Preferences help
enabled [disable] Abstract
Number of results
2008 | 55 | 3 | 507-510
Article title

Pullulanase from rice endosperm

Title variants
Languages of publication
Pullulanase (EC 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 β-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 β-limit dextrin more rapidly as their concentrations increased. The enzyme exhibited α-glucosyltransfer activity and produced an α-1,6-linked compound of two maltotriose molecules from pullulan.

Physical description
  • Research Institute for Bioresources, Okayama University, Kurashiki-shi, Okayama, Japan
  • Research Institute for Bioresources, Okayama University, Kurashiki-shi, Okayama, Japan
  • Research Institute for Bioresources, Okayama University, Kurashiki-shi, Okayama, Japan
  • Drummond GS, Smith EE, Whelan WJ (1970) On the specificity of starch debranching enzymes. FEBS Lett 9: 136-140.
  • Dunn G, Hardie DG, Manners DJ (1973) Observations on the action of limit dextrinases on amylopectin-like polysaccharides. Biochem J 133: 413-416.
  • Gordon RW, Manners DJ, Stark JR (1975) The limit dextrinase of the broad bean (Vicia faba L.). Carbohydr Res 42: 125-134.
  • Iwaki K, Fuwa H (1981) Purification and some properties of debranching enzyme of germinating rice endosperm. Agric Biol Chem 45: 2683-2688.
  • James MG, Robertson DS, Myers AM (1995) Characterization of the maize gene sugary 1, a determinant of starch composition in kernels. Plant Cell 7: 417-429.
  • Kubo A, Fujita N, Harada K, Matsuda T, Satoh H, Nakamura Y (1999) The starch-debranching enzymes isoamylase and pullulanase are both involved in amylopectin biosynthesis in rice endosperm. Plant Physiol 121: 399-410.
  • Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage. Nature 227: 680-685.
  • Lee Eyc, Marshall JJ, Whelan WJ (1971) The substrate specificity of amylopectin-debranching enzymes from sweet corn. Arch Biochem Biophys 143: 365-374.
  • Li B, Servaites JC, Geiger DR (1992) Characterization and subcellular localization of debranching enzyme and endoamylase from leaves of sugar beet. Plant Physiol 98: 1277-1284.
  • Ludwig I, Ziegler P, Beck E (1984) Purification and properties of spinach debranching enzyme. Plant Physiol 74: 856-861.
  • Maeda I, Nikuni Z (1978) Purification of a debranching enzyme (R-enzyme) from malted barley, and the role of the enzyme in the digestion of starch granules during the germination of barley seeds. Carbohyd Res 61: 309-320.
  • Mouille G, Maddelein M-L, Libessart N, Talaga P, Decq A, Delru B, Ball S (1996) Preamylopectin processing: a mandatory step for starch biosynthesis in plants. Plant Cell 8: 1353-1366.
  • Nakamura Y, Umemoto T, Takahata Y, Komae K, Amano E, Satoh S (1996) Changes in structure of starch and enzyme activities affected by sugary mutations in developing rice endosperm. Possible role of starch debranching enzyme (R-enzyme) in amylopectin biosynthesis. Physiol Plant 97: 491-498.
  • Nakamura Y, Kubo A, Shimamune T, Matsuda T, Harada K, Satoh H (1997) Correlation between activities of starch debranching enzyme and α-polyglucan structure in endosperms of sugary-1 mutants of rice. Plant J 12: 143-153.
  • Okita TW, Preiss J (1980) Starch degradation in spinach leaves. Isolation and characterization of the amylases and R-enzyme of spinach leaves. Plant Physiol 66: 870-876.
  • Pan D, Nelson OE (1984) A debranching enzyme deficiency in endosperms of the sugary-1 mutants of maize. Plant Physiol 74: 324-328.
  • Reisfeld RA, Lewis UJ, Williams DE (1962) Disc electrophoresis of basic proteins and peptides on polyacrylamide gels. Nature (London) 195: 281-283.
  • Schindler I, Renz A, Schmid FX, Beck E (2001) Activation of spinach pullulanase by reduction results in a decrease in the number of isomeric forms. Biochim Biophys Acta 1548: 175-186.
  • Somogyi M (1952) Notes on sugar determination. J Biol Chem 195: 19-23.
  • Warburg O, Christian W (1942) Isolation and crystallization of enolase. Biochem Z 310: 384-421.
  • Yamada J, Izawa M (1979) A debranching enzyme of rice seeds at milky stage, its purification and substrate specificities. Agric Biol Chem 43: 37-44.
  • Yamasaki Y (2003) β-Amylase in germinating millet seeds. Phytochemistry 64: 935-939.
  • Yamasaki Y, Kariya J, Nakashima S, Konno H (2007a) Purification and properties of pullulanase from germinated millet seeds. J Prep Chromatogr 2: 8-12.
  • Yamasaki Y, Nakashima S, Konno H (2007b) A novel α-glucosidase from the moss Scopelophila cataractae. Acta Biochim Polon 54: 401-406.
Document Type
Publication order reference
YADDA identifier
JavaScript is turned off in your web browser. Turn it on to take full advantage of this site, then refresh the page.