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 K_i 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.

Keywords:

• α-amylase inhibitor
• chitinase-like protein
• Musa (AAA group)

ACKNOWLEDGMENTS

The authors thank the Thailand Research Fund (Thai Fruits-Functional Fruits RDG5220038), the National Research University Project of CHE, the National Research University Project of CHE, the Ratchadaphiseksomphot Endowment Fund (AG001B, AM1019A, and AS613A), and the Thai Government Stimulus Package 2 (TKK2555) for financial support of this research, as well as the Institute of Biotechnology and Genetic Engineering for support and facilities. We also, thank Dr. Robert Butcher (Publication Counseling Unit, Chulalongkorn University) for his constructive comments in preparing this manuscript.