Purification and Characterization of Proteinaceous Thermostable α-Amylase Inhibitor from Sardinian Common Bean Nieddone Cultivar (Phaseolus vulgaris L.)

The increasing need for new treatments for obesity and diabetes has led to the development of new drugs and food supplements that could reduce carbohydrate absorption. Many starch blockers, based on common bean proteinaceous inhibitors against alpha-amylase (alpha-AI), are already present on the market. The extraction and purification of alpha-amylase inhibitor from a promising common bean cultivar from Sardinia (Nieddone) is described, highlighting the unique value of the Nieddone cultivar, particularly for its inhibitory activity on digestive enzymes and its complete lack of a hemagglutination effect on human red blood cells. The purification of alpha-AI involved two chromatographic steps (IEC and SEC) and was essential for revealing certain properties of the inhibitor. The purified inhibitor has a tetrameric structure (alpha(2)beta(2)) and a molecular weight of approximately 42 kDa, as determined by SEC and SDS-PAGE, confirming it as a lectin-like inhibitor. The identification of the alpha-AI sequence was obtained by bottom-up high-resolution mass spectrometry, which allowed us to identify a unique peptide from the alpha chain and six unique peptides from the beta chains. alpha-AI exhibited an optimum temperature of around 40 degrees C and two pH optima at 5 and 6.5, respectively. Its remarkable stability at high temperatures was measured (approximately 25% of activity retained even after 5 h at 100 degrees C), whereas the raw extract lost its activity entirely after just 10 min at 90 degrees C. Thus, the purification process significantly enhances the thermal stability of alpha-AI. The demonstrated effectiveness of the purified alpha-AI against the alpha-amylase enzyme in pigs, humans and insects underscores the protein's potential for treating obesity and diabetes, as well as for managing insect pests.