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Isolation, partial purification and characterization of sucrose synthase from thermotolerant wheat (Triticum aestivum L.)

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Title Isolation, partial purification and characterization of sucrose synthase from thermotolerant wheat (Triticum aestivum L.)
 
Creator Ekta
 
Contributor Singal, H.R.
 
Subject Planting, Biomass, Spacing, Pruning, Yields, Vegetative propagation, Drying, Developmental stages, Sowing, Biological development
 
Description Wheat 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.
 
Date 2016-11-15T14:55:13Z
2016-11-15T14:55:13Z
2009
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/85667
 
Language en
 
Format application/pdf
 
Publisher CCSHAU