Beta-Amylases from Alfalfa (Medicago sativa L.) Roots

Amylase was found in high activity (193 international units per milligram protein) in the tap root of alfalfa (Medicago sativa L. cv. Sonora). The activity was separated by gel filtration chromatography into two fractions with molecular weights of 65,700 (heavy amylase) and 41,700 (light amylase). Activity staining of electrophoretic gels indicated the presence of one isozyme in the heavy amylase fraction and two in the light amylase fraction. Three amylase isozymes with electrophoretic mobilities identical to those in the heavy and the light amylase fractions were the only amylases identified in crude root preparations. Both heavy and light amylases hydrolyzed amylopectin, soluble starch, and amylose but did not hydrolyze pullulan or beta-limit dextrin. The ratio of viscosity change to reducing power production during starch hydrolysis was identical for both alfalfa amylase fractions and sweet potato beta-amylase, while that of bacterial alpha-amylase was considerably higher. The identification of maltose and beta-limit dextrin as hydrolytic end-products confirmed that these alfalfa root amylases are all beta-amylases.The pH optimum for both beta-amylase fractions was 6.0. Both light and heavy beta-amylases showed normal Michaelis-Menten kinetics, with soluble starch as substrate, and had respectively K(m) values of 5.9 and 6.8 milligrams starch per milliliter and V(max) of 640 and 130 international units per milligram protein. Arrhenius plots indicated that the energy of activation for the heavy beta-amylase remained relatively unchanged (12.7 to 13.0 kilocalories per mole) from 0 to 30 degrees C, whereas the energy of activation for the light amylase increased from 12.0 to about 28.0 kilocalories per mole at 8.7 degrees C as temperature was lowered. The light amylase was shown to be inhibited by maltose.