Analysis of Beta-Glucosidases Active on Isoflavonoid Conjugates

GRANT=S06GM8003-33-0011 异黄酮是食物（“营养品”）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。在许多来源于大豆的食物中，大百分比的异黄烷酸最初以葡萄糖缀合物（7-0-葡糖苷）的形式存在，并且这些葡萄糖缀合物在消化之前或消化期间的水解可能极大地影响异黄烷酸的摄取、生物利用度和稳定性。尽管它们具有潜在的重要性，但很少有关于特异性切割β-葡糖苷酶缀合物的β-葡糖苷酶的报道。该提案的一个主要重点将涉及植物β-葡萄糖苷酶（BG 1和BG 2）的生物化学和分子分析，具有高特异性对葡聚糖缀合物，其克隆最近分离自苜蓿，模式豆科植物物种在世界范围内研究。经过初步研究，以提高这些新鉴定的植物酶的微生物表达，突变酶将产生和生化特性，以确定哪些结构要素影响其不同的底物特异性。将从大豆（Glycine max）中分离其他β-葡萄糖苷酶克隆，以揭示关于该豆类物种（作为主要饮食来源大豆）中底物特异性和大豆代谢的结构决定因素的其他信息。BG 1和BG 2在植物细胞内的位置及其翻译后修饰将通过生物化学和瞬时植物转化技术的组合进行检查。同时，将进行在毕赤酵母中扩大BG 1和BG 2表达的初步实验。如果成功的话，以后的目标将是寻找β-葡萄糖苷酶对葡萄糖摄取和随后的生物效应。这些研究还将为RISE资助的本科生提供现代分子和生物化学技术的培训机会。这些研究还将为ISE资助的本科生提供现代分子和生物化学技术的培训机会，为他们将来从事生物医学研究做好准备。

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.