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.
Highlights
Introduction bAmylase (E.C. 3.2.1.2), member of family 14 of glycosyl hydrolases, catalyses the release of successive b-maltose from the non-reducing ends of a-1,4 linked oligo and poly glucans
Exclusive maltose production is utilised in pharmaceutical industry for dispensing, production of maltose rich syrups, and non-digestible sweetner, maltitol [1]
De-novo synthesis of the enzyme is reported during early germination of rice [3]. b-Amylase has been shown to play a more important role as compared to a-amylase during early hours of germination in wheat scutella [4]
Summary
Amylase (E.C. 3.2.1.2), member of family 14 of glycosyl hydrolases, catalyses the release of successive b-maltose from the non-reducing ends of a-1,4 linked oligo and poly glucans. The enzyme is distributed in higher plants and some micro-organism. Exclusive maltose production is utilised in pharmaceutical industry for dispensing, production of maltose rich syrups, and non-digestible sweetner, maltitol [1]. In germination of cereal seeds, b-amylase is known to play a vital role, where it is present in free and bound forms [1]. De-novo synthesis of the enzyme is reported during early germination of rice [3]. B-Amylase has been shown to play a more important role as compared to a-amylase during early hours of germination in wheat scutella [4] De-novo synthesis of the enzyme is reported during early germination of rice [3]. b-Amylase has been shown to play a more important role as compared to a-amylase during early hours of germination in wheat scutella [4]
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