Abstract
Thaumatin-like protein from banana (designated BanTLP) has been purified by employing a simple protocol consisting of diethylaminoethyl Sephadex (DEAE–Sephadex) chromatography, gel filtration on Sephadex G50, and reversed-phase chromatography. The purified protein was identified by MALDI-TOF mass spectrometry, with an estimated molecular weight of 22.1 kDa. BanTLP effectively inhibited in vitro spore germination of Penicillium expansum, one of the main postharvest pathogens in fruits. This study further investigated the antifungal properties and underlying mechanisms of BanTLP against P. expansum. Results demonstrated that BanTLP exhibited antifungal activity in a wide pH range (4.0–10.0) at 20–50 °C. Propidium iodide (PI) influx and potassium release confirmed that BanTLP induced membrane disruption of the test pathogen, increasing the membrane permeability and disintegration of the cell. This led to cell death, as evidenced by the assays of thiobarbituric acid-reactive species (TBARS) content, the production of reactive oxygen species (ROS), and 1,6-diphenyl-1,3,5-hexatriene (DPH) fluorescence integrity. Ultrastructural alterations in P. expansum conidia after BanTLP treatment revealed severe damage to the cell wall. These results suggest that BanTLP purified from banana exerts antifungal activity against P. expansum by inducing plasma membrane disturbance and cell wall disorganization.
Highlights
Banana (Musa acuminate) is considered to be one of the most important and popular fruits in the human diet due to its pleasant taste and its high nutritional value, in addition to its anti-hypertension, anti-diabetic, anti-ulcerogenic, anti-cancer, anti-proliferative, and antioxidant functions [1]
BanTLP has been purified using a three-step chromatographic purification procedure consisting of anion-exchange chromatography, gel filtration, and reversed-phase chromatography, whereby the elutes were monitored by determining the absorbance at 280 nm
The purity of BanTLP was confirmed by SDS-PAGE, which revealed that the purified protein consisted exclusively of a single polypeptide (Figure 1C, insert)
Summary
Banana (Musa acuminate) is considered to be one of the most important and popular fruits in the human diet due to its pleasant taste and its high nutritional value, in addition to its anti-hypertension, anti-diabetic, anti-ulcerogenic, anti-cancer, anti-proliferative, and antioxidant functions [1]. Research has shown that the pulp of ripe banana contains a great quantity of thaumatin-like protein (TLP) [2]. TLP, which belongs to pathogenesis-related (PR) protein family 5 (PR5 protein), shares an amino acid sequence with thaumatin TLPs are the products of a large, highly complex gene family containing fungal, plant, and animal TLPs. TLPs appear to possess biological functions such as antifungal, glucanase, and xylanase inhibition activities, and are considered to be involved in host defense, stress tolerance, and cell signaling [4,5,6]. Many transgenic plants overexpressing TLPs have been developed for enhancing host resistance against pathogens, indicating that TLPs hold a great prospective for the molecular breeding of resistant plants [3]
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