ISOLATION AND CHARACTERIZATION OF RUMINAL PROTEASES

这项研究的长期目标是了解基因

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.