Mechanism of solvent-induced thermal stabilization of alpha-amylase from Bacillus amyloliquefaciens.
The transition temperature of irreversible thermal inactivation of alpha-amylase from Bacillus amyloliquefaciens was estimated to be 60 degrees C. At this temperature, the enzyme inactivation followed first-order kinetics, having a half-life (t 1/2) of 12 min with a rate constant (k) of 0.06 min-1. Conformational change was a prerequisite for this thermal inactivation. This is governed by stepwise temperature-dependent phenomena. Among the solvent stabilizers tested, the enzyme was thermally stable in presence of DMSO and PEG 300 and the stabilizing efficiency of these cosolvents was concentration-dependent. The enzyme was partially stabilized by 5.0 M DMSO and 1.9 M PEG 300 up to 78 degrees C. However, above 78 degrees C the enzyme was inactivated in these cosolvents also. The mechanism of stabilization has been explained by preferential hydration of the enzyme in these structure stabilizing solvents by exclusion from the protein surface and interface by measurement of partial specific volume in these cosolvents. The data suggest a high value of preferential interaction parameter, (delta g3/delta g2)tau, mu 1, mu 3 being -0.606/g/g g/g in 40% DMSO and a low value of -0.025 g/g in 5% glycerol. The preferential interaction parameters in sucrose and glycerol suggests that (delta g3/delta g2)tau, mu 1, mu 3m is highest of -0.420 g/g in 10% glycerol than any other cosolvent.
Rajendran
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S
Department of Protein Technology, Central Food Technological Research Institute, Mysore, India.
Radha
C
C
Prakash
V
V
D001407:Bacillus / Q000201:enzymology*; D002942:Circular Dichroism; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D004795:Enzyme Stability / Q000187:drug effects*; D005990:Glycerol / Q000494:pharmacology; D007700:Kinetics; D011092:Polyethylene Glycols / Q000494:pharmacology; D011487:Protein Conformation; D011489:Protein Denaturation; D012997:Solvents / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D013056:Spectrophotometry, Ultraviolet; D013395:Sucrose / Q000494:pharmacology; D013696:Temperature; D000516:alpha-Amylases / Q000737:chemistry / Q000378:metabolism*
D012997:Solvents; D011092:Polyethylene Glycols; D013395:Sucrose; D000516:alpha-Amylases; D005990:Glycerol; D004121:Dimethyl Sulfoxide