Thermal stability of alpha-amylase in aqueous cosolvent systems.
The activity and thermal stability of alpha-amylase were studied in the presence of different concentrations of trehalose, sorbitol, sucrose and glycerol. The optimum temperature of the enzyme was found to be 50 +/- 2 degrees C. Further increase in temperature resulted in irreversible thermal inactivation of the enzyme. In the presence of cosolvents, the rate of thermal inactivation was found to be significantly reduced. The apparent thermal denaturation temperature (Tm) app and activation energy (Ea) of alpha-amylase were found to be significantly increased in the presence of cosolvents in a concentration-dependent manner. In the presence of 40% trehalose, sorbitol, sucrose and glycerol, increments in the (Tm)app were 20 degrees C, 14 degrees C, 13 degrees C and 9 degrees C, respectively. The Ea of thermal denaturation of alpha-amylase in the presence of 20% (w/v) trehalose, sorbitol, sucrose and glycerol was found to be 126, 95, 90 and 43 kcal/mol compared with a control value of 40 kcal/mol. Intrinsic and 8-anilinonaphathalene-1-sulphonic acid (ANS) fluorescence studies indicated that thermal denaturation of the enzyme was accompanied by exposure of the hydrophobic cluster on the protein surface. Preferential interaction parameters indicated extensive hydration of the enzyme in the presence of cosolvents.
Yadav
Jay Kant
JK
Department of Protein Chemistry and Technology, Central Food Technological Research Institute, Mysore 570020, India.
Prakash
V
V
D002021:Buffers; D004795:Enzyme Stability / Q000528:radiation effects*; D005990:Glycerol / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D011489:Protein Denaturation; D012997:Solvents / Q000737:chemistry*; D013012:Sorbitol / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000379:methods; D013213:Starch / Q000737:chemistry; D013379:Substrate Specificity; D013395:Sucrose / Q000737:chemistry; D013499:Surface Properties; D013696:Temperature; D013816:Thermodynamics; D014199:Trehalose / Q000737:chemistry; D014867:Water / Q000737:chemistry*; D000516:alpha-Amylases / Q000032:analysis / Q000737:chemistry*
D002021:Buffers; D012997:Solvents; D014867:Water; D013012:Sorbitol; D013395:Sucrose; D013213:Starch; D014199:Trehalose; D000516:alpha-Amylases; D005990:Glycerol