Isolation of a thermostable trypsin inhibitor with exploitable potential

Area of Science:

• Biochemistry
• Plant Science
• Molecular Biology

Background:

• Leguminous seeds are a rich source of bioactive proteins, including protease inhibitors.
• Trypsin inhibitors play crucial roles in plant defense mechanisms and have potential therapeutic applications.
• Chinese black soybean (Glycine max) is a valuable crop with unexplored bioactive compounds.

Purpose of the Study:

• To isolate and characterize a novel trypsin inhibitor from Chinese black soybean seeds.
• To determine the physicochemical properties and inhibitory activity of the isolated inhibitor.
• To investigate the antiproliferative and antifungal activities of the novel inhibitor.

Main Methods:

• Purification using ammonium sulfate precipitation, ion-exchange chromatography (CM-Sephadex C-50), and gel filtration chromatography (Sephacryl S-200HR, POROS HS-20).
• N-terminal amino acid sequencing to determine homology with known inhibitors.
• SDS-PAGE and isoelectric focusing to estimate molecular mass and isoelectric point.
• Enzyme inhibition assays to determine trypsin inhibitory activity and stability under varying temperature and pH conditions.
• In vitro assays to evaluate antiproliferative activity against tumor cell lines and antifungal activity against plant pathogens.

Main Results:

• A novel trypsin inhibitor, Glytine, was successfully isolated and purified.
• Glytine exhibited significant thermal stability (up to 100°C) and pH stability (pH 2-12).
• The inhibitor has a molecular mass of 19.9 kDa and an isoelectric point of 6.2.
• Complete inhibition of trypsin was achieved at a weight ratio of 1.5.
• Glytine demonstrated antiproliferative effects on human liver hepatoma (Bel-7402) and neuroblastoma (SHSY5Y) cells.
• Antifungal activity was observed against Pythium aphanidermatum, Fusarium oxysporum, Alternaria alternata, Fusarium solani, and Botrytis cinerea.

Conclusions:

• Glytine is a novel, highly stable trypsin inhibitor isolated from Glycine max seeds.
• The inhibitor possesses significant antiproliferative and antifungal properties, expanding its potential applications.
• This study highlights the potential of Glytine as a therapeutic agent and a component in crop protection strategies.