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

Abstract

An a-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 //mol min" 1 mg 1 . The classification as a-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-lability in the presence of CaCI 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 a-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.

Germination Conditions Affect Physicochemical Properties of Germinated Brown Rice Flour

“…As steeping continued from 24 to 36 and to 48 h, α‐amylase activity of all GNBR significantly increased (SN ranged from 459 to 55). The difference in amylase activity of GNBR and BR during steeping was probably due to its granule structure and organization (Witt and Sauter 1996).…”

Similar temperature requirement for sugar accumulation and for the induction of new forms of sucrose phosphate synthase and amylase in cold‐stored potato tubers

“…Poplar trees contain increased maltose and maltase activity in their bark during autumn (Sauter & van Cleve 1993). The bark contains many starch‐degrading activities that can be resolved by native electrophoresis, and three endoamylase activities, one debranching enzyme and one D ‐enzyme form increase in the autumn during the period of rapid starch degradation (Witt & Sauter 1994; Witt, Buchholz & Sauter 1995; Witt & Sauter 1996).…”

The binding of α‐amylase to starch plays a decisive role in the initiation of storage starch degradation in turions of Spirodela polyrhiza

“…α‐Amylases have been proposed to commonly initiate starch granule degradation in plants (Beck and Ziegler 1989). This idea stems from the finding that α‐amylases, in contrast to most starch‐degrading enzymes, are able to degrade native starch granules without participation of other enzymes (see Witt and Sauter 1996 and Sauter et al 1998 for findings concerning potato tubers and poplar wood). The initial degradation products released by the attack of α‐amylase can be further degraded by other enzymes (Beck and Ziegler 1989, Ziegler 1995).…”