PURIFICATION AND CHARACTERIZATION OF ALPHA-AMYLASE FROM VINE SHOOT INTER-NODES

Soluble sugars arising from starch hydrolysis are thought to enhance f reezing resistance during the winter period. Wood a-amylase (EC 3.2.1. 1) is required for this hydrolysis process in the shoots of woody plan ts. One isoform has been purified from winter-resting vine shoots inte r-nodes (Vitis vinifera L.) by a factor of 1,400. It exhibited a molec ular mass of 71 kDa and an isoelectric point of 6.5. The fixation of c alcium ions by this amylase was sufficient to alter its net charge. He nce, its association with a strong anion exchanger could be reversed i n the presence of 6 mM calcium allowing its partial purification. Fina l purification was achieved by affinity chromatography on epoxy sephar ose 6B beads coated with cycloheptaamylose. The enzymatic activity of the purified enzyme was calcium-dependent with a pH-optimum of 7.0 and a temperature-optimum of 57 degrees C. It was totally inhibited by ED TA and a 50% loss of activity was observed after the addition of 6 mM zinc ions. The presence of calcium ions also conferred a greater therm o-stability to the enzyme.