Analysis of Beta-Glucosidases Active on Isoflavonoid Conjugates

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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.