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Partial purification and characterization of Hexokinase from developing grains of thermotolerant wheat (Triticum aestivum L. em. Thell)”

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Title Partial purification and characterization of Hexokinase from developing grains of thermotolerant wheat (Triticum aestivum L. em. Thell)”
 
Creator Fageria, Leena
 
Contributor Singal, H. R.
 
Subject fruits, vegetables, insecticides, extraction, biological interaction, yields, organic amendments, garlic, livestock, biological phenomena
 
Description Wheat grain is the dominant grain of world commerce and is the staple food of millions of
people worldwide. High temperature beyond 30
0
C, 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. As this sucrose enters the cell, it is
metabolized to produce fructose and glucose. Hexokinase (HXK) is an important enzyme as it
catalyzes the irreversible phosphorylation of hexoses to hexose-phosphates. The hexose-phosphates
produced catalyzes synthesis of starch. Keeping above in view, the present investigations were
conducted to purify and characterize hexokinase from developing grains of thermotolerant wheat.
Hexokinase 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. 30-60% ammonium sulphate fractionation, DEAE-cellulose ion
exchange chromatography and gel filtration through sephadex G-100. The enzyme was purified about
19.27 fold with 48.43 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 50 kDa,
indicating that enzyme is a monomer. The purified enzyme exhibited optimum activity at pH 8 and
35
0
C. It was thermostable upto 50
0
C. The enzyme followed Michaelis-Menten kinetics with Km value
of 2.5 mM and 1.82 mM for Glc and ATP as substrate, respectively. Amongst various nucleotides and
metabolites tested AMP, ADP, UDP, CTP and G-6-P were found to be the potent inhibitors of purified
hexokinase, inhibiting the enzyme activity by 13, 16, 5, 37 and 13%, respectively. However, GTP,
UTP, G-1,6-BP and F-1,6-BP acted as poor inhibitors of the enzyme. The enzyme activity was
stimulated by K
+
and Mg
2+
while Cu
2+
and Co
2+
inhibited the activity at 5 mM concentration. To
summarize, higher thermostability of enzyme is suggestive of enzyme’s adaptation to high temperature
stress.
 
Date 2016-09-14T10:39:22Z
2016-09-14T10:39:22Z
2013
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/76586
 
Language en
 
Format application/pdf
 
Publisher CCSHAU