Purification and Characterization of Pea Epicotyl β-Amylase

Abstract

The most abundant .beta.-amylase (EC 3.2.1.2) in pea (Pisum sativum L.) was purified greater than 880-fold from epicotyls of etiolated germinating seedlings by anion exchange and gel filtration chromatography, glycogen precipitation, and preparative electrophoresis. The electrophoretic mobility and relative abundance of this .beta.-amylase are the same as that of an exoamylase previously reported to be primarily vacuolar. The enzyme was determined to be a .beta.-amylase by end product analysis and by its inability to hydrolyze .beta.-limit dextrin and to release dye from starch azure. Pea .beta.-amylase is an approximate 55 to 57 kilodalton monomer with a pl of 4.35, a pH optimum of 6.0 (soluble starch substrate), an Arrhenius energy of activation of 6.28 kilocalories per mole, and a Km of 1.67 milligrams per milliliter (soluble starch). The enzyme is strongly inhibited by heavy metals, p-chloromercuriphenylsulfonic acid and N-ethylmaleimide, but much less strongly by iodoacetamide and iodoacetic acid, indicating cysteinyl sulfhydryls are not directly involved in catalysis. Pea .beta.-amylase is competitively inhibited by its end product, maltose, with a Ki of 11.5 millimolar. The enzyme is partially inhibited by Schardinger maltodextrins, with .alpha.-cyclohexaamylose being a stronger inhibitor than .beta.-cycloheptaamylose. Moderately branched glucans (e.g. amylopectin) were better substrates for pea .beta.-amylase than less branched or non-branched (amyloses) or highly branched (glycogens) glucans. The enzyme failed to hydrolyze native starch grains from pea and glucans smaller than maltotetraose. The mechanism of pea .beta.-amylase is the multichain type. Possible roles of pea .beta.-amylase in cellular glucan metabolism are discussed.