一株新型降解玉米赤霉烯酮的沙福芽孢杆菌及其关键酶分析

摘要/Abstract

摘要： 玉米赤霉烯酮（zearalenone, ZEN）是污染粮食谷物最广泛的真菌毒素之一。本研究以ZEN为主要碳源，采用富集培养法从小麦样品中分离获得一株快速降解ZEN的微生物菌株。通过形态学观察、16S rDNA及促旋酶B亚单位（gyrase B, gyrB）基因序列分析，初步鉴定该菌株为沙福芽孢杆菌（Bacillus safensis），命名为M7L4，该菌株在基础盐培养基中培养24 h可将10 μg/mL的 ZEN完全降解。质谱分析表明，M7L4的活细胞可将ZEN转化为质荷比为397.1的新型磷酸化衍生物玉米赤霉烯酮-磷酸盐（Zearalenone-phosphate, ZEN-P）。以菌株M7L4的基因组为模板，扩增出转化ZEN的关键酶Phosphoenolpyruvate(PEP)-utilizing enzyme（BsaPUE），在大肠杆菌BL21(DE3)中诱导表达并纯化。重组酶BsaPUE具有磷酸转移酶活性，在有ATP和Mg2+存在的情况下，30 min内可将10 μg/mL ZEN完全转化为ZEN-P。沙福芽孢杆菌及其磷酸转移酶可为生物去除ZEN提供有效的材料资源。

关键词: 玉米赤霉烯酮, 芽孢杆菌, 降解, 玉米赤霉烯酮-磷酸盐

Abstract: Zearalenone (ZEN) is one of the most widespread mycotoxins found in cereal crops. A bacterial strain with high efficiency of ZEN removal was isolated from wheat samples by enrichment culture method using ZEN as a main carbon source. Based on the morphology observation, 16S rDNA sequence and gyrase B (gyrB) sequence analysis, the strain was identified as Bacillus safensis and named as M7L4. 10 μg/mL of ZEN could be completely reduced by viable cells of M7L4 strain within 24 hours when cultured in minimal salt medium. The transformation product of ZEN by M7L4 strain was detected by UPLC-QTOF-MS/MS and identified as zearalenone-phosphate (ZEN-P) with a mass-to-charge ratio (m/z) of 397.1. A phosphoenolpyruvate(PEP)-utilizing enzyme gene of Bacillus safensis (named BsaPUE) was amplified from M7L4 strain and inserted to a pET-28(+) vector. The recombinant Escherichia coli BL21(DE3) harboring the plasmid pET-28a(+)-BsaPUE was induced with IPTG and purified by Ni-NTA. The recombinant BsaPUE exhibited phosphotransferase activity that could transform 100% of ZEN (10 μg/mL) to ZEN-P in the presence of ATP and Mg2+. Since the M7L4 strain and its phosphotransferase had high biotransformation capacity of ZEN, they could be promising resources for the detoxification of ZEN.

Key words: Zearalenone, Bacillus, Degradation, Zearalenone-phosphate

中图分类号:

TS207.7

赵程程孙长坡孙晶王峻刘虎军. 一株新型降解玉米赤霉烯酮的沙福芽孢杆菌及其关键酶分析[J]. 食品科学, 0, (): 0-0.

参考文献

[1] RAI A, DAS M, TRIPATHI A. Occurrence and Toxicity of a Fusarium Mycotoxin, Zearalenone[J]. Critical Reviews in Food Science and Nutrition, 2020, 60(16): 2710-2729. DOI: 10.1080/10408398.2019.1655388.
[2] LI M , Yang C , Mao Y , et al. Zearalenone Contamination in Corn, Corn Products, and Swine Feed in China in 2016–2018 as Assessed by Magnetic Bead Immunoassay[J]. Toxins, 2019, 11(8). DOI: 10.3390/toxins11080451.
[3] ROGOWSK A, POMASTOWSKI P, SAGANDYKOVA G, et al. Zearalenone and Its Metabolites: Effect on Human Health, Metabolism and Neutralization Methods[J]. Toxicon, 2019, 162: 46-55. DOI:10.1016/j.toxicon.2019.03.004.
[4] Han X, HUANGFU B, Xu T, et al. Research Progress of Safety of Zearalenone: A Review[J]. Toxins (Basel), 2022, 14(6): 386. DOI: 10.3390/toxins14060386.
[5] OSTRY V, MALIR F, TOMAN J, et al. Mycotoxins as Human Carcinogens—the IARC Monographs Classification[J]. Mycotoxin Research, 2016, 33(1): 65-73. DOI10.1007/s12550-016-0265-7.
[6] 中华人民共和国国家质量监督检验检疫总局, 中国国家标准化管理委员会. 饲料卫生标准: GB 13078—2017 [S]. 北京: 中国标准出版社, 2017: 4
[7] 国家卫生和计划生育委员会, 国家食品药品监督管理总局. 食品中真菌毒素限量: GB 2761—2017[S]. 北京: 中国标准出版社, 2017: 5.
[8] 雷元培, 周建川, 郑文革, 等. 2019-2020年中国饲料原料和饲料中霉菌毒素污染调查报告[J]. 饲料工业, 2022, 43(20): 59-64. DOI: 10.13302/j.cnki.fi.2022.20.010
[9] WANG N, WU W, PAN J, et al. Detoxification Strategies for Zearalenone Using Microorganisms: A Review[J]. Microorganisms, 2019, 7(7): 208. DOI: 10.3390/microorganisms7070208.
[10] MURTAZA B, LI X, DONG L, et al. Microbial and Enzymatic Battle with Food Contaminant Zearalenone (ZEN)[J]. Applied Microbiology and Biotechnology, 2022, 106(12): 4353-4365. DOI: 10.1007/S00253-022-12009-7
[11] 王晓璐, 罗会颖, 姚斌. 饲料霉菌毒素的生物降解研究进展[J]. 动物营养学报, 2022, 34(10): 6152-6159. DOI: 10.3969/j.issn.1006-267x.2022.10.004.
[12] KAKEYA H, TAKAHASHI-ANDO N, KIMURA M, et al. Biotransformation of the Mycotoxin, Zearalenone, to a Non-Estrogenic Compound by a Fungal Strain of Clonostachys sp.[J]. Bioscience Biotechnology and Biochemistry, 2002, 66(12): 2723-2726. DOI: 10.1271/bbb.66.2723.
[13] TAKAHASHI-ANDO N, KIMURA M, KAKEYA H, et al. A Novel Lactonohydrolase Responsible for the Detoxification of Zearalenone: Enzyme Purification and Gene Cloning[J]. Biochemical Journal, 2002, 365: 1-6. DOI: 10.1042/bj20020450.
[14] KOSAWANG C, KARLSSON M, VéL?Z H, et al. Zearalenone Detoxification by Zearalenone Hydrolase is Important for the Antagonistic Ability of Clonostachys rosea against Mycotoxigenic Fusarium graminearum[J]. Fungal Biology, 2014, 118(4): 364-373. DOI: 10.1016/j.funbio.2014.01.005.
[15] ALTALHI A D, EL-DEEB B. Localization of Zearalenone Detoxification Gene(s) in pZEA-1 Plasmid of Pseudomonas putida ZEA-1 and Expressed in Escherichia coli[J]. Journal of Hazardous Materials, 2009, 161: 1166-1172. DOI: 10.1016/j.jhazmat.2008.04.068.
[16] MOLNAR O, SCHATZMAYR G, FUCHS E, et al. Trichosporon mycotoxinivorans sp. nov., a New Yeast Species Useful in Biological Detoxification of Various Mycotoxins[J]. Systematic and Applied Microbiology, 2004, 27(6):661-671. DOI: 10.1078/0723202042369947.
[17] VEKIRU E, HAMETNER C, MITTERBAUER R, et al. Cleavage of Zearalenone by Trichosporon mycotoxinivorans to a Novel Nonestrogenic Metabolite[J]. Applied and Environmental Microbiology. 2010, 76(7): 2353-2359. DOI: 10.1128/AEM.01438-09.
[18] Z?OCH M, ROGOWSKA A, POMASTOWSKI P, et al. Use of Lactobacillus paracasei Strain for Zearalenone Binding and Metabolization[J]. Toxicon, 2020, 181: 9-18. DOI: 10.1016/j.toxicon.2020.03.011.
[19] CHEN SW, WANG HT, SHIH WY, et al. Application of Zearalenone (ZEN)-Detoxifying Bacillus in Animal Feed Decontamination through Fermentation[J]. Toxins (Basel), 2019, 11(6): 330. DOI: 10.3390/toxins11060330.
[20] 段锦, 丁轲, 余祖华, 等. 玉米赤霉烯酮降解菌株的筛选及降解特性研究[J]. 动物营养学报, 2021, 33(09): 5266-5276. DOI: 10.3969/j.issn.1006-267x.2021.09.046.
[21] ZHU Y, DROUIN P, LEPP D, et al. A Novel Microbial Zearalenone Transformation through Phosphorylation[J]. Toxins, 2021, 13, 294. DOI: 10.3390/toxins13050294.
[22] YANG S, ZHENG H, Xu J, et al. New Biotransformation Mode of Zearalenone Identified in Bacillus subtilis Y816 Revealing a Novel ZEN Conjugate[J]. Journal of Agricultural and Food Chemistry, 2021, 69(26): 7409-7419. DOI: 10.1021/acs.jafc.1c01817.
[23] YAMAMOTO S, HARAYAMA S. PCR Amplification and Direct Sequencing of gyrB Genes with Universal Primers and Their Application to the Detection and Taxonomic Analysis of Pseudomonas putida Strains[J]. Applied Environmental Microbiology, 1995, 61(3): 1104-1109. DOI: 10.1016/j.ijms.2010.08.029.
[24] 陈海念, 冯蓉, 杨胜竹, 等. 1株生防菌的鉴定及其发酵条件优化[J]. 浙江大学学报(农业与生命科学版), 2020, 46(02): 177-188. DOI: 10.3785/j.issn.1008-9209.2019.05.301
[25] 傅奇, 林俊杰, 冯亚栋. 16S与gyrB基因联合建树快速鉴定一株解淀粉芽胞杆菌[J].食品与发酵工业, 2020, 46(12): 116-120. DOI: 10.13995/j.cnki.11-1802/ts.023253.
[26] 李献梅, 王小芬, 杨洪岩, 等. 促旋酶(gyrase)B亚单位基因gyrB在鉴别细菌近缘种中的应用[J].微生物学报, 2008, 48(5): 701-706. DOI: 10.3321/j.issn:0001-6209.2008.05.023.
[27] ABDELLI F, JARDAK M, ELLOUMI J, et al. Antibacterial, Anti-adherent and Cytotoxic Activities of Surfactin(s) from a Lipolytic Strain Bacillus safensis F4[J]. Biodegradation, 2019, 30: 287–300. DOI: 10.1007/s10532-018-09865-4.
[28] RONG S, XU H, LI L, et al. Antifungal Activity of Endophytic Bacillus safensis B21 and its Potential Application as a Biopesticide to Control Rice Blast[J]. Pesticide Biochemistry and Physiology, 2020, 162: 69-77. DOI: 10.1016/j.pestbp.2019.09.003.