实验方法与实验动物 EXPERIMENTAL METHOD AND ANIMAL

瘤胃细菌脲酶抑制剂瑞香素的筛选与效果评价

  • 贺越 ,
  • 赵圣国 ,
  • 张晓音 ,
  • 郑楠 ,
  • 王加启
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  • 1. 中国农业科学院北京畜牧兽医研究所, 动物营养学国家重点实验室, 北京 100193;
    2. 中国农业科学院北京畜牧兽医研究所, 农业农村部奶产品质量安全风险评估实验室(北京), 北京 100193
贺越(1996-),女,重庆人,硕士研究生,动物营养与饲料科学专业。E-mail:heyueh@163.com

收稿日期: 2021-01-21

  网络出版日期: 2021-09-18

基金资助

中国农业科学院农业科技创新工程重大产出科研选题(CAAS-ZDXT2019004);中国农业科学院科技创新工程(ASTIP-IAS12);现代农业产业技术体系专项(CARS-36)

Screening and Effect Evaluation of Daphnetin as an Inhibitor of Ruminal Microbial Urease

  • HE Yue ,
  • ZHAO Shengguo ,
  • ZHANG Xiaoyin ,
  • ZHENG Nan ,
  • WANG Jiaqi
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  • 1. State Key Laboratory of Animal Nutrition, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China;
    2. Laboratory of Quality and Safety Risk Assessment for Dairy Products of Ministry of Agriculture and Rural Affairs, Institute of Animal Sciences, Chinese Academy of Agricultural Sciences, Beijing 100193, China

Received date: 2021-01-21

  Online published: 2021-09-18

摘要

本试验旨在获得植物来源的脲酶抑制剂并探究其对瘤胃细菌脲酶抑制的基本特征。首先试验以奶牛瘤胃脲酶为靶标,利用分子对接虚拟筛选植物源化合物库,结合瘤胃细菌脲酶活性抑制试验,探究抑制效果最好的化合物对瘤胃细菌脲酶的半抑制浓度(IC50)值、酶动力学特征以及作用位点,最后通过瘤胃微生物体外厌氧培养试验评价该化合物对尿素分解的影响。结果表明:1)在同浓度条件下,瑞香素对瘤胃细菌脲酶的抑制效果最好。2)瑞香素对瘤胃细菌脲酶的IC50值为(93.43±3.87)μmol/L,是瘤胃细菌脲酶的混合型抑制剂。3)硫醇试剂存在时,瑞香素对瘤胃细菌脲酶的抑制效果显著降低(P<0.05),分子对接结果显示瑞香素与氨基酸残基Ala-166、His-245、Lys-216、Ala-362、Cys-318形成相互作用。4)瑞香素能显著减缓瘤胃微生物体外厌氧培养2、4、6 h后体系中尿素的分解(P<0.05)。综上所述,瑞香素能通过与瘤胃细菌脲酶活性中心的氨基酸相互作用来抑制脲酶的活性,减缓尿素的分解。

本文引用格式

贺越 , 赵圣国 , 张晓音 , 郑楠 , 王加启 . 瘤胃细菌脲酶抑制剂瑞香素的筛选与效果评价[J]. 动物营养学报, 2021 , 33(9) : 5256 -5265 . DOI: 10.3969/j.issn.1006-267x.2021.09.045

Abstract

This study was designed to obtain an urease inhibitor that belonged to plants, and then to explore the basic characteristics of its inhibition to ruminal microbial urease. Firstly, selected plant-sourced compound library was screened via molecular docking with ruminal microbial urease as target, combining ruminal microbial urease inhibition assay to gain the compound with the best inhibition. And then, The half inhibitory concentration (IC50), enzyme kinetic characteristics and acting sites of this compound to ruminal microbial urease were test. Finally, using anaerobic culture test of rumen microorganism in vitro to evaluate its influence to urea. The results showed as follows:1) daphnetin was the compound with the best inhibition under the same concentration. 2) The IC50 of daphnetin to ruminal microbial urease was (93.43±3.87) μmol/L. What's worse, daphnetin was mixed urease inhibitor to ruminal microbial urease. 3) With the interact of thiol reagents, the inhibitory capacity of daphnetin to ruminal microbial urease had decreased significantly (P<0.05), and molecular docking results indicated that daphnetin could interact with amino acid Ala-166, His-245, Lys-216, Ala-362 and Cys-318. 4) Daphnetin could decreased decomposition of urea of anaerobic culture system for rumen microorganisms in vitro markedly after addition of 2, 4 and 6 h (P<0.05). In conclusion, daphnetin inhibits ruminal microbial urease through interacting with some amino acids belong to active site of urease, and reduces urea decomposition.

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