Structural stability of soybean (Glycine max) α-amylase: properties of the unfolding transition studied with fluorescence and CD spectroscopy.
Stability and unfolding of mammalian and microbial α-amylases have been intensively investigated. However, there is only limited information available on the structural stability of plant α-amylases, namely of the two isoenzymes from barley AMY1 and AMY2, of the α-amylase from mung bean (Vigna radiata), and of the α-amylase from malted sorghum (Sorghum bicolor). We report here the stability of soyabean α-amylase (GMA), against elevated temperatures and chemical denaturants (GndHCl) by employing circular dichroism and fluorescence spectroscopy. Since it is well-known that calcium ions play a crucial role for enzymatic activity and stability of a-amylases, we performed our studies with calcium bound and calcium free GMA. The thermal unfolding transition temperature decreased from 72°C for calcium saturated samples to 57°C for the case of calcium depleted GMA. Similarly, the GndHCl transition concentration was lowered from 0.70 M for calcium bound GMA to 0.41 M in the absence of calcium. Thermal unfolding of GMA irreversible due to aggregation of the unfolded state. GMA unfolded in 6 M GndHCl shows high degree of reversibility after diluting the unfolded enzyme in native buffer containing 7 M glycerol. Furthermore, the refolded enzyme showed 93% of activity.
Kumari
Arpana
A
School of Biotechnology, Faculty of Science, Banaras Hindu University, Varanasi, India.
Rosenkranz
Tobias
T
Fitter
Jörg
J
Kayastha
Arvind M
AM
D002942:Circular Dichroism; D004305:Dose-Response Relationship, Drug; D004795:Enzyme Stability / Q000187:drug effects; D019791:Guanidine / Q000494:pharmacology; D006358:Hot Temperature; D007700:Kinetics; D017510:Protein Folding / Q000187:drug effects; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding / Q000187:drug effects; D013025:Glycine max / Q000201:enzymology*; D013050:Spectrometry, Fluorescence; D000516:alpha-Amylases / Q000737:chemistry*
D000516:alpha-Amylases; D019791:Guanidine