[feed] Atom [feed] RSS 1.0 [feed] RSS 2.0

Structure, Function and Stability Of Alpha-Amylase From Jowar(Sorghum Bicolor)

Siva Sai Kumar, R. (2008) Structure, Function and Stability Of Alpha-Amylase From Jowar(Sorghum Bicolor). PhD thesis, University of Mysore.

[img] PDF
Siva_Sai_Kumar.pdf

Download (2MB)

Abstract

The focus of this investigation has been to screen cereals for thermostable a-amylases, which retain activity when hot water is added to supplementary foods to reconstitute them. The isolation, characterization and stability parameters of the a-amylase from sorghum are reported for the first time. The thermostability of a-amylase from malted sorghum is higher compared to a-amylases from barley (Hordeum jubatum) and ragi (Eleusine coracana). The digestion of gelatinized starch, by this enzyme, results in maltose, glucose and maltotriose, which increase nutrient density by decreasing its water absorption capacity. The major amylase from malted jowar, a 47 kDa a-amylase, is rich in b structure (~60%) like other cereal amylases. a-Amylase from Sorghum bicolor is reversibly unfolded, by GuHCl and urea at pH 7.0 in 50 mM Hepes containing 13.6 mM calcium and 15 mM DTT. The isothermal equilibrium unfolding at 27°C is characterized by two-state transition with DG(H2O) of 16.5 kJ mol-1 and 22 kJ mol-1 for GuHCl at pH 4.8 and 7.0, respectively and DG(H2O) of 25.2 kJ mol-1 for urea at pH 4.8. The pH dependent stability described by DG(H2O) and the effect of salt on urea induced unfolding confirm the dominant role of electrostatic interactions in enzyme stability. Homology modeling studies of sorghum a-amylase, using barley v AMY1 as a template, have shown a structure similar to that expected from the high sequence identity. The reactivity of the conserved cysteine residues, during unfolding, suggests that unfolding starts from the ‘B’ domain of the enzyme. The conserved cysteine residues are essential for enzyme activity but not for the secondary and tertiary fold acquired during refolding of the denatured enzyme. Tm, the midpoint of thermal inactivation, is found to be 69.6 ± 0.3 °C. Thermal inactivation of a-amylase follows first-order kinetics at pH 4.8, the pH optimum of the. NaCl is a destabilizer, while sucrose is a stabilizer against thermal inactivation. Calcium content of sorghum a-amylase is determined to be 0.8 mole of Ca2+/ mole of protein. Removal of calcium leads to decreased thermo stability and enzymatic activity, and increased susceptibility to proteolytic degradation. The loss of sorghum amylase activity upon removal of calcium suggests that Ca2+ is important for maintenance of the configuration of the active site. Activity recovery of the EDTA inactivated sorghum a-amylase in presence of barium is similar to that of calcium. These studies on structure function and stability of sorghum a-amylase may lead to the development of enzymes with improved stability and in help in understanding the folding and refolding of a multi – domain protein. vi

Item Type: Thesis (PhD)
Uncontrolled Keywords: alpha-amylases cereals jowar isolation characterization stability
Subjects: 600 Technology > 08 Food technology > 21 Cereals > 06 Sorghum
500 Natural Sciences and Mathematics > 04 Chemistry and Allied Sciences > 16 Enzyme Chemistry
Divisions: Protein Chemistry and Technology
Depositing User: Food Sci. & Technol. Information Services
Date Deposited: 07 May 2009 09:28
Last Modified: 07 May 2009 09:28
URI: http://ir.cftri.com/id/eprint/8931

Actions (login required)

View Item View Item