Detoxification Of Zearalenone Research Articles

Characteristics of a Novel Zearalenone Lactone Hydrolase ZHRnZ and Its Thermostability Modification.

Zearalenone (ZEN) is a toxic secondary metabolite produced by the Fusarium fungi, which widely contaminates grains, food, and feed, causing health hazards for humans and animals. Therefore, it is essential to find effective ZEN detoxification methods. Enzymatic degradation of ZEN is believed to be an eco-friendly detoxification strategy, specifically thermostable ZEN degradation enzymes are needed in the food and feed industry. In this study, a novel ZEN lactone hydrolase ZHRnZ from Rosellinia necatrix was discovered using bioinformatic and molecular docking technology. The recombinant ZHRnZ showed the best activity at pH 9.0 and 45 °C with more than 90% degradation for ZEN, α-zearalenol (α-ZOL), β-zearalenol (β-ZOL) and α-zearalanol (α-ZAL) after incubation for 15 min. We obtained 10 mutants with improved thermostability by single point mutation technology. Among them, mutants E122Q and E122R showed the best performance, which retained more than 30% of their initial activity at 50 °C for 2 min, and approximately 10% of their initial activity at 60 °C for 1 min. The enzymatic kinetic study showed that the catalytic efficiency of E122R was 1.3 times higher than that of the wild-type (WT). Comprehensive consideration suggests that mutant E122R is a promising hydrolase to detoxify ZEN in food and feed.

Actinobacteria as Promising Biocontrol Agents for In Vitro and In Planta Degradation and Detoxification of Zearalenone.

Zearalenone (ZEN) is a prevalent mycotoxin found in grains and grain-derived products, inducing adverse health effects in both animals and humans. The in-field application of microorganisms to degrade and detoxify ZEN is a promising strategy to enhance the safety of food and feed. In this study, we investigated the potential of three actinobacterial strains to degrade and detoxify ZEN in vitro and in planta on wheat ears. The residual ZEN concentration and toxicity in the samples were analysed with UHPLC-MS/MS and a bioluminescence BLYES assay, respectively. Streptomyces rimosus subsp. rimosus LMG19352 could completely degrade and detoxify 5 mg/L ZEN in LB broth within 24 h, along with significant reductions in ZEN concentration both in a minimal medium (MM) and on wheat ears. Additionally, it was the only strain that showed a significant colonisation of these ears. Rhodococcus sp. R25614 exhibited partial but significant degradation in LB broth and MM, whereas Streptomyces sp. LMG16995 degraded and detoxified ZEN in LB broth after 72 h by 39% and 33%, respectively. Although all three actinobacterial strains demonstrated the metabolic capability to degrade and detoxify ZEN in vitro, only S. rimosus subsp. rimosus LMG19352 showed promising potential to mitigate ZEN in planta. This distinction underscores the importance of incorporating in planta screening assays for assessing the potential of mycotoxin-biotransforming microorganisms as biocontrol agents.

Application of atmospheric cold plasma for zearalenone detoxification in cereals: Kinetics, mechanisms, and cytotoxicity analysis

IntroductionZearalenone (ZEN) is one of the most widely contaminated mycotoxins in world, posing a severe threat to human and animal health. Atmospheric cold plasma (ACP) holds great penitential in mycotoxin degradation. ObjectivesThis study aimed to investigate the degradation efficiency and mechanisms of ACP on ZEN as well as the cytotoxicity of ZEN degradation products by ACP. Additionally, this study also investigated the degradation efficiency of ACP on ZEN in cereals and its effect on cereal quality. MethodsThe degradation efficiency and products of ZEN by ACP was analyzed by HPLC and LC-MS/MS. The human normal liver cells and mice were employed to assess the cytotoxicity of ZEN degradation products. The ZEN artificially contaminated cereals were used to evaluate the feasibility of ACP detoxification in cereals. ResultsThe results showed that the degradation rate of ZEN was 96.18 % after 30-W ACP treatment for 180 s. The degradation rate was dependent on the discharge power, and treatment time and distance. Four major ZEN degradation products were produced after ACP treatment due to the oxidative destruction of CC double bond, namely C18H22O7 (m/z = 351.19), C18H22O8 (m/z = 367.14), C18H22O6 (m/z = 335.14), and C17H20O6 (m/z = 321.19). L02 cell viability was increased from 52.4 % to 99.76 % with ACP treatment time ranging from 0 to 180 s. Mice results showed significant recovery of body weight and depth of colonic crypts as well as mitigation of glomerular and liver damage. Additionally, ACP removed up to 50.55 % and 58.07 % of ZEN from wheat and corn. ConclusionsThis study demonstrates that ACP could efficiently degrade ZEN in cereals and its cytotoxicity was significantly reduced. Therefore, ACP is a promising effective method for ZEN detoxification in cereals to ensure human and animal health. Future study needs to develop large-scale ACP device with high degradation efficiency.

Site-directed display of zearalenone lactonase on spilt-intein functionalized nanocarrier for green and efficient detoxification of zearalenone

In this study, we prepared a functional organic–inorganic hybrid nanoflower (InHNF) via split intein moiety in a biomineralization process without using organic solvents. InHNF could specifically bind the target enzymes from crude cell lysates within seconds and site-directedly display them on the surface by forming a peptide bond with enzyme’s terminal amino acid residue. This unique feature enabled InHNF to increase the specific activity of zearalenone detoxifying enzyme ZHD518 by 40 ∼ 60% at all tested temperatures and prevented enzyme denaturation even under extreme pH conditions (pH 3–11). Furthermore, it exhibited excellent operational stability, with a residual activity of over 70% after eight reaction cycles. Strikingly, InHNF-ZHD518 achieved above 50% ZEN degradation despite the near inactivation of free ZHD518 in beer sample. Overall, InHNF nanocarriers can achieve environmentally friendly, purification-free, and site-directed immobilization of food enzymes and enhance their catalytic properties, making them suitable for a wide range of industrial applications.

Current review of biodegradation and detoxification strategies for zearalenone contaminated food and feed

Mycotoxins are toxic metabolites produced by fungi that may cause serious health problems in humans and animals. Zearalenone is an estrogenic mycotoxin produced by Fusarium species that leads to huge economic losses in the food industry and livestock husbandry. Contamination of food and feed with zearalenone has reproductive problems, carcinogenicity, immunotoxicity, and other cytotoxic effects. At present, microorganisms and enzymes derived from microbial strains have been widely used for the degradation of zearalenone in food and feed. Researchers have developed biodegradation of zearalenone by the use of microbial and their enzyme derivatives, which offers harmless products and is environmentally friendly. Development of recombinant enzymes improves enzymatic detoxification of zearalenone to a non-toxic product without damaging the nutritional content. This review summarizes biodegradation and detoxification strategies of zearalenone using microorganisms and enzyme derivatives to nontoxic products.

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

Zearalenone (ZEN), a nonsteroidal estrogenic mycotoxin produced by Fusarium spp., contaminates cereals and threatens human and animal health by inducing hepatotoxicity, immunotoxicity, and genotoxicity. In this study, a new Bacillus subtilis strain, YQ-1, with a strong ability to detoxify ZEN, was isolated from soil samples and characterized. YQ-1 was confirmed to degrade more than 46.26% of 20 μg mL-1 ZEN in Luria-Bertani broth and 98.36% in fermentation broth within 16 h at 37 °C; one of the two resulting products was ZEN-diglucoside. Under optimal reaction conditions (50 °C and pH 5.0-9.0), the reaction mixture generated by YQ-1 catalyzing ZEN significantly reduced the promoting effect of ZEN on MCF-7 cell proliferation, effectively eliminating the estrogenic toxicity of ZEN. In addition, a new glycosyltransferase gene (yqgt) from B. subtilis YQ-1 was cloned with 98% similarity to Bs-YjiC from B. subtilis 168 and over-expressed in E. coli BL21 (DE3). ZEN glycosylation activity converted 25.63% of ZEN (20 μg mL-1) to ZEN-diG after 48 h of reaction at 37 °C. The characterization of ZEN degradation by B. subtilis YQ-1 and the expression of YQGT provide a theoretical basis for analyzing the mechanism by which Bacillus spp. degrades ZEN.

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.

A Probiotic Bacillus amyloliquefaciens D-1 Strain Is Responsible for Zearalenone Detoxifying in Coix Semen.

Zearalenone (ZEN) is a mycotoxin produced by Fusarium spp., which commonly and severely contaminate food/feed. ZEN severely affects food/feed safety and reduces economic losses owing to its carcinogenicity, genotoxicity, reproductive toxicity, endocrine effects, and immunotoxicity. To explore efficient methods to detoxify ZEN, we identified and characterized an efficient ZEN-detoxifying microbiota from the culturable microbiome of Pseudostellaria heterophylla rhizosphere soil, designated Bacillus amyloliquefaciens D-1. Its highest ZEN degradation rate reached 96.13% under the optimal condition. And, D-1 can almost completely remove ZEN (90 μg·g-1) from coix semen in 24 h. Then, the D-1 strain can detoxify ZEN to ZEM, which is a new structural metabolite, through hydrolyzation and decarboxylation at the ester group in the lactone ring and amino acid esterification at C2 and C4 hydroxy. Notably, ZEM has reduced the impact on viability, and the damage of cell membrane and nucleus DNA and can significantly decrease the cell apoptosis in the HepG2 cell and TM4 cell. In addition, it was found that the D-1 strain has no adverse effect on the HepG2 and TM4 cells. Our findings can provide an efficient microbial resource and a reliable reference strategy for the biological detoxification of ZEN.

Effective degradation of zearalenone by dye-decolorizing peroxidases from Pleurotus ostreatus and its metabolic pathway and toxicity analysis

The widespread detection of zearalenone (ZEN) in cereal crops and feeds poses a significant threat to both humans and animals. Consequently, the urgency for the international community to address this issue is evident in the demand for safe and effective measures to mitigate zearalenone contamination and explore detoxification methods. In this study, a dye-decolorizing peroxidase (PoDyP4) from Pleurotus ostreatus is characterized for its impressive ZEN degradation effectiveness. PoDyP4 was demonstrated that the ability to almost completely degrade ZEN at pH 6.0 and 40 °C for 2 h, even at high concentrations of 1 mM. The promotion of enzymatic degradation of ZEN was most pronounced in the presence of Mg2+, while Cu2+ and Fe2+ exhibited a notable inhibitory effect. The degradation mechanism elucidated the detoxification of ZEN by PoDyP4 through hydroxylation and polymerization reactions. The resulting metabolic products displayed significantly reduced toxicity and minimal impact on the viability and apoptosis of mouse spermatocytes GC-2 cells, in comparison to the original ZEN. Hydrophobic contacts and hydrogen bonds were found to be crucial for ZEN-PoDyP4 stability via molecular docking. This finding suggests that PoDyP4 may have a promising application in the field of food and feed for zearalenone detoxification.

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 ℃ 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 ℃). 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.

Characterization of modified rice straw biochar in immobilizing Bacillus subtilis 168 and evaluation on its role as a novel agent for zearalenone-removal delivery

Microbial remediation of environmental pollutants can be advanced by carrier based cells immobilization. Whereas the effects of microorganisms immobilized on biochar for removal of zearalenone (ZEN) still remain unknown. Herein, this work presented the characterization of rice straw biochar (RSB) around modification in immobilizing Bacillus subtilis 168 and the role in fighting ZEN in vitro. Specifically, 10% of RSB with pH 5 condition were optimal for bearing cells, where majority of cells loaded inside the pore and minority on surface with agglomeration or scattering status. Octadecyl trimethyl ammonium chloride-inclusion RSB showed better performances including over 93% of ZEN detoxification rate (32.48% in free cells), cells preservation, and stability of detoxification in simulated gastrointestinal environment. RSB treated with sulphuric acid made nutrients adsorption generally less than 6.5%. No residues of α-ZEL and α-ZAL were found in ZEN biotransformation process whether by free cells or composites. Mechanism discussion implied that predominant monolayer chemisorption by RSB and subsequent biodegradation by extracellular enzymes from microorganism involved in ZEN-removal process. Collectively, these findings contribute to provide an applying strategy for coordination of biochar and microorganisms as potentially mycotoxin detoxifying agent in agricultural feed bioremediation and environmental decontamination processes.

The N-terminal hydrophobicity modulates a distal structural domain conformation of zearalenone lacton hydrolase and its application in protein engineering

Zearalenone (ZEN) is one of the most common mycotoxins in maize, wheat, barley, sorghum, rye and other grains. ZEN contamination in feed is an international health issue due to its estrogenicity by competitively binding to estrogen receptors. Enzymatic detoxification of ZEN is superior to physical and chemical methods in terms of safety, environmental impact and preserving nutritional value and palatability, but is hampered by both the currently limited repertoire of detoxifying enzymes and the lack of knowledge about their structure-function relationships. In this study, a ZEN lacton hydrolase candidate (ZHD11C) was identified from thermo-tolerant Fonsecaea multimorphosa CBS 102226, and characterized to be more thermostable than these reported homologues. An intriguing feature of ZHD11C is that the N-terminal hydrophobicity affects its thermal stability and causes conformational change of a domain far from the N-terminal. This finding was successfully applied to enhance the thermostability of the most active ZEN lacton hydrolase ZHD518 through rationally tailoring its N-terminal hydrophobicity. Our results not only provide more insights into the structure-function relationships of ZEN lacton hydrolases, but generate better candidate for bio-decontamination of zearalenone in feed industries.

Combined in silico investigation and in vitro characterization of the zearalenone detoxification potential of dye-decolorizing peroxidase from Bacillus subtilis 168

Zearalenone (ZEN), one of the most hazardous mycotoxins commonly present in food, causes severe safety risks to human health. In this work, the ZEN detoxification potential of dye-decolorizing peroxidase BsDyP from Bacillus subtilis 168 was investigated by a combined computational and experimental study. Molecular docking and dynamics simulation suggested that BsDyP could allow the binding of ZEN at the γ-edge of heme and surface exposed redox-active Tyr335 residue. Consistently, the recombinant BsDyP expressed in Escherichia coli was found to be capable of directly oxidizing 83% of ZEN with H2O2 as an electron acceptor. Moreover, the presence of 2, 2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) as a redox mediator of BsDyP could enhance ZEN degradation to more than 98% in the pH range of 5.0–10.0. BsDyP-catalyzed ZEN degradation could be regarded as detoxification as the transformation product displayed much less estrogenicity. Moreover, bi-enzymes system including glucose oxidase (GOD) and BsDyP was applied to degrade ZEN in contaminated corn steep liquor and achieved a 33% degradation percentage. The findings suggested that BsDyP could be developed as a biocatalyst for detoxification of ZEN in contaminated food and feed.

Transcriptomic responses of the zearalenone (ZEN)-detoxifying yeast Apiotrichum mycotoxinivorans to ZEN exposure

Zearalenone (ZEN) is a potent oestrogenic mycotoxin that is mainly produced by Fusarium species and is a serious environmental pollutant in animal feeds. Apiotrichum mycotoxinivorans has been widely used as a feed additive to detoxify ZEN. However, the effects of ZEN on A. mycotoxinivorans and its detoxification mechanisms remain unclear. In this study, transcriptomic and bioinformatic analyses were used to investigate the molecular responses of A. mycotoxinivorans to ZEN exposure and the genetic basis of ZEN detoxification. We detected 1424 significantly differentially expressed genes (DEGs), of which 446 were upregulated and 978 were downregulated. Functional and enrichment analyses showed that ZEN-induced genes were significantly associated with xenobiotic metabolism, oxidative stress response, and active transport systems. However, ZEN-inhibited genes were mainly related to cell division, cell cycle, and fungal development. Subsequently, bioinformatic analysis identified candidate ZEN-detoxification enzymes. The Baeyer–Villiger monooxygenases and carboxylesterases, which are responsible for the formation and subsequent hydrolysis of a new ZEN lactone, respectively, were significantly upregulated. In addition, the expression levels of genes related to conjugation and transport involved in the xenobiotic detoxification pathway were significantly upregulated. Moreover, the expression levels of genes encoding enzymatic antioxidants and those related to growth and apoptosis were significantly upregulated and downregulated, respectively, which made it possible for A. mycotoxinivorans to survive in a highly toxic environment and efficiently detoxify ZEN. This is the first systematic report of ZEN tolerance and detoxification in A. mycotoxinivorans. We identified the metabolic enzymes that were potentially involved in detoxifying ZEN in the GMU1709 strain and found that ZEN-induced transcriptional regulation of genes is key to withstanding highly toxic environments. Hence, our results provide valuable information for developing enzymatic detoxification systems or engineering this detoxification pathway in other species.

Detoxification of the mycoestrogen zearalenone by Bacillus licheniformis spore CotA laccase and application of immobilized laccase in contaminated corn meal

Zearalenone (ZEN) is an estrogen-like mycotoxin posing a great threat to human and animal health. The present study showed that the induction of spore CotA laccase production of Bacillus licheniformis by ions Mn2+ and Cu2+ promoted the ZEN-degrading ability of the strain. The spore CotA laccase heterologously expressed in Escherichia coli was capable of directly oxidizing ZEN with Km, Kcat and Vmax values of 90.43 μg mL−1, 0.11s−1 and 35.48 μg min−1 mg−1, respectively. The use of 2, 2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), acetosyringone, ferulic acid, caffeic acid and methyl syringate as redox mediators could enhance ZEN degradation by CotA laccase. The human breast cancer MCF-7 cell proliferation assay showed that CotA laccase and CotA laccase-mediator systems eliminated the estrogenicity of ZEN. Immobilized CotA laccase onto chitosan microspheres exhibited higher thermal stability over free CotA laccase, maintaining about 87% of its initial activity after heat treatment at 80 °C for 30 min. The removal rate of ZEN in corn meal by free and immobilized CotA laccase was 70% and 90%, respectively. Meanwhile, immobilized enzyme could achieve a 54% ZEN degradation in corn meal after three consecutive cycles. Thus, CotA laccase could be a promising biocatalyst for detoxification of ZEN in corn meal.

Excavation, expression, and functional analysis of a novel zearalenone-degrading enzyme.

Zearalenone (ZEN) is a toxic secondary metabolite of Fusarium sp. commonly found in wheat, corn, and other crops. In addition to economic losses, ZEN can seriously endanger the health of both humans and livestock, thus presenting an urgent need for ZEN-detoxifying enzymes that function in the extreme heat or pH conditions of industrial fermenters. Here, we identify and characterize the activity of the ZEN-degrading enzyme from Exophiala spinifera, ZHD_LD, which shares 60.15% amino acid identity and a conserved catalytic triad with the well-characterized ZEN-detoxifying protein ZHD101 from Clonostachys rosea. Biochemical activity and stability assays indicated that purified recombinant ZHD_LD exhibited high activity against ZEN with optimal reaction conditions of 50 ℃ and pH 7.0-10.0. Structural modeling of the ZHD_LD active site and comparison with ZHD101 revealed its likely mechanism of ZEN degradation. This research provides an industrially valuable candidate enzyme for ZEN detoxification in food and livestock feed.

The replacement of main cap domain to improve the activity of a ZEN lactone hydrolase with broad substrate spectrum

Zearalenone (ZEN) and its derivatives is a major kind of mycotoxins that being commonly detected in contaminated grain products. ZEN lactone hydrolase (ZHD) can hydrolyze ZEN to produce non-toxic product, providing an environment-friendly way for ZENs detoxification. However, most of ZEN derivatives are more toxic, which leads to a high demand for the novel ZHD with high degradation activity towards ZEN derivatives. Here, a new ZEN lactone hydrolase Zhd11B from Fonsecaea monophora was characterized to efficiently hydrolyze ZEN and more toxic derivatives α-ZAL and β-ZAL. The broad substrate spectrum of Zhd11B may attribute to the improved protein flexibility. Through cap-domain swap, the activity towards ZEN, α-ZAL and β-ZAL was further improved by 1.5-, 1.6- and 2.9-fold, respectively, which suggested that the conserved cap domain could be a structural element for ZHDs engineering, providing a new strategy for molecular modification of ZHDs. Structure comparison and molecular dynamic analysis showed that the protonation of catalytic histidine residue (H245 in Zhd11B) and a more stable combination between “goalkeeper” of the catalytic pocket are main determinant factors of ZHDs’ catalytic efficiency. Our results provide a good experimental material for better understanding of the mechanism of substrate specificity and cap domain swapping would provide a new strategy for the molecular modification.

Evaluation of nano-silica, microwave heating, and ultraviolet irradiation effects on zearalenone detoxification in sunflower oils

In the present study, we report three methods of silica nanoparticles (SNPs) as adsorbent, ultraviolet (UV) irradiation, and microwave heating and evaluate their capabilities in reducing and eliminating zearalenone (ZEN). The offered method not only was used for ZEN detoxification, but also greatly enhanced the sensitivity of ZEN measurement. The aim of this study was to evaluate ZEN concentration in sunflower oil samples by high-performance liquid chromatography (HPLC) method. This method was successfully validated for sunflower oil samples while the limit of detection (LOD) method (signal-to-noise ratio of 3:1) was 0.5 μg/l. The acquired removal data with the HPLC method through SNPs were fitted well with Freundlich isotherm, denoting that the multi-layer adsorption took place on the adsorbent. The equilibrium adsorption capacity of ZEN was 61.02 μg/g in an optimum time of 240 min on SNPs. The experimental results were evaluated by the adsorption kinetic model, which specified the adsorption kinetics of ZEN on SNPs, obeying the pseudo-second order model. This model demonstrated that the sorption rate depended on the sorption capacity but not the concentration of the sorbate. Moreover, the method presented to determine ZEN based on the use of SNPs in sunflower oil was accomplished by the adsorption process. Furthermore, the removal efficiencies of ZEN by SNPs, UV irradiation, and microwave heating were compared and obtained to be 92.1, 96.22, and 37.30%, respectively for determined times. These results confirm the removal efficiency of these methods is sensitive enough to ZEN analysis in sunflower oil samples.

Cloning and Characterization of Three Novel Enzymes Responsible for the Detoxification of Zearalenone.

Zearalenone is a common mycotoxin contaminant in cereals that causes severe economic losses and serious risks to health of human and animals. Many strategies have been devised to degrade ZEN and keep food safe. The hydrolase ZHD101 from Clonostachys rosea, which catalyzes the hydrolytic degradation of ZEN, has been studied widely. In the current research, three new enzymes that have the capacity to detoxify ZEN were identified, namely CLA, EXO, and TRI, showing 61%, 63%, and 97% amino acids identities with ZHD101, respectively. Three coding genes was expressed as heterologous in Escherichia coli BL21. Through biochemical analysis, the purified recombinant CLA, EXO, TRI, and ZHD101 exhibited high activities of degrading ZEN with the specific activity of 114.8 U/mg, 459.0 U/mg, 239.8 U/mg, and 242.8 U/mg. The optimal temperatures of CLA, EXO, TRI, and ZHD101 were 40 °C, 40 °C, 40 °C, and 45 °C, and their optimum pH were 7.0, 9.0, 9.5, and 9.0, respectively. Our study demonstrated that the novel enzymes CLA, EXO, and TRI possessed high ability to degrade ZEN from the model solutions and could be the promising candidates for ZEN detoxification in practical application.

Screening of zearalenone-degrading bacteria and analysis of degradation conditions

Zearalenone(ZEN) is a mycotoxin produced by Fusarium, possessing estrogen-like effects, carcinogenicity, and multiple toxicities. To seek more efficient and practical agents for biological detoxification and broaden their application, this study isolated 194 bacterial strains from the moldy tuberous root of Pseudostellaria heterophylla, which were co-cultured with ZEN. An efficient ZEN-degrading strain H4-3-C1 was screened out by HPLC and identified as Acinetobacter calcoaceticus by morphological observation and molecular identification. The effects of culture medium, inoculation dose, culture time, pH, and temperature on the degradation of ZEN by H4-3-C1 strain were investigated. The mechanism of ZEN degradation and the degrading effect in Coicis Semen were discussed. The degradation rate of 5 μg·mL~(-1) ZEN by H4-3-C1 strain was 85.77% in the LB medium(pH 6) at 28 ℃/180 r·min~(-1) for 24 h with the inoculation dose of 1%. The degradation rate of ZEN in the supernatant of strain culture was higher than that in the intracellular fluid and thalli. The strain was inferred to secret extracellular enzymes to degrade ZEN. In addition, the H4-3-C1 strain could also degrade ZEN in Coicis Semen. If the initial content of ZEN in Coicis Semen was reduced from 90 μg·g~(-1) to 40.68 μg·g~(-1), the degradation rate could reach 54.80%. This study is expected to provide a new strain and application technology for the biological detoxification of ZEN in food processing products and Chinese medicinal materials.