A Chitinase-Like Protein with α-Amylase Inhibitory Activity from Kluai Hom Thong Banana Fruit: Musa (AAA group)

Abstract

Diabetes is a syndrome of disordered metabolism resulting in abnormally high blood sugar levels (hyperglycemia). The one possibility of lowering postprandial glucose levels is by the inhibition of α-amylase activity. In this study, aqueous extracts from local Thai fruits were tested for their inhibitory effect on α-amylase. The results showed that the fruits of Kluai Hom Thong; Musa (AAA group) showed significant reduction in α-amylase activity. An α-amylase inhibitor was purified by using a combination of ammonium sulfate precipitation, ion exchange chromatography with DEAE cellulose, and gel filtration chromatography with Superdex 75, respectively. The enriched preparation had a specific activity of 355.7 AI U/mg proteins for a yield of 18.5% total protein. The molecular weight of this α-amylase inhibitor was estimated by SDS-15% (w/v) PAGE to be about ∼20 kDa. Periodic staining and phenol-sulfuric assay showed that the α-amylase inhibitor was a glycoprotein and contained 10.74 ± 0.35% (by weight) carbohydrate moiety. The pH stability of this α-amylase inhibitor was between pH 4 and 7, and it is stable up to 40°C but is totally inactivated after exposure to 40°C for 120 min. The α-amylase inhibitory activity was stimulated by Ca2+ and Mn2 at less than 25 mM but not by Mg2+, Fe3+, Hg2+, Co2+, and EDTA. Determination of the kinetics of the inhibition of α-amylase by standard enzymatic methods indicated the Ki of this protein was 0.89 mg protein/mL. The amino acid sequence of an internal fragment of this purified α-amylase inhibitor had a similarity to the sequence from the plant lectin/endochitnase1 family.