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.

School of Biological and Pharmaceutical Engineering Nanjing Tech University Nanjing China

School of Food Science and Pharmaceutical Engineering Nanjing Normal University Nanjing China

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Afriyie-Gyawu E, Wiles MC, Huebner HJ, Richardson MB, Fickey C, Phillips TD (2005) Prevention of zearalenone-induced hyperestrogenism in prepubertal mice. J Toxicol Environ Health A 68:353–368. https://doi.org/10.1080/15287390590900822 PubMed DOI

Azam MS, Yu D, Liu N, Wu A (2019) Degrading ochratoxin A and zearalenone mycotoxins using a multifunctional recombinant enzyme. Toxins 11:301. https://doi.org/10.3390/toxins11050301 DOI PMC

Bi K, Zhang W, Xiao ZZ, Zhang DW (2018) Characterization, expression and application of a zearalenone degrading enzyme from Neurospora crassa. AMB Express 8:1–10. https://doi.org/10.1186/s13568-018-0723-z DOI

Bolechová M, Benešová K, Běláková S, Čáslavský J, Pospíchalová M, Mikulíková R (2015) Determination of seventeen mycotoxins in barley and malt in the Czech Republic. Food Control 47:108–113. https://doi.org/10.1016/j.foodcont.2014.06.045 DOI

Bullerman LB, Bianchini A (2007) Stability of mycotoxins during food processing. Int J Food Microbiol 119:140–146. https://doi.org/10.1016/j.ijfoodmicro.2007.07.035 PubMed DOI

Cheng B, Shi WT, Luo J, Peng FZ, Wan CX, Wei H (2010) Cloning of zearalenone-degraded enzyme gene (ZEN-jjm) and its expression and activity analysis. Int J Food Microbiol 18:225–230. https://doi.org/10.3969/j.issn.1674-7968.2010.02.004 DOI

Du J, Zhao W, Peng GR, Li QL, Yin CS, Yao WS, Zhang XK, Yang L, Fu LZ, Chen XY, Liu Y (2021) Expression and immunogenicity of No-toxin recombinant Clostridium septicum alpha toxin. Acta Vet Zootech Sin 52:202–209. https://doi.org/10.11843/j.issn.0366-6964.2021.021

Higa-Nishiyama A, Takahashi-Ando N, Shimizu T, Kudo T, Yamaguchi I, Kimura M (2005) A model transgenic cereal plant with detoxification activity for the estrogenic mycotoxin zearalenone. Transgenic Res 14:713–717. https://doi.org/10.1007/s11248-005-6633-2 PubMed DOI

Hui R, Hu X, Liu W, Zheng Y, Chen Y, Guo G-T, Jin J, Chen C-C (2017) Characterization and crystal structure of a novel zearalenone hydrolase from Cladophialophora bantiana. Acta Crystallogr F-Struct Biol Commun 73:515–519. https://doi.org/10.1107/S2053230X17011840 PubMed DOI PMC

Ismail A, Gonçalves BL, de Neeff DV, Ponzilacqua B, Coppa FSCC, Hintzsche HH, Sajid M, Cruz AG, Corassin CH, Oliveira CAF (2018) Aflatoxin in foodstuffs: occurrence and recent advances in decontamination. Food Res Int 113:74–85. https://doi.org/10.1016/j.foodres.2018.06.067 PubMed DOI

Kakeya H, Takahashi-Ando N, Kimura M, Onose R, Yamaguchi I, Osada H (2002) Biotransformation of the mycotoxin, zearalenone, to a non-estrogenic compound by a fungal strain of Clonostachys sp. Biosci Biotechnol Biochem 66:2723–2726. https://doi.org/10.1271/bbb.66.2723 PubMed DOI

Kouadio JH, Dano SD, Moukha S, Mobio TA, Creppy EE (2007) Effects of combinations of Fusarium mycotoxins on the inhibition of macromolecular synthesis, malondialdehyde levels, DNA methylation and fragmentation, and viability in Caco-2 cells. Toxicon 49:306–317. https://doi.org/10.1016/j.toxicon.2006.09.029 PubMed DOI

Lin M, Tan J, Xu ZB, Huang J, Tian Y, Chen B, Wu YD, Tong Y, Zhu YS (2019) Computational design of enhanced detoxification activity of a zearalenone lactonase from Clonostachys rosea in acidic medium. RSC Adv 9:31284–31295. https://doi.org/10.1039/C9RA04964A PubMed DOI PMC

Lu QJ, Liang XC, Chen F (2011) Detoxification of Zearalenone by viable and inactivated cells of Planococcus sp. Food Control 22:191–195. https://doi.org/10.1016/j.foodcont.2010.06.019 DOI

Ma HX, Zhang X, Yao JB, Cheng SH (2019) Breeding for the resistance to Fusarium head blight of wheat in China. Front Agric Sci Eng 6:251–264. https://doi.org/10.15302/J-FASE-2019262

Marin DE, Taranu I, Pistol G, Stancu M (2013) Effects of zearalenone and its metabolites on the swine epithelial intestinal cell line: IPEC 1. Proc Nutr Soc. https://doi.org/10.1017/S0029665113000426 DOI

Molina-Molina J-M, Real M, Jimenez-Diaz I, Belhassen H, Hedhili A, Torné P, Fernández MF, Olea N (2014) Assessment of estrogenic and anti-androgenic activities of the mycotoxin zearalenone and its metabolites using in vitro receptor-specific bioassays. Food Chem Toxicol 74:233–239. https://doi.org/10.1016/j.fct.2014.10.008 PubMed DOI

Mustapha MB, Bousselmi M, Jerbi T, Bettaïeb NB, Fattouch S (2014) Gamma radiation effects on microbiological, physico-chemical and antioxidant properties of Tunisian millet (Pennisetum glaucum LR Br.). Food Chem 154:230–237. https://doi.org/10.1016/j.foodchem.2014.01.015 PubMed DOI

Park GY, Park SJ, Choi MY, Koo IG, Byun JH, Hong JW, Sim JY, Collins GJ, Lee JK (2012) Atmospheric-pressure plasma sources for biomedical applications. Plasma Sources Sci Technol 21:043001. https://doi.org/10.1088/0963-0252/21/4/043001 DOI

Ruzzini AC, Ghosh S, Horsman GP, Foster LJ, Bolin JT, Eltis LD (2012) Identification of an acyl-enzyme intermediate in a meta-cleavage product hydrolase reveals the versatility of the catalytic triad. J Am Chem Soc 134:4615–4624. https://doi.org/10.1021/ja208544g PubMed DOI

Takahashi-Ando N, Kimura M, Kakeya H, Osada H, Yamaguchi I (2002) A novel lactonohydrolase responsible for the detoxification of zearalenone: enzyme purification and gene cloning. Biochem J 365:1–6. https://doi.org/10.1042/bj20020450 PubMed DOI PMC

Takahashi-Ando N, Ohsato S, Shibata T, Hamamoto H, Yamaguchi I, Kimura M (2004) Metabolism of zearalenone by genetically modified organisms expressing the detoxification gene from Clonostachys rosea. Appl Environ Microbiol 70:3239–3245. https://doi.org/10.1128/AEM.70.6.3239-3245.2004 PubMed DOI PMC

Vekiru E, Hametner C, Mitterbauer R, Rechthaler J, Adam G, Schatzmayr G, Krska R, Schuhmacher R (2010) Cleavage of zearalenone by Trichosporon mycotoxinivorans to a novel nonestrogenic metabolite. Appl Environ Microbiol 76:2353–2359. https://doi.org/10.1128/AEM.01438-09 PubMed DOI PMC

Wang MY, Huang S, Chen J, Chen S, Long M (2021) Complete genome sequence of zearalenone degrading bacteria Bacillus velezensis A2. Curr Microbiol 78:347–350. https://doi.org/10.1007/s00284-020-02234-6 PubMed DOI

Wang MY, Wu WW, Li L, He JB, Huang S, Chen S, Chen J, Miao L, Yang SS, Li P (2019) Analysis of the miRNA expression profiles in the zearalenone-exposed TM3 Leydig cell line. Int J Mol Sci 20:635. https://doi.org/10.3390/ijms20030635 DOI PMC

Wang MX, Yin LF, Hu HZ, Selvaraj JN, Zhou YL, Zhang GM (2018) Expression, functional analysis and mutation of a novel neutral zearalenone-degrading enzyme. Int J Biol Macromol 118:1284–1292. https://doi.org/10.1016/j.ijbiomac.2018.06.111 PubMed DOI

Wang SC, Wang QH, Zhou YL, Lu ZH, Zhang GM, Ma YH (2017) A new GH13 α-glucosidase from alkaliphilic Bacillus pseudofirmus 703 with both exo-α-l, 4-glucosidase and oligo-l, 6-glucosidase activities toward amylopectin. Int J Biol Macromol 101:973–982. https://doi.org/10.1016/j.ijbiomac.2017.03.129 PubMed DOI

Wang SW, Hou QQ, Guo QQ, Zhang J, Sun YM, Wei H, Shen LX (2020) Isolation and characterization of a deoxynivalenol-degrading bacterium Bacillus licheniformis YB9 with the capability of modulating intestinal microbial flora of mice. Toxins 12:184. https://doi.org/10.3390/toxins12030184 DOI PMC

Wu N, Ou W, Zhang ZD, Wang YW, Xu Q, Huang H (2021) Recent advances in detoxification strategies for zearalenone contamination in food and feed. Chin J Chem Eng 30:168–177. https://doi.org/10.1016/j.cjche.2020.11.011 DOI

Xiang L, Wang QH, Zhou YL, Yin LF, Zhang GM, Ma YH (2016) High-level expression of a ZEN-detoxifying gene by codon optimization and biobrick in Pichia pastoris. Microbiol Res 193:48–56. https://doi.org/10.1016/j.micres.2016.09.004 PubMed DOI

Xu ZX, Liu WD, Chen C-C, Li Q, Huang J-W, Ko T-P, Liu GZ, Liu WT, Peng W, Cheng Y-S, Chen Y, Jin J, Li HZ, Zheng YY, Guo R-T (2016) Enhanced α-zearalenol hydrolyzing activity of a mycoestrogen-detoxifying lactonase by structure-based engineering. ACS Catal 6:7657–7663. https://doi.org/10.1021/acscatal.6b01826 DOI

Zhang HY, Dong MJ, Yang QY, Apaliya MT, Li J, Zhang XY (2016) Biodegradation of zearalenone by Saccharomyces cerevisiae: possible involvement of ZEN responsive proteins of the yeast. J Proteomics 143:416–423. https://doi.org/10.1016/j.jprot.2016.04.017 PubMed DOI

Zhang Y, Jia Z, Yin S, Shan A, Gao R, Qu A, Liu M, Nie S (2014) Toxic effects of maternal zearalenone exposure on uterine capacity and fetal development in gestation rats. Reprod Sci 21:743–753. https://doi.org/10.1177/1933719113512533 PubMed DOI PMC

Zheng YZ, Hossen SM, Sago YK, Yoshida M, Nakagawa H, Nagashima H, Okadome H, Nakajima T, Kushiro M (2014) Effect of milling on the content of deoxynivalenol, nivalenol, and zearalenone in Japanese wheat. Food Control 40:193–197. https://doi.org/10.1016/j.foodcont.2013.11.043 DOI

Zhou JZ, Zhu LD, Chen JF, Wang W, Zhang RM, Li YW, Zhang QZ, Wang WX (2020) Degradation mechanism for zearalenone ring-cleavage by zearalenone hydrolase RmZHD: a QM/MM study. Sci Total Environ 709:135897. https://doi.org/10.1016/j.scitotenv.2019.135897 PubMed DOI