大麦枝切り酵素の精製と澱粉粒分解におけるその役割 (アミラ-ゼシンポジウム(1978)特集)
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概要
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A debranching enzyme (R-enzyme or pullulan-6-glucanohydrolase, EC 3.2.1.41) was purified from malted barley by subjecting crude extract to ammonium sulfate precipitation, ion-exchange chromatography on DEAE-cellulose, gel filtration on Sephadex G-150 and affinity chromatography on a-cyclodextrin-Sepharose 6B, successively, to give a final preparation with specific activity of 10 units/mg of protein. This preparation showed a single band on polyacrylamide disc-gel electrophoresis and had optimum pH and temperature of 5.0-5.5 and 50°C, respectively. Molecular weight was determined to be 103, 000 by SDS-disc-gel electrophoresis, whereas a value of 80, 000 was obtained by gel filtration using Sephadex G-150. This enzyme had a K<SUB>m</SUB> of 0.07 mg/ml and V<SUB>max</SUB> of 9.6 μmoles of maltotriose/min per mg protein with pullulan as substrate. Amylopectin and glycogens were poor substrates both in terms of K<SUB>m</SUB> and V<SUB>max</SUB>. Generally, Q-limit dextrins were better substrates than respective amylopectin and glycogens. In addition to several branched oligosaccharides which are substrates to the bacterial pullulanase and isoamylase, this enzyme could hydrolyze isopanose (6-maltosylglucose). α-and β-Amylases, R-enzyme and a-glucosidase, all known to be present in malted barley and prepared from barley or malted barley, were incubated, separately or in combination, with barley starch granules and the digestion rates were compared to that obtained by an extract from malted barley. It was revealed that a-amylase alone digested barley starch with considerable rate, which was, however, slower than that by malted barley extract. Addition of β-amylase to the a-amylase incubation mixture increased the digestion rate but a further increase was obtained on addition of R-enzyme. However, only in the presence of all of four enzymes, the digestion rate reached the same value that effected by malted barley extract. The two incubation mixtures (malted barley extract and the reconstituted enzyme system) gave quite similar oligosaccharide patterns on paper chromatography. It was suggested that α-amylase played a central role in the digestion of barley starch during germination by hydrolyzing insoluble starch granules to soluble oligosaccharides. /-Amylase hydrolyzed these oligosaccharides into maltose. R-enzyme hydrolyzed branch linkages in oligosaccharides thus helping β-amylase to act further on. α-Glucosidase converted maltose into glucose which, in turn, might be transported and utilized by young seedlings.
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