Open AccessΒ-Amylase from Starchless Seeds of Trigonella Foenum-Graecum and Its Localization in Germinating Seeds
Author(s) -
Garima Srivastava,
Arvind M. Kayastha
Publication year - 2014
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0088697
Subject(s) - trigonella , amylase , germination , starch , enzyme , enzyme assay , hydrolysis , cotyledon , chemistry , amyloplast , biochemistry , biology , food science , botany , plastid , chloroplast , gene
Fenugreek ( Trigonella foenum-graecum ) seeds do not contain starch as carbohydrate reserve. Synthesis of starch is initiated after germination. A β -amylase from ungerminated fenugreek seeds was purified to apparent electrophoretic homogeneity. The enzyme was purified 210 fold with specific activity of 732.59 units/mg. M r of the denatured enzyme as determined from SDS-PAGE was 58 kD while that of native enzyme calculated from size exclusion chromatography was 56 kD. Furthermore, its identity was confirmed to be β-amylase from MALDI-TOF analysis. The optimum pH and temperature was found to be 5.0 and 50°C, respectively. Starch was hydrolyzed at highest rate and enzyme showed a K m of 1.58 mg/mL with it. Antibodies against purified Fenugreek β -amylase were generated in rabbits. These antibodies were used for localization of enzyme in the cotyledon during different stages of germination using fluorescence and confocal microscopy. Fenugreek β -amylase was found to be the major starch degrading enzyme depending on the high amount of enzyme present as compared to α -amylase and also its localization at the periphery of amyloplasts. A new finding in terms of its association with protophloem was observed. Thus, this enzyme appears to be important for germination of seeds.