ISOLATION AND CHARACTERIZATION OF RUMINAL PROTEASES

这项研究的长期目标是了解基因 调节瘤胃微生物活动的因素。这将增强我们的 确定膳食蛋白质需求的能力是最昂贵的 组件。这项提议的具体目的是1)隔离和 瘤胃主要蛋白水解酶的生化特性 细菌和2)克隆负责该酶表达的基因。 这将是第一次分离和鉴定这种蛋白水解酶。 来自一种瘤胃细菌。主要的蛋白水解物将是 通过收集培养物，或许还可以通过隔离获得。这些遗嘱 通过生长在不同的蛋白质上筛选蛋白质分解活性 来源和粗提物的蛋白酶活性。一种似乎是 将选择优势种进行生化分析。使用一系列 各种吸附凝胶，将蛋白质提纯至均一。 生化特征将包括但不限于， 分子大小(SDS-PAGE、凝胶过滤)、比活性和动力学 (v/max，K/m)。此外，该蛋白质将受到N-末端的影响 测序以允许基因探针的开发。然后这个基因就会变成 将其克隆到大肠杆菌载体pBluescrip中，并进行测序。数列 分析将产生关于基因的有价值的信息，可能提供 对监管的洞察。这项研究的几个方面 都与健康有关。将涵盖的学科包括营养学、 生物化学和微生物学。具体地说，反刍动物的区域 营养；碳水化合物、蛋白质和核酸生物化学； 课程内容包括胃肠微生物学。所采用的技术 将包括厌氧微生物、层析(柱、高效液相、气相色谱)、 差速离心法克隆、测序和电泳法。这个 对无菌技术的需求将不断得到强调。这些学科 和技术都与生物医学科学高度相关。一个 在这一领域接受培训的学生将发展基本的理解 以及从事与健康相关的职业所需的资格。

The long-term objective of this research is to understand the genetic factors that regulate rumen microbial activity. This will enhance our ability to determine dietary protein requirements, the most expensive component. The specific aims of this proposal are to 1) isolate and biochemically characterize an enzyme from a predominant proteolytic rumen bacterium and 2) clone the gene responsible for expression of the enzyme. This will be the first such proteolytic enzyme isolated and characterized from a ruminal bacterium. Predominant proteolytic species will be obtained through culture collections and, perhaps, isolation. These will be screen for proteolytic activities through growth on various protein sources and crude extract protease activity. An enzyme that appears to be predominant will be selected for biochemical analysis. Using a series of various adsorption gels, the protein will be purified to homogeneity. Biochemical characterization will include, but not be limited to, molecular size (SDS-PAGE, gel filtration), specific activity, and kinetics (V/max, K/m). In addition, the protein will be subject to N-terminal sequencing to allow development of gene-probes. The gene will then be cloned into an E. coli vector pBluescript, and sequenced. Sequence analysis will yield valuable information about the gene that may provide insight into regulation. There are several aspects of this research that are health-related. The disciplines to be covered include nutrition, biochemistry, and microbiology. Specifically, the areas of ruminant nutrition; carbohydrate, protein, and nucleic acid biochemistry; and gastrointestinal microbiology will be addressed. The techniques employed will include anaerobic microbiology, chromatography (column, HPLC, GC), differential centrifugation cloning, sequencing, and electrophoresis. The need for sterile technique will be constantly stressed. These disciplines and techniques are all highly relevant to the biomedical sciences. A student trained in this area will develop the fundamental understanding and qualifications necessary for a health-related career.