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Structural Stability of Soybean (Glycine max) α-Amylase: Properties of the Unfolding Transition Studied with Fluorescence and CD Spectroscopy

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Abstract:

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 soybean 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 α-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 is 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.





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  • Protein & Peptide Letters publishes short papers in all important aspects of protein and peptide research, including structural studies, recombinant expression, function, synthesis, enzymology, immunology, molecular modeling, drug design etc. Manuscripts must have a significant element of novelty, timeliness and urgency that merit rapid publication. Reports of crystallisation, and preliminary structure determinations of biologically important proteins are acceptable. Purely theoretical papers are also acceptable provided they provide new insight into the principles of protein/peptide structure and function.
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