title: Β-amylase from starchless seeds of Trigonella foenum-graecum and its localization in germinating seeds.
abstract: 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. Mr 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 Km 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.
authors.affiliation: School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi, India.
authors.forename: Garima
authors.initials: G
authors.lastname: Srivastava
authors.affiliation: School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi, India.
authors.forename: Arvind M
authors.initials: AM
authors.lastname: Kayastha
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D018548:Cotyledon / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D018525:Germination; D007150:Immunohistochemistry; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010936:Plant Extracts / Q000378:metabolism; D021381:Protein Transport; D011817:Rabbits; D012639:Seeds / Q000201:enzymology*; D013213:Starch / Q000378:metabolism*; D013379:Substrate Specificity; D029910:Trigonella / Q000201:enzymology*; D000516:alpha-Amylases / Q000378:metabolism; D001614:beta-Amylase / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*
chemical_list: D010455:Peptides; D010936:Plant Extracts; D013213:Starch; D000516:alpha-Amylases; D001614:beta-Amylase