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Biuret hydrolase and cyanuric acid hydrolase: Enzymes for metabolism and detection of s-triazines.

机译：缩二脲水解酶和氰尿酸水解酶：用于代谢和检测s-三嗪的酶。

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摘要

There is an increasing need to test for chemicals present in adulterated foods or food products. There is also an increasing need to degrade chemicals present in water. The s-triazine compounds compose a major class of industrial chemicals. The research in our labs focuses on developing microbial enzyme-based bioassays for s-triazines and understanding the enzymatic biodegradation of them. Cyanuric acid is an intermediate in bacterial metabolism of common s-triazine compounds.;In 2008, intentional adulteration of milk and milk products (e.g., infant formula) with the s-triazine melamine affected 300,000 infants & youth in China, resulting in 50,000 hospitalizations and 6 deaths. In 2010, tons of melamine-tainted milk products were again for sale. Melamine is sometimes co-contaminated with cyanuric acid. When ingested together, they form insoluble crystals in kidneys, which may lead to renal failure. This scenario caused >13,000 pet deaths in the U.S. (2007) due to adulterated pet food. There is interest in detecting cyanuric acid and melamine, individually or together, in foods.;With regards to s-triazines in water, cyanuric acid is a stable compound that accumulates over time in swimming pools disinfected with N-chloroisocyanuric acids. With high concentrations of cyanuric acid, disinfection is ineffective and public health is not protected from disease-causing microorganisms. To address this concern, the pool is drained, refilled with fresh water, and treated again.;The research here identifies and characterizes microbial enzymes involved in s-triazine metabolism that could be used to 1) develop sensitive bioassays for cyanuric acid and/or 2) biodegrade cyanuric acid in water. Cyanuric acid hydrolase acts on cyanuric acid. Using 13C nuclear magnetic resonance (NMR) and mass spectrometry (MS), this research establishes the product of this reaction is carboxybiuret. This work also demonstrates carboxybiuret spontaneously decarboxylates to generate biuret. In the subsequent reaction, biuret hydrolase acts on biuret. Using an interdisciplinary approach combining bioinformatics, microbiology, molecular biology, protein separations, enzymology, quantitative analyses, 13C NMR, and MS, this research identifies and characterizes the first biuret hydrolase to be purified in active form (from R. leguminosarum bv. viciae 3841). Together, cyanuric acid and biuret hydrolases release ammonia, which is detected via the colorimetric Berthelot Assay.;Cyanuric acid hydrolase belongs to rare family of unstable proteins. In an effort to identify a stable protein scaffold that could be modified to hydrolyze cyanuric acid, an array of amidohydrolase family members were analyzed for low-level activity with cyanuric acid to use as a starting point. Guanine deaminase from Bradyrhizobium diazoefficiens USDA110 was observed to have such activity. This is the only known metallo-enzyme to hydrolyze a ring-opening amide bond of cyanuric acid.
机译：越来越需要测试掺假食品或食品中存在的化学物质。也越来越需要降解水中存在的化学物质。 S-三嗪化合物构成一类主要的工业化学品。我们实验室的研究重点是针对S-三嗪开发基于微生物酶的生物测定方法，并了解它们的酶促生物降解能力。氰尿酸是常见的s-三嗪化合物细菌代谢的中间体。2008年，中国将300,000婴幼儿故意掺入牛奶和奶制品（例如婴儿配方奶粉）和s-三嗪三聚氰胺，导致50,000住院治疗和6人死亡。 2010年，再次出售了三吨受三聚氰胺污染的奶制品。三聚氰胺有时会与氰尿酸一起被污染。一起摄入时，它们在肾脏中形成不溶性晶体，可能导致肾衰竭。在这种情况下，由于宠物食品掺假，在美国（2007年）导致超过13,000例宠物死亡。对于食品中的氰尿酸和三聚氰胺，无论是单独还是一起检测，都有兴趣。关于水中的三嗪，氰尿酸是一种稳定的化合物，随着时间的推移会累积在用N-氯异氰尿酸消毒的游泳池中。氰尿酸浓度高时，消毒效果不佳，并且未保护公共卫生免受致病微生物的侵害。为了解决这一问题，将池中的水排干，重新装满淡水，然后再次进行处理。；此处的研究确定并表征了与S-三嗪代谢有关的微生物酶，这些酶可用于1）开发氰尿酸和/或氰尿酸的敏感生物测定法2）在水中生物降解氰尿酸。三聚氰酸水解酶作用于三聚氰酸。利用13 C核磁共振（NMR）和质谱（MS），这项研究确定了该反应的产物是羧基缩二脲。这项工作还证明了羧基缩二脲自发地脱羧生成缩二脲。在随后的反应中，缩二脲水解酶作用于缩二脲。使用跨学科的方法，结合了生物信息学，微生物学，分子生物学，蛋白质分离，酶学，定量分析，13C NMR和MS，该研究鉴定并鉴定了第一种以活性形式纯化的缩二脲水解酶（来自豆科植物豌豆（R. leguminosarum bv。viciae 3841）） ）。氰尿酸和缩二脲水解酶一起释放出氨，这可通过比色Berthelot测定法检测。氰尿酸水解酶属于稀有不稳定蛋白家族。为了确定可以被修饰以水解氰尿酸的稳定的蛋白质支架，分析了一系列酰胺水解酶家族成员的氰尿酸的低水平活性，以此作为起点。观察到重氮根瘤菌USDA110的鸟嘌呤脱氨酶具有这种活性。这是唯一已知的水解氰尿酸的开环酰胺键的金属酶。

著录项

  • 作者

    Cameron, Stephan Mary.;

  • 作者单位

    University of Minnesota.;

  • 授予单位 University of Minnesota.;
  • 学科 Chemistry Biochemistry.;Biology Molecular.;Biology Microbiology.
  • 学位 Ph.D.
  • 年度 2013
  • 页码 326 p.
  • 总页数 326
  • 原文格式 PDF
  • 正文语种 eng
  • 中图分类
  • 关键词

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