative stress and apoptosis in follicular granulosa cells. These findings provide novel insights into the prevention and control of mycotoxins in livestock.

Caspase-8 dependent apoptosis contributes to dyskinesia caused by muscle defects and neurotoxicity in zebrafish exposed to zearalenone.

Zearalenone (ZEA), one of the usual mycotoxins, has been recognized in many areas and crops, posing a significant threat to the living organisms even to human beings. However, the mechanisms of locomotive defects remain unknown. Herein, zebrafish larvae was employed to investigate ZEA effects on developmental indexes, muscle and neural toxicity, apoptosis, transcriptome and motor behaviors of zebrafish larvae. Zebrafish larvae exposed to ZEA (0, 0.5, 1, 2 and 4 µM) showed no change in survival rate, but the malformation rate of zebrafish larvae increased dramatically manifesting with severe body bending and accomplished with adverse effects on hatching rate and body length. Moreover, the larvae manifested with defective muscle and abnormal neural development, resulting in decreased swimming ability, which probably due to the abnormal overactivation of apoptosis. And this was confirmed by enriched caspase 8-mediated apoptosis signaling pathway in the following transcriptome analysis. Meanwhile, there was a recovery in swimming behaviors in the larvae co-exposed in ZEA and caspase 8 inhibitor. These findings provide an important evidence for risk assessment and potential treatment target of ZEA exposure.

Zearalenone Promotes Uterine Hypertrophy through AMPK/mTOR Mediated Autophagy.

Zearalenone (ZEN), a non-steroidal Fusarium graminearum with an estrogen effect, can cause damage to the gastrointestinal tract, immune organs, liver, and reproductive system. Further analysis of the mechanism of ZEN has become an important scientific issue. We have established in vivo and in vitro models of ZEN intervention, used AMPK/mTOR as a targeted pathway for ZEN reproductive toxicity, and explored the molecular mechanism by which ZEN may induce uterine hypertrophy in weaned piglets. Our study strongly suggested that ZEN can activate the phosphorylation of AMPK in uterine endometrial epithelium cells, affect the phosphorylation level of mTOR through TSC2 and Rheb, induce autophagy, upregulate the expression of proliferative genes PCNA and BCL2, downregulate the expression of apoptotic gene BAX, promote uterine endometrial epithelium cells proliferation, and ultimately lead to thickening of the endometrial and myometrium, increased density of uterine glands, and induce uterine hypertrophy.

Enhancing the activity of zearalenone lactone hydrolase toward the more toxic α-zearalanol via a single-point mutation.

Zearalenone (ZEN) and its derivatives are estrogenic mycotoxins known to pose significant health threats to humans and animals. Especially, the derivative α-zearalanol (α-ZAL) is over 10 times more toxic than ZEN. Simultaneous degradation of ZEN and its derivatives, especially α-ZAL, using ZEN lactone hydrolases (ZHDs) is a promising solution to eliminate their potential hazards to food safety. However, most available ZHDs exhibit limited activity toward the more toxic α-ZAL compared to ZEN. Here, we identified a broad-substrate spectrum ZHD, named ZHDAY3, from Exophiala aquamarina CBS 119918, which could not only efficiently degrade ZEN but also exhibited 73% relative activity toward α-ZAL. Through rational design, we obtained the ZHDAY3(N153H) mutant, which exhibited the highest specific activity (253.3 ± 4.3 U/mg) reported so far for degrading α-ZAL. Molecular docking, structural comparative analysis, and kinetic analysis collectively suggested that the shorter distance between the side chain of the catalytic residue His242 and the lactone bond of α-ZAL and the increased binding affinity to the substrate were mainly responsible for the improved catalytic activity of ZHDAY3(N153H) mutant. This mechanism was further validated through additional molecular docking of 18 mutants and experimental verification of six mutants.IMPORTANCEThe mycotoxins zearalenone (ZEN) and its derivatives pose a significant threat to food safety. Here, we present a highly promising ZEN lactone hydrolase (ZHD), ZHDAY3, which is capable of efficiently degrading both ZEN and the more toxic derivative α-ZAL. Next, the ZHDAY3(N153H) mutant obtained by single-point mutation exhibited the highest specific activity for degrading α-ZAL reported thus far. We further elucidated the molecular mechanisms underlying the enhanced hydrolytic activity of ZHDAY3(N153H) toward α-ZAL. These findings represent the first investigation on the molecular mechanism of ZHDs against α-ZAL and are expected to provide a significant reference for further rational engineering of ZHDs, which will ultimately contribute to addressing the health risks and food safety issues posed by ZEN-like mycotoxins.

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.

Identification of the hub genes linked to zearalenone-induced hepatotoxicity in broiler chickens.

Zearalenone (ZEN) is a mycotoxin found in food and feed that impairs the function of multiple organs, especially the liver. However, the specific mechanisms through which ZEN induces liver damage in broiler chickens are not well understood. Therefore, this study aimed to identify the key genes linked to the hepatotoxicity induced by ZEN exposure in broiler chickens. Gene expression data from ZEN-treated and control chicken embryo primary hepatocytes (CEPHs) were used to implement differential expression analysis. Totally, 436 differentially expressed genes (DEGs) were detected, in which 223 and 213 genes were up- and down-regulated in ZEN-treated CEPHs, respectively. Gene ontology analysis suggested that these DEGs were involved in various biological processes, including chromosome segregation, mitotic cytokinesis, mitotic cell cycle, cell division, and mitotic spindle organization. Pathway analysis showed that the DEGs were associated with p53, FoxO, ubiquitin-mediated proteolysis, cell cycle, and mismatch repair signaling pathways. Furthermore, the hub genes, including BRCA1, CDC45, CDCA3, CDKN3, CENPE, CENPF, CENPI, CENPM, CENPU, and CEP55, potentially contributed to ZEN-induced hepatotoxicity. In conclusion, our study provides the valuable insight into the mechanism underlying ZEN-induced hepatotoxicity in broiler chickens.

Quercetin Attenuates the Combined Effects of Zearalenone and Lipopolysaccharide on IPEC-J2 Cell Injury through Activating the Nrf2 Signaling Pathway.

Zearalenone (ZEA) is a mycotoxin with an estrogen-like effect that is widely found in feed. Lipopolysaccharides (LPS) derived from Gram-negative bacteria are a common endotoxin, and both toxins have effects on human and livestock health. During animal feeding, ZEA as an exotoxin and LPS as an endotoxin have the potential to co-exist in organisms. At present, other studies have only focused on the hazards of single toxins, but there are fewer studies on the coexistence and interaction between ZEA and LPS. Therefore, a further study to investigate the combined toxic effects of different concentrations of ZEA and LPS is warranted. Quercetin (QUE) is a natural flavonoid compound with strong antioxidant and anti-inflammatory properties. It is unclear whether QUE can mitigate the combined effects of ZEA and LPS. IPEC-J2, isolated from the jejunum of non-breastfed neonatal piglets, is an ideal model for the study of epithelial cell transport, intestinal bacterial interactions, and the nutrient modulation of intestinal function. Therefore, the purpose of the present study was to demonstrate the effect of QUE in alleviating the combined toxic effect of ZEA and LPS on IPEC-J2 cell damage. Cell viability was measured after treating IPEC-J2 cells sequentially with 10, 20, 30, 40, 60, 80, and 100 µM ZEA, 1, 10, 50, and 100 µg/mL LPS, and 20, 40, 60, 80, 100, and 200 µM QUE for 24 h. Based on the cell viability results, 20 µM ZEA and 1 µg/mL LPS were selected as the most suitable concentrations for further analysis. For QUE, 20 µM increased the cell viability, while 40-200 µM QUE decreased the cell viability. Therefore, for the subsequent study, 20 µM QUE was selected in combination with 20 µM ZEA and 1 µg/mL LPS. The results showed that QUE increased the cellular viability and decreased the LDH content more compared to the effects of the ZEA+LPS group. At the gene level, QUE addition up-regulated the expression of Nrf2, HO-1, SOD2, and NQO1 at the gene or protein level compared to those of the ZEA+LPS group. The measurement of tight junction-related genes and proteins showed QUE up-regulated the expression of Claudin, ZO-1, and Occludin genes and proteins more than in the ZEA+LPS group. QUE addition reduced the rate of apoptosis more than that in the ZEA+LPS group. The expressions of Bcl-2 and Bax were examined at the gene level, and QUE addition significantly reduced the Bax gene expression level compared to that of the ZEA+LPS group, but there was no apparent variation in the expression level of Bcl-2. In summary, QUE can alleviate the combined toxic effects of ZEA and LPS on IPEC-J2 cells via modulating the Nrf2 signaling pathway, up-regulating the expression of antioxidative genes, and enhancing the intestinal barrier.

A Hydrolase Produced by Rhodococcus erythropolis HQ Is Responsible for the Detoxification of Zearalenone.

Zearalenone (ZEN), an estrogenic mycotoxin, is one of the prevalent contaminants found in food and feed, posing risks to human and animal health. In this study, we isolated a ZEN-degrading strain from soil and identified it as Rhodococcus erythropolis HQ. Analysis of degradation products clarified the mechanism by which R. erythropolis HQ degrades ZEN. The gene zenR responsible for degrading ZEN was identified from strain HQ, in which zenR is the key gene for R. erythropolis HQ to degrade ZEN, and its expression product is a hydrolase named ZenR. ZenR shared 58% sequence identity with the hydrolase ZenH from Aeromicrobium sp. HA, but their enzymatic properties were significantly different. ZenR exhibited maximal enzymatic activity at pH 8.0-9.0 and 55 °C, with a Michaelis constant of 21.14 µM, and its enzymatic activity is 2.8 times that of ZenH. The catalytic triad was identified as S132-D157-H307 via molecular docking and site-directed mutagenesis. Furthermore, the fermentation broth of recombinant Bacillus containing ZenR can be effectively applied to liquefied corn samples, with the residual amount of ZEN decreased to 0.21 µg/g, resulting in a remarkable ZEN removal rate of 93%. Thus, ZenR may serve as a new template for the modification of ZEN hydrolases and a new resource for the industrial application of biological detoxification. Consequently, ZenR could potentially be regarded as a novel blueprint for modifying ZEN hydrolases and as a fresh resource for the industrial implementation of biological detoxification.

Mining Lactonase Gene from Aflatoxin B1-Degrading Strain Bacillus megaterium and Degrading Properties of the Recombinant Enzyme.

Mycotoxins are toxic secondary metabolites mainly produced by filamentous fungal species that commonly contaminate food and feed. Aflatoxin B1 (AFB1) is extremely toxic and seriously threatens the health of humans and animals. In this work, the Bacillus megaterium HNGD-A6 was obtained and showed a 94.66% removal ability of AFB1 by employing extracellular enzymes as the degrading active substance. The degradation products were P1 (AFD1, C16H14O5) and P2 (C14H16N2O2), and their toxicity was greatly reduced compared to that of AFB1. The AttM gene was mined by BlastP comparison and successfully expressed in Escherichia coli BL21. AttM could degrade 86.78% of AFB1 at pH 8.5 and 80 °C, as well as 81.32% of ochratoxin A and 67.82% of zearalenone. The ability of AttM to degrade a wide range of toxins and its resistance to high temperatures offer the possibility of its use in food or feed applications.

Identification and Modification of Enzymatic Substrate Specificity through Residue Alteration in the Cap Domain: A Thermostable Zearalenone Lactonase.

Zearalenone (ZEN) and its derivatives are prevalent contaminants in cereal crops. This study investigated a novel thermostable ZEN lactonase (ZENM) from Monosporascus sp. GIB2. ZENM demonstrated its highest activity at 60 °C, maintaining over 90% relative activity from 50 to 60 °C. Notably, efficient hydrolysis of ZEN and its two derivatives was achieved using ZENM, with specific activities of 333 U/mg for ZEN, 316 U/mg for α-zearalenol (α-ZOL), and 300 U/mg for α-zearalanol (α-ZAL). The activity of ZENM toward α-ZOL is noteworthy as most ZEN lactonases rarely achieve such a high degradation rate of α-ZOL. Based on the sequence-structure analysis, five residues (L123, G163, E171, S199, and S202) conserved in other ZEN lactonases were substituted in ZENM. Of interest was the G163S mutant in the cap domain that displayed enhanced activity toward α-ZOL compared to the wild-type enzyme. Notably, the mutant G163S exhibited higher catalytic activity toward α-ZOL (kcat/Km 0.223 min-1 µM-1) than ZEN (kcat/Km 0.191 min-1 µM-1), preferring α-ZOL as its optimum substrate. In conclusion, a thermostable ZEN lactonase has been reported, and the alteration of residue G163 in the cap domain has been shown to modify the substrate specificity of ZEN lactonase.

Smart-seq2 Technology Reveals a Novel Mechanism That Zearalenone Inhibits the In Vitro Maturation of Ovine Oocytes by Influencing TNFAIP6 Expression.

Zearalenone (ZEN), a non-steroidal estrogenic fungal toxin widely present in forage, food, and their ingredients, poses a serious threat to animal and human reproductive health. ZEN also threatens ovine, a major source of human food and breeding stock. However, the mechanisms underlying the impact of ZEN on the in vitro maturation (IVM) of ovine oocytes remain unclear. This study aimed to elucidate these mechanisms using the Smart-seq2 technology. A total of 146 differentially expressed genes were obtained, using Smart-seq2, from sheep oocytes cultured in vitro after ZEN treatment. ZEN treatment inhibited RUNX2 and SPP1 expression in the PI3K signaling pathway, leading to the downregulation of THBS1 and ultimately the downregulation of TNFAIP6; ZEN can also decrease TNFAIP6 by reducing PTPRC and ITGAM. Both inhibit in vitro maturation of ovine oocytes and proliferation of cumulus cells by downregulating TNFAIP6. These findings provide data and a theoretical basis for elucidating ZEN's toxicity mechanisms, screening therapeutic drugs, and reducing ZEN-related losses in the ovine industry.

Zearalenone promotes porcine ESCs apoptosis by enhancing Drp1-mediated mitochondrial fragmentation and activating the JNK pathway.

Zearalenone (ZEA) is widely present in food and feed, and pigs are susceptible to its effects. This study explored the underlying function of ZEA-induced apoptosis in porcine endometrial stromal cells (ESCs) through activation of the JNK signaling pathway and mitochondrial division. This study utilized ESCs to explore the impact of exposure to ZEA. A mitochondrial division inhibitor (Mdivi) was also included as a reference. The results indicated a gradual decrease in cell viability with increasing ZEA concentration. In addition, ZEA can modify the growth status of porcine ESCs, disrupt their ultrastructure, and lead to apoptosis of porcine ESCs via the mitochondrial division pathway and JNK signaling pathway. In summary, our study found the critical targets of ZEA infected with pig ESCs, which provided a conceptual foundation to prevent and control ZEA.

Emerging Zein-Bound Zearalenone in Maize: Thermal-Induced Mechanism of Binding or Releasing.

The emerging zein-bound zearalenone (ZEN) in maize could affect its nutrition and health. Besides, thermal processing could affect the zein-ZEN interaction, causing the binding or release of ZEN. To control the harm of zein-bound ZEN on the quality of maize, the thermal-induced mechanism of binding or releasing of zein-bound ZEN were studied. Results showed that thermal processing decreased the binding constant from 1.70 to 0.27 × 104 L mol-1, and binding energy from -78.41 to -32.51 kJ mol-1, with the decreased hydrogen bonds, hydrophobic, and electrostatic interactions of ZEN with Leu81 and Arg85, Val125, Ala129, and Gln132. Furthermore, thermal processing destroyed the interactions among zein molecules and caused the unwinding of zein, releasing the ZEN from the hydrophobic cavity of zein. This paper provided theoretic insights into the heat-induced binding/releasing mechanism of ZEN with zein, which helped to perfect the exposure risk evaluation of ZEN (including free and zein-bound ZEN) in maize-based products.

chi-miR-130b-3p regulates the ZEA-induced oxidative stress damage through the KEAP1/NRF2 signaling pathway by targeting SESN2 in goat GCs.

As a dominant mycotoxin, zearalenone (ZEA) has attracted extensive attention due to its estrogen-like effect and oxidative stress damage in cells. In order to find a way to relieve cell oxidative stress damage caused by ZEA, we treated goat granulosa cells (GCs) with ZEA and did a whole transcriptome sequencing. The results showed that the expression level of Sesterin2 (SESN2) was promoted extremely significantly in the ZEA group (p < .01). In addition, our research demonstrated that SESN2 could regulate oxidative stress level in GCs through Recombinant Kelch Like ECH Associated Protein 1 (KEAP1)/Nuclear factor erythroid 2-related factor 2 (NRF2) signaling pathway. The overexpression of SESN2 could reduce the oxidative damage, whereas knockdown of SESN2 would aggravate the oxidative damage caused by ZEA. What's more, microRNA (miRNA) chi-miR-130b-3p can bind to SESN2 3'-untranslated region (3'UTR) to regulate the expression of SESN2. The mimics/inhibition of chi-miR-130b-3p would have an effect on oxidative damage triggered by ZEA in GCs as well. In summary, these results elucidate a new pathway by which chi-miR-130b-3p affects the KEAP1/NRF2 pathway in GCs by modulating SESN2 expression in response to ZEA-induced oxidative stress damage.

LncRNA profiles of Cyanidin-3-O-glucoside ameliorated Zearalenone-induced damage in porcine granulosa cells.

Long non-coding RNA (lncRNA), a class of RNA molecules with transcripts longer than 200 nt, is crucial for maintaining animal reproductive function. Zearalenone (ZEN) damaged animal reproduction by targeting ovarian granulosa cells (GCs), especially in pigs. Nonetheless, it is not quite clear that whether Cyanidin-3-O-glucoside (C3G) exert effects on porcine GCs (pGCs) after ZEN exposure by altering lncRNA expression. Here, we sought to gain novel information regarding C3G protect against damages induced by ZEN in pGCs. The pGCs were divided into control (Ctrl), ZEN, ZEN + C3G (Z + C), and C3G groups. Results revealed that C3G effectively increased cell viability and suppressed ZEN-induced apoptosis in pGCs. 87 and 82 differentially expressed lncRNAs (DELs) were identified in ZEN vs. Ctrl and Z + C vs. ZEN group, respectively. Gene Ontology (GO) analysis observed that the DELs were related to cell metabolism and cell-matrix adhesion biological processes. The Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis found that the DELs were associated with the phosphatidylinositide 3-kinases (PI3K)-protein kinase B (AKT) signaling pathway. In brief, we demonstrated that C3G could shield apoptosis induced by ZEN, which may be connected with the changes of lncRNA expression profiles in pGCs. This study complemented our understanding of the genetic basis and molecular mechanisms by which C3G mitigated the toxicity of ZEN in pGCs.

Short Culture of Bovine Hepatocytes Biopsied from Dairy Cows as a Model for Toxicological Studies-CYP 1A1 Activity Response to Zearalenone Treatment.

This study presents a simple and cost-effective method for isolating hepatocytes from liver biopsies obtained from healthy and ketotic dairy cows, which can be utilized for studying cellular metabolism, drug toxicity, and hepatocyte-specific gene function and regulation. The expression of hepatocyte marker genes (G6PC, ALB, CYP1A2) was measured and found to be highest at 6 h post-isolation, with a subsequent decrease over time. Cells isolated from ketotic livers exhibited lower expression levels than those from healthy livers. Furthermore, for the functional characterization of ketotic hepatocytes, the cells were exposed to varying doses of zearalenone (ZEA). While doses of 10-50 µM did not affect cell viability, the highest dose of ZEA (100 µM) significantly decreased cell viability, as measured using XTT assay. Additionally, the potential induction of cytochrome P450 A1 (CYP1A1) by ZEA was found. Despite limitations such as a short-term culture, this model provides a useful tool for conducting toxicological research.

Protective Effect of Fucoxanthin on Zearalenone-Induced Hepatic Damage through Nrf2 Mediated by PI3K/AKT Signaling.

Hepatotoxic contaminants such as zearalenone (ZEA) are widely present in foods. Marine algae have a wide range of potential applications in pharmaceuticals, cosmetics, and food products. Research is ongoing to develop treatments and products based on the compounds found in algae. Fucoxanthin (FXN) is a brown-algae-derived dietary compound that is reported to prevent hepatotoxicity caused by ZEA. This compound has multiple biological functions, including anti-diabetic, anti-obesity, anti-microbial, and anti-cancer properties. Furthermore, FXN is a powerful antioxidant. In this study, we examined the effects of FXN on ZEA-induced stress and inflammation in HepG2 cells. MTT assays, ROS generation assays, Western blots, and apoptosis analysis were used to evaluate the effects of FXN on ZEA-induced HepG2 cell inflammation. Pre-incubation with FXN reduced the cytotoxicity of ZEA toward HepG2 cells. FXN inhibited the ZEA-induced production of pro-inflammatory cytokines, including IL-1 ß, IL-6, and TNF-α. Moreover, FXN increased HO-1 expression in HepG2 by activating the PI3K/AKT/NRF2 signaling pathway. In conclusion, FXN inhibits ZEA-induced inflammation and oxidative stress in hepatocytes by targeting Nrf2 via activating PI3K/AKT signaling.

Biochemical characterization and molecular modification of a zearalenone hydrolyzing enzyme Zhd11D from Phialophora attinorum.

ZEN lactone hydrolase (ZHD) can hydrolyze zearalenone (ZEN) to less or non-toxic product, providing an environment-friendly way for food or feeds-containing ZENs detoxification. Here, a newly identified ZHD from Phialophora attinorum, annotated as Zhd11D, was characterized to exhibit highest activity against ZEN at pH 8.0 and 35 â„ƒ with a specific activity of 304.7 U/mg, which was far higher than most of the reported ZHDs. A nonspecific protein engineering method was introduced through fusing a segment of amphiphilic short peptide S1 at the N-terminus of Zhd11D, resulting in both improved activity (1.5-fold) and thermostability (2-fold at 40 â„ƒ). Biochemical analysis demonstrated that self-aggregation caused by intermolecular interactions between S1 contributed to the improvement of the enzymatic properties of Zhd11D. Additionally, S1-Zhd11D showed a higher hydrolysis rate of ZEN than Zhd11D in peanut oil.

Mutation, food-grade expression, and characterization of a lactonase for zearalenone degradation.

Zearalenone (ZEN) is a mycotoxin that causes serious threats to human health. People are exposed to ZEN contamination externally and internally through many ways, while environmental-friendly strategies for efficient elimination of ZEN are urgently needed worldwide. Previous studies revealed that the lactonase Zhd101 from Clonostachys rosea can hydrolyze ZEN to low toxicity compounds. In this work, the enzyme Zhd101 was conducted with combinational mutations to enhance its application properties. The optimal mutant (V153H-V158F), named Zhd101.1, was selected and introduced into the food-grade recombinant yeast strain Kluyveromyces lactis GG799(pKLAC1-Zhd101.1), followed by induced expression and secretion into the supernatant. The enzymatic properties of this mutant were extensively examined, revealing a 1.1-fold increase in specific activity, as well as improved thermostability and pH stability, compared to the wild-type enzyme. The ZEN degradation tests and the reaction parameters optimization were carried out in both solutions and the ZEN-contaminated corns, using the fermentation supernatants of the food-grade yeast strain. Results showed that the degradation rates for ZEN by fermentation supernatants reached 96.9% under optimal reaction conditions and 74.6% in corn samples, respectively. These new results are a useful reference to zearalenone biodegradation technologies and indicated that the mutant enzyme Zhd101.1 has potential to be used in food and feed industries. KEY POINTS: • Mutated lactonase showed 1.1-fold activity, better pH stability than the wild type. • The strain K. lactis GG799(pKLAC1-Zhd101.1) and the mutant Zhd101.1 are food-grade. • ZEN degradation rates by supernatants reached 96.9% in solution and 74.6% in corns.