Singal, H.R.Ekta2016-11-152016-11-152009http://krishikosh.egranth.ac.in/handle/1/85667Wheat grain is the dominant grain of world commerce and is the staple food of millions of people world wide. High temperature beyond 300C which is usually encountered during later part of grain filling period, affects grain yield (reduction by 20-50 per cent) and grain quality. Starch is the major storage carbohydrate in wheat grains. It is synthesized from sucrose which is the principal product of leaf photosynthesis and transported to the wheat grain. Sucrose synthase is the first enzyme and an important link in sucrose-starch conversion pathway. Keeping above in view, the present investigation was conducted to purify and characterize sucrose synthase from thermotolerant wheat. Sucrose synthase was purified to near homogeneity (as revealed by single band on Native-PAGE) from immature grains (21 days after anthesis) of thermotolerant wheat WH-1021 by using conventional protein purification techniques viz. ammonium sulphate fractionation, gel filtration through sephadex G-100 and DEAE-cellulose ion exchange chromatography. The enzyme was purified about 27 fold with approximately 37 per cent recovery. The molecular weight as determined by gel filtration and subunit molecular weight as determined by SDS-PAGE (single band) were found to be 269 KDa and 63 KDa respectively indicating that enzyme is a homotetramer. The purified enzyme exhibited optimum activity at 370C temperature and pH 6.5. It was thermostable upto 500C. The activity followed Michaelis-Menten kinetics with Km value of 14.28 mM and 1.18 mM for sucrose and UDP, respectively. Among the various nucleotides tested the enzyme was highly specific for UDP as substrate. The kinetic studies revealed that sucrose synthase catalysed the sucrose degradation by ping-pong mechanism. The enzyme activity was inhibited by Mn2+ (38.5 % inhibition) while NO3- stimulated (20.8% stimulation) the activity at 2 mM concentration. Amongst various metabolites tested NADP+ and G-6-P were found to be the potent inhibitors of purified sucrose synthase (inhibiting the enzyme activity by 16 and 34%), respectively. To summarize, higher thermostability of enzyme is suggestive of enzyme’s adaptation to high temperature stress.enPlanting, Biomass, Spacing, Pruning, Yields, Vegetative propagation, Drying, Developmental stages, Sowing, Biological developmentThesis