Isolation and Purification of Amylase from Sugarcane Leaves

Investigations were made concerning the number and properties of amylase in sugarcane leaves. Enzymes were extracted froin leaf samples which had been frozen, lyophilized, and ground to a fine powder. Techniques of differential solubihty, gel filtiation, and papei electrophoresis were employed to remove amylase from the crude extracts. Solubility studies showed that amylase was moderately precipitated between 30and 38-percent saturation by ammonium sulfate, and more heavlly between 46and 62-percent saturation. Filtration experiments with Sephadex G-zoo succeeded in separating amylases from the bullt of non-catalytic piotein. This was possible when gel columns were paclred with water 01 0.10 M NaC1, but not when paclred with 0.10 M phosphate buffer. Amylase action was not reta~ded by filtration or pleliin~nary dialysis. No evidence of cofactors or endogenous inhibitors was found. Electrophoresis experiments revealed that most of the salt-fractionated protein bore a . negative charge, and that amylases were predoiniilantly positive. However, gel filtration changed the electrophoretic behavior of amylases so that negative, positive, and essentially neutral catalysts were obtained. Significance of these phenomena are discussed.