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

Abstract

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 β-limit dextrin. The ratio of viscosity change to reducing power production during starch hydrolysis was identical for both alfalfa amylase fractions and sweet potato β-amylase, while that of bacterial α-amylase was considerably higher. The identification of maltose and β-limit dextrin as hydrolytic end-products confirmed that these alfalfa root amylases are all β-amylases.

The pH optimum for both β-amylase fractions was 6.0. Both light and heavy β-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 β-amylase remained relatively unchanged (12.7 to 13.0 kilocalories per mole) from 0 to 30°C, whereas the energy of activation for the light amylase increased from 12.0 to about 28.0 kilocalories per mole at 8.7°C as temperature was lowered. The light amylase was shown to be inhibited by maltose.