ISOLATION AND CHARACTERIZATION OF RUMINAL PROTEASES

这项研究的长期目标是了解遗传 调节瘤胃微生物活性的因素。 这将提高我们的 确定膳食蛋白质需求的能力，最昂贵的 成分 该提案的具体目标是：1）隔离和 生物化学表征来自主要蛋白水解瘤胃的酶 细菌和2）克隆负责酶表达的基因。 这将是第一个这样的蛋白水解酶分离和特点 来自瘤胃细菌 主要的蛋白水解物质将是 通过培养收集，也许，隔离。 这些将 通过在各种蛋白质上生长筛选蛋白水解活性 来源和粗提物蛋白酶活性。 一种酶， 将选择占优势的进行生化分析。 使用一系列 各种吸附凝胶，蛋白质将被纯化至均一。 生化表征将包括但不限于， 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 测序以允许开发基因探针。 然后，基因将 克隆到E. coli载体pBluescript，并进行测序。 序列 分析将产生有关基因的有价值的信息， 对监管的洞察。 这项研究有几个方面， 与健康有关。 涵盖的学科包括营养， 生物化学和微生物学。 具体来说，反刍动物 营养;碳水化合物、蛋白质和核酸生物化学;以及 胃肠道微生物学将得到解决。 采用的技术 将包括厌氧微生物学，色谱法（柱，HPLC，GC）， 差速离心克隆、测序和电泳。 的 对无菌技术的需求将不断受到强调。 这些学科 和技术都与生物医学高度相关。 一 在这方面受过训练的学生将发展基本的理解 和健康相关职业所需的资格。

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.