来源于<i>Exophiala aquamarina</i>的新型玉米赤霉烯酮水解酶的性质及底物结合中心关键氨基酸的功能研究<sup>*</sup>

doi:10.13523/j.cb.2105018

中国生物工程杂志

2021, Vol. 41

Issue (10): 19-27 DOI: 10.13523/j.cb.2105018

研究报告

来源于Exophiala aquamarina的新型玉米赤霉烯酮水解酶的性质及底物结合中心关键氨基酸的功能研究^*

梁爱玲^1,²,刘文婷^1,²,武攀^1,²,李倩²,高健²,张洁²,刘卫东²,贾士儒^1,^**(

),郑迎迎^2,^**(

)

1 天津科技大学生物工程学院工业微生物教育部重点实验室天津 300457
2 中国科学院天津工业生物技术研究所天津 300308

Characterization and Function of Key Amino Acids in Substrate Bingding Center of a Novel Zearalenone Hydrolase from Exophiala aquamarina

LIANG Ai-ling^1,²,LIU Wen-ting^1,²,WU Pan^1,²,LI Qian²,GAO Jian²,ZHANG Jie²,LIU Wei-dong²,JIA Shi-ru^1,^**(

),ZHENG Ying-ying^2,^**(

)

1 Key Lab of Industrial Microbiology, College of Biotechnology, Tianjin University of Science & Technology, Tianjin 300457, China
2 Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China

全文: PDF(1634 KB) HTML

摘要：

目的:开发一种对α-玉米赤霉烯醇(α-zearalenol,α-ZOL)催化活性高的水解酶,以促进玉米赤霉烯酮的酶法完全脱毒,推动玉米赤霉烯酮降解酶在饲料业和畜牧业的应用。方法:在NCBI数据库中通过基因挖掘获得一个来源于Exophiala aquamarina CBS 119918的新型玉米赤霉烯酮水解酶EaZHD,基因全长792 bp。构建重组质粒pET46-Eazhd,并在大肠杆菌中成功表达。经Ni-NTA亲和层析和DEAE阴离子交换柱纯化后,利用HPLC测定残余底物浓度的方法来表征其酶学性质。结果:EaZHD对玉米赤霉烯酮(zearalenone,ZEN)的酶活力为0.764 U/mg,而对α-ZOL的酶活力为1.529 U/mg,是对底物ZEN降解活力的2倍。EaZHD在pH 8.6和温度40℃条件下的活性最高,并具有较好的热稳定性和碱性条件下较高的pH稳定性。通过对EaZHD活性中心附近的氨基酸进行突变和活性分析,确定了对催化活性起关键作用的三个氨基酸,以及对底物特异性有影响的两个关键氨基酸。结论:为推动饲料业和畜牧业中玉米赤霉烯酮的酶法脱毒奠定了基础。

关键词： 新型玉米赤霉烯酮水解酶; α-玉米赤霉烯醇; 酶学性质; 酶法脱毒

Abstract:

Objective: In order to promote the enzymatic complete detoxification of zearalenone and the application in feed industry, we proposed to develop a new hydrolase exhibiting higher activity to α-ZOL. Methods: A novel zearalenone hydrolase EaZHD sequence from Exophiala aquamarina CBS 119918 with a total length of 263 amino acids was obtained. The recombinant plasmid pET46-Eazhd was constructed and expressed in E. coli. Then EaZHD was purified with Ni-NTA affinity chromatography and DEAE ion exchange column. The enzymatic properties and activity analysis were evaluated with HPLC. Results: The results showed that the activity of EaZHD was 0.764 U/mg against ZEN and 1.529 U/mg against α-ZOL, which is 2-fold higher. The optimal pH of EaZHD was 8.6 and the optimal temperature was 40 °C. It had a better stability at alkaline pH. The thermal stability is better than ZHD101. Three amino acid residues around active site were identified to play a key role in the catalysis activity and the other two residues were found to affect the substrate specificity. Conclusion: The study provides the basis for the enzymatic detoxification of zearalenone in the feed and stock raising industries.

Key words: Novel zearalenone hydrolase α-Zearalenol Enzymatic properties Enzymatic detoxification

收稿日期: 2021-05-10 出版日期: 2021-11-08

ZTFLH:

Q819

基金资助: * 国家重点研发计划(2019YFA0905100);* 国家重点研发计划(2018YFA0901201);天津市科学基金(18JCYBJC24300);天津市合成生物技术创新能力提升行动(TSBICIP-KJGG-002-06);天津市合成生物技术创新能力提升行动(TSBICIP-KJGG-001)

通讯作者: 贾士儒,郑迎迎 E-mail: jiashiru@tust.edu.cn;zheng_yy@tib.cas.cn

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引用本文:

梁爱玲,刘文婷,武攀,李倩,高健,张洁,刘卫东,贾士儒,郑迎迎. 来源于Exophiala aquamarina的新型玉米赤霉烯酮水解酶的性质及底物结合中心关键氨基酸的功能研究^*[J]. 中国生物工程杂志, 2021, 41(10): 19-27.

LIANG Ai-ling,LIU Wen-ting,WU Pan,LI Qian,GAO Jian,ZHANG Jie,LIU Wei-dong,JIA Shi-ru,ZHENG Ying-ying. Characterization and Function of Key Amino Acids in Substrate Bingding Center of a Novel Zearalenone Hydrolase from Exophiala aquamarina. China Biotechnology, 2021, 41(10): 19-27.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2105018 或 https://manu60.magtech.com.cn/biotech/CN/Y2021/V41/I10/19

图1 EaZHD与其他同源蛋白的序列比对

表1 引物列表

图2 Eazhd的PCR结果

图3 EaZHD的SDS-PAGE分析

图4 EaZHD与其他常见ZHD的活性比对

图5 pH对EaZHD活性及稳定性的影响

图6 温度对EaZHD活性及其稳定性的影响

图7 EaZHD的模拟结构与ZHD101结构的对比

图8 ZEN和α-ZOL的化学结构式

图9 EaZHD突变体的活性测定

图10 EaZHD野生型与突变体的分子结构比较

[1]	Moss M O. The environmental factors controlling mycotoxin formation//Smith J E, Anderson R A. Mycotoxins and Animal Foods. Boca Raton FL: CRC Press, 1991: 37-56.
[2]	Speight N. Mycotoxin-related illness//Kohlstadt I. Advancing Medicine with Food and Nutrients, 2nd ed. Boca Raton FL: CRC Press, 2012: 821-850.
[3]	Hussein H S, Brasel J M. Toxicity, metabolism, and impact of mycotoxins on humans and animals. Toxicology, 2001, 167(2): 101-134. pmid: 11567776
[4]	Shi H T, Li S L, Bai Y Y, et al. Mycotoxin contamination of food and feed in China: occurrence, detection techniques, toxicological effects and advances in mitigation technologies. Food Control, 2018, 91: 202-215. doi: 10.1016/j.foodcont.2018.03.036
[5]	Shang S H. Economic issues associated with aflatoxins//Eaton D L, Groopman J D. The Toxicology of Aflatoxins: Human Health, Veterinary, and Agricultural Significance. San Diego: Academic Press, 1994: 513-527.
[6]	Vasanthi S, Bhat R V. Mycotoxins in foods-occurrence, health & economic significance & food control measures. The Indian Journal of Medical Research, 1998, 108: 212-224.
[7]	Pandya J P, Arade P C. Mycotoxin: a devil of human, animal and crop health. Advances in Life Sciences, 2016, 5: 3937-3941.
[8]	Liew W P P, Mohd-Redzwan S. Mycotoxin: its impact on gut health and microbiota. Frontiers in Cellular and Infection Microbiology, 2018, 8: 60. doi: 10.3389/fcimb.2018.00060
[9]	Ates E, Mittendorf K, Stroka J, et al. Determination of Fusarium mycotoxins in wheat, maize and animal feed using on-line clean-up with high resolution mass spectrometry. Food Additives & Contaminants: 2013, 30(1): 156-165.
[10]	Kotowicz N K, Frac M, Lipiec J. The importance of Fusarium fungi in wheat cultivation-pathogenicity and mycotoxins production: a review. Journal of Animal & Plant Sciences, 2014, 21: 3326-3243.
[11]	Kolb E. Recent knowledge on the mechanism of action and metabolism of mycotoxins. Zeitschrift Fur Die Gesamte Innere Medizin Und Ihre Grenzgebiete, 1984, 39(15): 353-358.
[12]	Diekman M A, Green M L. Mycotoxins and reproduction in domestic livestock. Journal of Animal Science, 1992, 70(5): 1615-1627. pmid: 1388147
[13]	Zhang Y Y, Jia Z Q, Yin S T, et al. Toxic effects of maternal Zearalenone exposure on uterine capacity and fetal development in gestation rats. Reproductive Sciences (Thousand Oaks, Calif), 2014, 21(6): 743-753.
[14]	Rogowska A, Pomastowski P, Sagandykova G, et al. Zearalenone and its metabolites: effect on human health, metabolism and neutralisation methods. Toxicon, 2019, 162: 46-56. doi: S0041-0101(19)30065-0 pmid: 30851274
[15]	Zhou H Y, George S, Hay C, et al. Individual and combined effects of aflatoxin B1, deoxynivalenol and zearalenone on HepG2 and RAW 264.7 cell lines. Food and Chemical Toxicology, 2017, 103: 18-27. doi: 10.1016/j.fct.2017.02.017
[16]	Shier W T, Shier A C, Xie W, et al. Structure-activity relationships for human estrogenic activity in zearalenone mycotoxins. Toxicon, 2001, 39(9): 1435-1438. pmid: 11384734
[17]	Jouany J P. Methods for preventing, decontaminating and minimizing the toxicity of mycotoxins in feeds. Animal Feed Science and Technology, 2007, 137(3-4): 342-362. doi: 10.1016/j.anifeedsci.2007.06.009
[18]	Kalagatur N K, Kamasani J R, Mudili V. Assessment of detoxification efficacy of irradiation on zearalenone mycotoxin in various fruit juices by response surface methodology and elucidation of its in-vitro toxicity. Frontiers in Microbiology, 2018, 9: 2937-2950. doi: 10.3389/fmicb.2018.02937 pmid: 30555450
[19]	Vila-Donat P, Marín S, Sanchis V, et al. A review of the mycotoxin adsorbing agents, with an emphasis on their multi-binding capacity, for animal feed decontamination. Food and Chemical Toxicology, 2018, 114: 246-259. doi: S0278-6915(18)30118-2 pmid: 29476792
[20]	Xu Y, Wang Y F, Ji J, et al. Chemical and toxicological alterations of zearalenone under ozone treatment. Food Additives & Contaminants, 2019, 36(1): 163-174.
[21]	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. BBioscience, iotechnology & Biochemistry, 2002, 66(12): 2723-2726.
[22]	Utermark J, Karlovsky P. Role of zearalenone lactonase in protection of Gliocladium roseum from fungitoxic effects of the mycotoxin zearalenone. Applied and Environmental Microbiology, 2007, 73(2): 637-642. pmid: 17114328
[23]	Takahashi-Ando N, Kimura M, Kakeya H, et al. A novel lactonohydrolase responsible for the detoxification of zearalenone: enzyme purification and gene cloning. Biochemical Journal, 2002, 365(1): 1-6. doi: 10.1042/bj20020450
[24]	Peng W, Ko T P, Yang Y Y, et al. Crystal structure and substrate-binding mode of the mycoestrogen-detoxifying lactonase ZHD from Clonostachys rosea. RSC Advances, 2014, 4(107): 62321-62325. doi: 10.1039/C4RA12111B
[25]	Xu Z X, Liu W D, Chen C C, et al. Enhanced α-zearalenol hydrolyzing activity of a mycoestrogen-detoxifying lactonase by structure-based engineering. ACS Catalysis, 2016, 6(11): 7657-7663. doi: 10.1021/acscatal.6b01826
[26]	Zheng Y Y, Liu W D, Chen C C, et al. Crystal structure of a mycoestrogen-detoxifying lactonase from Rhinocladiella mackenziei: molecular insight into ZHD substrate selectivity. ACS Catalysis, 2018, 8(5): 4294-4298. doi: 10.1021/acscatal.8b00464
[27]	Wang M X, Yin L F, Hu H Z, et al. Expression, functional analysis and mutation of a novel neutral zearalenone-degrading enzyme. International Journal of Biological Macromolecules, 2018, 118: 1284-1292. doi: 10.1016/j.ijbiomac.2018.06.111

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