Pullulanase from rice endosperm.

Abstract

Pullulanase (EC 3.2.1.41) in non-germinating seeds was compared with that in germinating seeds. Moreover, pullulanase from the endosperm of rice (Oryza sativa L., cv. Hinohikari) seeds was isolated and its properties investigated. The pI value of pullulanase from seeds after 8 days of germination was almost equal to that from non-germinating seeds, which shows that these two enzymes are the same protein. Therefore, the same pullulanase may play roles in both starch synthesis during ripening and starch degradation during germination in rice seeds. The enzyme was isolated by a procedure that included ammonium sulfate fractionation, DEAE-cellulofine column chromatography, preparative isoelectric focusing, and preparative disc gel electrophoresis. The enzyme was homogeneous by SDS/PAGE. The molecular weight of the enzyme was estimated to be 100 000 based on its mobility on SDS/PAGE and 105 000 based on gel filtration with TSKgel super SW 3000, which showed that it was composed of a single unit. The isoelectric point of the enzyme was 4.7. The enzyme was strongly inhibited by beta-cyclodextrin. The enzyme was not activated by thiol reagents such as dithiothreitol, 2-mercaptoethanol or glutathione. The enzyme most preferably hydrolyzed pullulan and liberated only maltotriose. The pullulan hydrolysis was strongly inhibited by the substrate at a concentration higher than 0.1%. The degree of inhibition increased with an increase in the concentration of pullulan. However, the enzyme hydrolyzed amylopectin, soluble starch and beta-limit dextrin more rapidly as their concentrations increased. The enzyme exhibited alpha-glucosyltransfer activity and produced an alpha-1,6-linked compound of two maltotriose molecules from pullulan.

Production of a Saccharifying Rice Malt for Brewing Using Different Rice Varieties and Malting Parameters

“…Therefore, the improved saccharification in tests 5 and 6 was probably due to the lower pH value. The very low value of the diastatic power could not be decisive for assessing good saccharification, but there should be other important amylolytic enzymes in rice malt such as limit dextrinase, pullulanase, and α-glucosidase that significantly contribute to the degradation of the rice malt starch. The filtration time of the laboratory worts last more than an hour despite rice wort’s low viscosity.…”

“…Rice malt, however, shows lower diastatic power values, and for this it seems less suitable for brewing. But rice malt contains other amylotytic enzymes which can act synergistically with α- and β-amylases. ,, This could mean that the required diastatic power for sufficient starch degradation in rice malt could be lower than in barley malt. One of these amylolytic enzymes is limit dextrinase, a debranching enzyme which catalyzes the hydrolysis of the α-1,6-glucosidic linkages in starch and related oligosaccharides and exhibits activity levels in rice several-fold higher than in barley malt. , Another enzyme, α-glucosidase, shows high activity in rice malt and an optimum temperature of 55 °C, while in barley malt its activity is low and the optimum temperature is 35–40 °C.…”

“…But rice malt contains other amylotytic enzymes which can act synergistically with αand β- amylases. 5,16,17 This could mean that the required diastatic power for sufficient starch degradation in rice malt could be lower than in barley malt. One of these amylolytic enzymes is limit dextrinase, a debranching enzyme which catalyzes the hydrolysis of the α-1,6-glucosidic linkages in starch and related oligosaccharides and exhibits activity levels in rice several-fold higher than in barley malt.…”

The Use of Rice in Brewing

“…Rice malt, however, shows lower diastatic power values, and for this it seems less suitable for brewing [17]. But it contains other amylolytic enzymes that can act synergistically with α-and β-amylases [9,18,19]. One of these amylolytic enzymes is limit dextrinase, a debranching enzyme, which catalyzes the hydrolysis of the α-1,6-glucosidic linkages in starch and related oligosaccharides and exhibits activity levels in rice several fold higher than in barley malt [20].…”

Amylolytic Yeasts: Producers of α-amylase and Pullulanase

“…Pullulanases are produced by plants (the endosperm of the rice seed; [94]) and Sorghum bicolor var. F-2-20 [95] and by microorganisms such as bacteria, fungi and certain yeasts [59].…”