Purification and characterization of a lipopeptide produced by Bacillus thuringiensis CMB26

Abstract

Aims:To isolate an antagonist for use in the biological control of phytopathogenic fungi including Colletotrichum gloeosporioides, then to purify and characterize the biocontrol agent produced by the antagonist. Methods and Results: Bacteria that exhibited antifungal activity against the causative agent pepper anthracnose were isolated from soil, with Bacillus thuringiensis CMB26 showing the strongest activity. A lipopeptide produced by B. thuringiensis CMB26 was precipitated by adjusting the pH 2 with 3 N N HCl and extracted using chloroform/ methanol (2 : 1, v/v) and reversed-phase HPLC. The molecular weight was estimated as 1447 Da by MALDI-TOF mass spectrometry. Scanning electron and optical microscopies showed that the lipopeptide has activity against Escherichia coli O157:ac88, larvae of the cabbage white butterfly (Pieris rapae crucivora) and phytopathogenic fungi. The lipopeptide had cyclic structure and the amino acid composition was LL-Pro, and L L-Ile in a molar ratio of 3 : 1 : 2 : 1 : 1 : 2 : 1 : 1. The purified lipopeptide showed the same amino acid composition as fengycin, but differed slightly in fatty acid composition, in which the double bond was at carbons 13-14 (m/z 303, 316) and there was no methyl group. Conclusion: A lipopeptide was purified and characterized from B. thuringiensis CMB26 and found to be similar to the lipopeptide fengycin. This lipopeptide can function as a biocontrol agent, and exhibits fungicidal, bactericidal, and insecticidal activity. Significance and Impact of the Study: Compared with surfactin and iturin, the lipopeptide from B. thuringiensis CMB26 showed stronger antifungal activity against phytopathogenic fungi. This lipopeptide is a candidate for the biocontrol of pathogens in agriculture.

Screening concepts, characterization and structural analysis of microbial-derived bioactive lipopeptides: a review

“…[125] GC-MS was utilized in the identification of fatty acid moieties in purified LP where the LP was discovered to contain a C 17 fatty acid with one double bond between carbons 13 and 14. [125] In a study using GC-MS to analyze TLCpurified LP, the GC analysis spectrum indicated that fatty acids portions of the LP contained 3-hydroxy tridecanoate (3-OH-C 13 ), tetradecanoate (3-OH-C 14 ), pentadecanoate (3-OH-C 15 ) and hexadecanoate (3-OH-C 16 ). [126] A subsequent study identified GC-MS peaks that were characteristic of b-hydroxy fatty acid methyl derivatives.…”

Antifungal activity and bioactive compounds produced by Bacillus mojavensis and Bacillus subtilis

“…These amphiphilic cyclic biosurfactants have many advantages: low toxicity, high biodegradability and environmentally friendly characteristics (Kim et al, 2004). In literature, several approaches for lipopeptide identification have been proposed but their biological activity needs to be determined after isolation procedures (Price et al, 2007).…”

Antifungal Activity of Bacillus amyloliquefaciens NJN-6 Volatile Compounds against Fusarium oxysporum f. sp. cubense

“…Previous research has shown that abundant nutrients in the soil increased the production of bacterial VOCs, suggesting that the amount of organic matter in the soil might affect the ability of NJN-6 to release VOCs into the soil (5, 6). Nonvolatile antibiotics, including lipopeptides, have strong antifungal activities (8,12). However, these nonvolatile antibiotics cannot spread over long distances, and only when these antagonists directly colonize the plant roots can they prevent pathogenic fungi from infecting plants.…”