Analysis of Beta-Glucosidases Active on Isoflavonoid Conjugates

对异黄酮类化合物具有活性的 β-葡萄糖苷酶的分析

• 批准号: 7271902
• 负责人: Nancy Paiva
• 金额: $ 11.28万
• 依托单位: SOUTHEASTERN OKLAHOMA STATE UNIVERSITY
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7271902
• 关键词: Ascomycetes; alfalfa; beta glucosidases; chemical conjugate; dietary constituent; enzyme mechanism; enzyme structure; flavonoids; glycosides; isozymes; plant physiology; plant proteins; protein engineering; soybeans

GRANT=S06GM8003-33-0011 Isoflavonoids are potentially important constituent of foods ("nutriceuticals"), in that diets rich in isoflavinoids are thought to decrease rates of certain cancers, osteoporosis, and other human diseases. In many foods derived from soybean, a large percentage of the isoflavinoids are initially present as glucose conjugates (7-0-glucosides), and hydrolysis of these glucose conjugates before or during digestion may greatly affect the uptake, bioavailability, and stability of isoflavinoids. Despite their potential importance, little has been published on beta-glucosidases which specifically cleave isoflavonoid conjugates. A major focus of this proposal will involve the biochemical and molecular analysis of plant beta-glucosidases (BG1 and BG2) with high specificity towards isoflavonoid conjugates, for which clones were recently isolated from Medicago truncatula a model legume species studied world-wide. After preliminary studies to improve microbial expression of these newly identified plant enzymes, mutant enzymes will be generated and biochemically characterized to determine which structural elements influence their differing substrate specificities. Additional beta-glucosidase clones will be isolated from soybean (Glycine max), to reveal additional information about structural determinants of substrate specificity and isoflavonoid metabolism in this legume species which serves as the major diet source isoflavonoids. The location of BG1 and BG2 within plant cells and their post-translational modifications will be examined by a combination of biochemical and transient plant transformation techniques. In parallel, preliminary experiments on the scale-up of the expression of BG1 and BG2 in Pichia will be carried out. If successful, a later goal will be to seek beta-glucosidases on isoflavonoid uptake and subsequent biological effects. These studies will also provide training opportunities for RISE-funded undergraduates in modern molecular and biochemical techniques. These studies will also provide training opportunities for ISE-funded undergraduates in modern molecular and biochemical techniques, preparing them for future careers in biomedical research.

格兰特= S06GM8003-33-0011 异黄素可能是食品的重要组成部分（“营养学家”），因为富含异黄素的饮食被认为会降低某些癌症，骨质疏松症和其他人类疾病的速度。在许多源自大豆的食物中，很大一部分的异黄素最初以葡萄糖结合物（7-0-葡萄糖剂）的形式存在，并且在消化之前或期间对这些葡萄糖结合物的水解可能会极大地影响摄取，生物利用度和同类药物的稳定性。尽管具有潜在的重要性，但在β-葡萄糖苷酶方面几乎没有发表，这些β-葡萄糖苷酶特别裂解异黄素结合物。该提案的主要重点将涉及对植物β-葡萄糖苷酶（BG1和BG2）的生化和分子分析，这些植物β-葡萄糖苷酶（BG1和BG2）具有很高的特异性对异类球体共轭物，最近将克隆从Medicalago truncatula分离出来，从Medicalago truncatula分离出一种全球所研究的模型群。在初步研究以改善这些新鉴定的植物酶的微生物表达后，将产生突变酶并在生化上表征以确定哪些结构元素会影响其不同的底物特异性。将从大豆（甘氨酸最大）中分离出其他β-葡萄糖苷酶克隆，以揭示有关这种豆类物种中有关底物特异性的结构决定因素和异黄素代谢的其他信息，这些豆类物种是主要饮食来源的饮食来源异黄素。 BG1和BG2在植物细胞中及其翻译后修饰的位置将通过生化和瞬时植物转化技术的组合来检查。同时，将进行有关BG1和BG2在Pichia中表达的扩大的初步实验。如果成功的话，以后的目标将是在异黄体摄取和随后的生物学作用上寻求β-葡萄糖苷酶。这些研究还将为现代分子和生化技术的上升资助的本科生提供培训机会。这些研究还将为ISE资助的现代分子和生化技术提供培训机会，为生物医学研究的未来职业做好准备。