Purification and properties of a starch granule-degrading α-amylase from potato tubers

Abstract

An α-amylase (EC 3.2.1.1) was purified to apparent electrophoretic homogeneity from potato (Solanum tuberosum L.) tubers by affinity chromatography on a starch granule column, Q-Sepharose chromatography, and gel filtration. The enzyme was purified 24 300-fold over the crude extract of soluble proteins with a yield of 13.2% to a specific activity of 824 μmol min -1 mg -1 . The classification as α-amylase was verified by substrate specificity and identification by HPLC of the degradation products. This amylase migrated on SDS-PAGE gels at 44 kDa, while it was detected by native PAGE as a band on top of separation gels comprising amylopectin. The optimum pH value was between 7.2 and 8.0. The increase of the pronounced heat-stability in the presence of CaCl 2 as well as the lack of response to EDTA indicated that the activity was not stabilized, but rather inhibited by Ca 2+ . The purified α-amylase from potato tubers was reversibly bound to starch granules from the same source, and the enzyme was able to catalyse the degradation of native granules in vitro. The observation that an amylase activity with the same characteristics as the soluble form was also associated to freshly prepared granules indicated that the enzyme was at least partially localized in plastids. The results suggest that this starch hydrolase is involved in the initiation of reserve starch dissolution in potato tubers.