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Partial purification and characterization of UDP-glucose pyrophosphorylase from developing grains of thermotolerant wheat (T. aestivum L.)

KrishiKosh

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Title Partial purification and characterization of UDP-glucose pyrophosphorylase from developing grains of thermotolerant wheat (T. aestivum L.)
 
Creator Balan, Deepika
 
Contributor Singal, H.R.
 
Subject Enzymes, Grain, Wheats, Planting, Concentrates, Proteins, Electrophoresis, Starch, Chromatography, Developmental stages
 
Description Wheat grain is the dominant grain of world commerce and is the staple food of millions of people
worldwide. High temperature beyond 30°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.The cytosolic UDPglucose
pyrophosphorylase is an essential enzyme responsible for production of G-1-P which is
an important intermediate for starch biosynthesis. Keeping above in view, the present
investigation was conducted to purify and characterize UDP-glucose pyrophosphorylase from
developing grains of thermotolerant wheat WH-1021, the variety having highest activity of this
enzyme. UDP-glucose pyrophosphorylase was purified to near homogeneity (as revealed by
Native-PAGE) from immature grains (21 days after anthesis) of thermotolerant wheat WH-1021
by using conventional protein purification techniques viz. ammonium sulphate fractionation ( 30-
70%), gel filtration through sephadex G-100 and DEAE-cellulose ion exchange chromatography.
The enzyme was purified about 35 fold with 43.3 per cent recovery. The molecular weight as
determined by gel filtration and subunit molecular weight as determined by SDS-PAGE were
found to be 82 kDa and 39 kDa respectively, indicating that enzyme is a homodimer. The purified
enzyme exhibited optimum activity at pH 8.5 and 350C temperature. It was thermostable upto
500C. The enzyme followed Michaelis-Menten kinetics with Km value of 1.66 mM and 0.9 mM
for PPi and UDPG as substrate, respectively. The enzyme activity was inhibited by UTP
(19.66% inhibition) while UDPG stimulated (17.68% stimulation) the activity at 2 mM
concentration.The enzyme activity was stimulated by Mg2+ while Na+ inhibited the activity at 2
mM concentration. To summarize, higher thermostability of enzyme is suggestive of enzyme’s
adaptation to high temperature stress.
 
Date 2016-10-05T13:53:06Z
2016-10-05T13:53:06Z
2015
 
Type Thesis
 
Identifier http://krishikosh.egranth.ac.in/handle/1/80005
 
Language en
 
Format application/pdf
 
Publisher CCSHAU