ABSTRACT
Nutrient dynamics in storage organs is a complex developmental process that requires coordinated interactions of environmental, biochemical, and genetic factors. Although sink organ developmental events have been identified, understanding of translational and post-translational regulation of reserve synthesis, accumulation, and utilization in legumes is limited. To understand nutrient dynamics during embryonic and cotyledonary photoheterotrophic transition to mature and germinating autotrophic seeds, an integrated proteomics and phosphoproteomics study in six sequential seed developmental stages in chickpea is performed. MS/MS analyses identify 109 unique nutrient-associated proteins (NAPs) involved in metabolism, storage and biogenesis, and protein turnover. Differences and similarities in 60 nutrient-associated phosphoproteins (NAPPs) containing 93 phosphosites are compared with NAPs. Data reveal accumulation of carbon-nitrogen metabolic and photosynthetic proteoforms during seed filling. Furthermore, enrichment of storage proteoforms and protease inhibitors is associated with cell expansion and seed maturation. Finally, combined proteoforms network analysis identifies three significant modules, centered around malate dehydrogenase, HSP70, triose phosphate isomerase, and vicilin. Novel clues suggest that ubiquitin-proteasome pathway regulates nutrient reallocation. Second, increased abundance of NAPs/NAPPs related to oxidative and serine/threonine signaling indicates direct interface between redox sensing and signaling during seed development. Taken together, nutrient signals act as metabolic and differentiation determinant governing storage organ reprogramming.
Subject(s)
Cicer/metabolism , Phosphoproteins/analysis , Plant Proteins/analysis , Seeds/physiology , Carbon/metabolism , Cicer/growth & development , Cicer/physiology , Enzymes/metabolism , Germination , Nitrogen/metabolism , Oxidation-Reduction , Phosphoproteins/metabolism , Plant Proteins/metabolism , Proteome/analysis , Proteome/metabolism , Reproducibility of Results , Seeds/growth & development , Seeds/metabolism , Signal TransductionABSTRACT
A novel therapeutic agent in the form of beta galactosidase immobilized on the surface of concanavalin A layered calcium alginate-starch beads has been developed. Immobilized beta galactosidase exhibited significantly very high stability against conditions of digestive system such as pH, salivary amylase, pepsin and trypsin. Soluble and immobilized beta galactosidase exhibited same pH-optima. However, the immobilized enzyme retained greater fraction of catalytic activity at higher and lower pH to pH-optima as compared to soluble enzyme. Immobilized enzyme preparation was quite stable under conditions present in mouth, stomach and intestine. Immobilized beta galactosidase retained 65% activity even after its sixth repeated use.
Subject(s)
Alginates/chemistry , Concanavalin A/chemistry , Enzymes, Immobilized/chemistry , beta-Galactosidase/chemistry , Amylases/metabolism , Aspergillus oryzae/enzymology , Enzyme Stability , Enzymes, Immobilized/isolation & purification , Glucuronic Acid/chemistry , Hexuronic Acids/chemistry , Humans , Hydrogen-Ion Concentration , Lactose Intolerance , Pepsin A/metabolism , Starch/chemistry , Trypsin/metabolism , beta-Galactosidase/isolation & purificationABSTRACT
Insoluble concanavalin A-beta galactosidase complex was obtained by using jack bean extract and this complex was crosslinked with glutaraldehyde, in order to maintain the integrity of complex in the presence of its substrate or products. Concanavalin A-beta galactosidase complex retained 92% of the initial enzyme activity whereas crosslinked complex showed 88% activity. Entrapment of concanavalin A-beta galactosidase complex into calcium alginate beads provided suitability to use this preparation in reactors. Temperature- and pH-optima of the various immobilized beta galactosidase preparations were the same as its soluble counterpart. Entrapped crosslinked concanavalin A-beta galactosidase complex retained more than 50% activity after 1h exposure with 4.0 M urea at room temperature. Moreover, entrapped crosslinked concanavalin A-beta galactosidase complex retained 81 and 62% of the original enzymatic activity in the presence of 5% calcium chloride and 5% galactose, respectively. Entrapped crosslinked concanavalin A-beta galactosidase complex preparation was more superior in the continuous hydrolysis of lactose in a batch process as compared to the other entrapped preparations. This entrapped crosslinked concanavalin A-beta galactosidase complex retained 95% activity after seventh repeated use and 93% of its original activity even after 2 months storage at 4 degrees C.
