Analysis of Beta-Glucosidases Active on Isoflavonoid Conjugates

GRANT=S06GM8003-33-0011 异黄酮类化合物是食物(营养食品)的潜在重要成分，因为富含异黄酮类化合物的饮食被认为可以降低某些癌症、骨质疏松症和其他人类疾病的发病率。在许多大豆食品中，很大比例的异黄酮类化合物最初是以葡萄糖结合物(7-0-葡萄糖苷)的形式存在的，这些葡萄糖结合物在消化前或消化过程中的水解会极大地影响异黄酮类化合物的摄取、生物利用度和稳定性。尽管它们具有潜在的重要性，但关于专门裂解异黄酮类结合物的β-葡萄糖苷酶的研究还很少。这项建议的一个主要焦点将涉及对异黄酮类结合物具有高度专一性的植物β-葡萄糖苷酶(Bg1和Bg2)的生化和分子分析，这些克隆是最近从元胞紫花苜蓿(Medicago Truncatula)中分离出来的，紫花苜蓿是世界范围内研究的模式豆科植物。在对这些新发现的植物酶进行微生物表达的初步研究后，将产生突变酶并进行生化特征研究，以确定哪些结构元素影响它们不同的底物特异性。从大豆中分离出更多的β-葡萄糖苷酶克隆，以揭示该豆科植物底物专一性和异黄酮类代谢的结构决定因素的更多信息，该豆科植物是大豆异黄酮类的主要食物来源。Bg1和Bg2在植物细胞中的位置和它们的翻译后修饰将通过生化和瞬时植物转化技术相结合的方法进行研究。同时，将对Bg1和Bg2在毕赤酵母中的表达进行放大的初步实验。如果成功，更晚的目标将是寻找β-葡萄糖苷酶对异黄酮类化合物的摄取和随后的生物学效应。这些研究还将为RISE资助的本科生提供现代分子和生化技术方面的培训机会。这些研究还将为ISE资助的本科生提供现代分子和生化技术方面的培训机会，为他们未来在生物医学研究中的职业生涯做好准备。

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.