Recent Topics Posters
Ihonor, Pamela , Patrick, Denise , Gana, Joyce Ache .
Alfalfa Recombinant Beta Amylase Activity in Vitro.
The exposure of alfalfa to defoliation stress causes large quantities of starch stored in the taproot to be degraded. Starch is degraded to maltose moieties by beta amylase. The involvement of beta amylase in the break down of starch in alfalfa is still uncertain because of its characteristic accumulation and then disappearance following defoliation. The purpose of this research is to purify alfalfa recombinant beta amylase from E-coli, test for activity as well as the rate of reaction. To accomplish this goal, the coding region of the alfalfa beta amylase cDNA was amplified by PCR, cloned into a PET expression vector, and verified for sequence accuracy. Protein expression was performed in E. coli lDE3 lysogen strain using IPTG to induce the target DNA containing beta amylase. Electrophoresis followed by Western blot analysis using sweet potato anti-beta-amylase to detect alfalfa recombinant beta amylase was used to determine if induction had occurred. Alfalfa recombinant beta amylase was purified by affinity chromatography, and its activity was tested with the beta amylase specific substrate p-nitrophenyl-alpha-D-maltopentaoside (PNPG5) containing alpha-glucosidase. Michealis Menten equation was used to study the kinetics of alfalfa recombinant beta amylase. Western blot analysis showed that sweet potato anti-beta- amylase cross-reacted with alfalfa recombinant beta amylase. Also, beta amylase hydrolyzed its specific substrate obeying the Michealis Menten kinetics with a Vmax of 3.4 micro moles per min. Our result shows clearly that alfalfa recombinant beta amylase has high in vitro activity.
Log in to add this item to your schedule
1 - Chicago state university, Biological sciences, Sci 310, Chicago, Illinois, 60628, United states of america
2 - Chicago State University, Biological Sciences, Sci 310, Chicago, IL, 60628
3 - Chicago State University, Biological Sciences, Sci 310, Chicago, IL, 60628, USA
Presentation Type: Recent Topics Poster
Location: Exhibit Hall (Northeast, Southwest & Southeast)/Hilton
Date: Sunday, July 8th, 2007
Time: 8:00 AM