Electrophoretically mediated reaction of glycosidases at a nanoliter scale.
We have investigated electrophoretically mediated microanalysis (EMMA) for the assay of a native glyco-enzyme. As a representative of this class of enzyme, beta-glucosidase was selected, and the reaction was analyzed. Our EMMA was based on the plug-plug interaction of enzyme and substrate plugs, which is essential to reduce quantities of materials. Furthermore, we have addressed the problem of incompatibility of the enzymatic reaction and separation of the reactants. As a result, EMMA of native glycosidase was achieved with a reaction volume of approximately 20 nL and the Michaelis constant was estimated according to the Lineweaver-Burk plot. The current method may have advantages over traditional assay methods, especially in terms of the amount of enzyme (ng order) and substrate (pmol order) required for a reaction.
Kanie
Yoshimi
Y
Mitsubishi Kagaku Institute of Life Sciences (MITILS), Tokyo, Japan.
Kanie
Osamu
O
D001236:Aspergillus oryzae / Q000201:enzymology; D019075:Electrophoresis, Capillary / Q000379:methods*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D036103:Nanotechnology / Q000379:methods*; D009994:Osmolar Concentration; D027861:Prunus / Q000201:enzymology; D001617:beta-Glucosidase / Q000378:metabolism*
D001617:beta-Glucosidase