Purification of peptides from Bacillus strains with biological activity

Abstract

Postharvest decays of fruits and vegetables account for significant levels of postharvest losses. It is estimated that about 20–25% of the harvested fruits and vegetables are decayed by pathogens during postharvest handling even in developed countries (El-Ghaouth et al., 2004; Droby, 2006; Zhu, 2006; Singh and Sharma, 2007). Synthetic fungicides are primarily used to control postharvest diseases of fruits and vegetables (El-Ghaouth et al., 2004; Korsten, 2006; Singh and Sharma, 2007; Zhu, 2006; Sharma et al, 2009). However, the global trend appears to be shifting towards reduced use of fungicides on produce and hence, there is a strong public and scientific desire to seek safer and eco-friendly alternatives for reducing the decay loss in the harvested commodities (Mari et al., 2007). One of the biggest ecological challenges facing microbiologists and plant pathologists in the near future is the development of environmentally friendly alternatives to the extensive use of chemical pesticides for combatting crop diseases. The use of beneficial microorganisms (biopesticides) is considered one of the most promising methods for more rational and safe crop-management practices (Fravel, 2005). Among different biological approaches, use of the microbial antagonists like yeasts, fungi, and bacteria is quite promising and gaining popularity (Eckert and Ogawa, 1988; Droby et al., 1991; Wisniewski and Wilson, 1992; Droby, 2006; Korsten, 2006). Production of antibiotics is the second important mechanism by which microbial antagonists suppress the pathogens of harvested fruits and vegetables. For instance, bacterial antagonists like Bacillus subtilis and Pseudomonas cepacia Burkh are known to kill pathogens by producing the antibiotic iturin (Gueldner et al., 1988; Pusey, 1989). The antagonism so produced by Bacillus subtilis was effective in controlling fungal rot in citrus (Singh and Deverall 1984) and Monilinia fructicola (Winter) Honey in peaches and cherries (Pusey and Wilson, 1984; Utkhede and Sholberg, 1986). Further, Pseudomonas cepacia inhibited the growth of postharvest pathogens like Botrytis cinerea and Penicillium expansum in apple by producing an antibiotic, pyrrolnitrin (Janisiewicz and Roitman, 1988; Janisiewicz et al., 1991). Pseudomona cepacia was also effective in controlling green mold (Penicillium digitatum) in lemon (Citrus limon L.) by producing antibiotics (Smilanick and Denis-Arrue, 1992). Similarly, the bacterial antagonist, Pseudomona syringae Van Hall, controlled green mold of citrus and gray mold of apple, by producing an antibiotic syringomycin (Bull et al., 1998). Members of the Bacillus genus are often considered microbial factories for the production of a vast array of biologically active molecules potentially inhibitory for phytopathogen growth, such as kanosamine or zwittermycin A from B. cereus. Their spore-forming ability also makes these bacteria some of the best candidates for developing efficient biopesticide products from a technological point of view. Bacillus spores have a high level of resistance to the dryness necessary for formulation into stable products. Bacillus subtilis strains are a rich source of antimicrobial peptides with a high potential for biological control applications. Lipopeptides are classified into three families depending on their amino acid sequence: iturins, fengycins and surfactins (Perez García, et al, 2011). The surfactins are powerful biosurfactants, which show antibacterial activity but no marked fungitoxicity (with some exceptions) (Ongena, and Jacques, 2007). These antibiotics are either cyclopeptides (iturins) or macrolactones (fengycins and surfactins) characterized by the presence of L and D amino acids and variable hydrophobic tails. Iturins display strong antifungal action against a wide variety of yeasts and fungi but only limited antibacterial activity. Fengycins also show a strong fungitoxic activity, specifically against filamentous fungi (Ongena and Jacques, 2007). The ability of various Bacillus strains to control fungal soil borne, foliar and postharvest diseases has been attributed mostly to iturins and fengycins (Ongena and Jacques, 2007; Romero et al, 2007; Arrebola et al, 2010). In order to identify the compounds responsible for antimicrobial activity, extracts of cell-free culture filtrates Bacillus strains generally are separated in silica thin-layer chromatography (TLC) sheets, using purified iturin A, fengycin, and surfactin as standards Spots with Rf values similar to the standarts fengycin (0.09), iturin A (0.3), and surfactin (0.7) will be observed (Romero et al, 2007). After this preliminary TLC, the extracts should be analyzed by reverse-phase high-performance liquid chromatography (RP-HPLC), three main groups of peak will be observed at elution times comparable with those observed for standard lipopeptides. To verify the purity of different fractions, analytical RP-HPLC performed. For an accurate characterization of the different purified compounds, mass spectra are recorded by matrix-assisted laser desorption-ionization time of flight mass spectrometry (MALDI-TOF-MS). The peaks are at 1008.7 m/z (surfactin), 1065.4 m/z (iturin A), and 1464 m/z (fengycin). All fractions will be studied by the Fourier transform-infrared spectrum(FT-IR) analysis showing bands in the range of 1,630 to 1,680cm–1, resulting from the stretching mode of the CO-N bond (amide I band), and at 1,570 to 1,515 cm–1, resulting from the deformation mode of the N-H bond combined with C-N stretching mode (amide II band), both indicating the presence of a peptide component; and also bands at 2,855 to 2,960 cm-1,resulting from typical CH stretching vibration in the alkyl chain (Romero et al, 2007; Maldonado et al, 2009). Finally the amino acid sequences are determined by electrospray ionization ion trap mass spectrometry (ESI-MS-MS). Usually, several homologues of these peptides coexist in the fermentation culture of Bacillus strains and culture conditions have significant influence on the amount and types of these homologues (Akpa et al. 2001). These features suggest a high degree of regulatory expression of the genes involved in the biosynthesis of these antimicrobial peptides. However, when studying the influence of culture conditions on the production of fengycins by Bacillus strains, it is difficult to discriminate the homologues (Akpa et al. 2001; Wang et al. 2004). Fengycin homologues have a similar moiety of β-hydroxy fatty acid L-Glu, D-Orn, L-Tyr, D-allo-Thr, L-Glu, D-Ala (D-Val), L-Pro, L-Gln, D-Tyr, L-Ile with a lactone bond connecting L-Tyr and L-Ile and are divided into two types (fengycin A and B) based on their amino acid sequences. For each homologue, if the amino acid at position 6 is D-Ala, it is fengycin A, and D-Val for fengycin B. As the length of hydrophobic fatty acid tails is variable, a fengycin A will have the same nominal mass as that of fengycin B with a fatty acid tail of two –CH2– groups shorter (for the molecular weight difference between Val and Ala is 28 Da while the molecular weight of two –CH2– groups is 28 Da too) (Wang et al. 2004). In surfactin family the variants are Esperin, Lichenycin, Pumilacidin and surfactin. In iturin family Bacillomycin D, F, L, LC, Iturin A, AL, C and Mycosubtilin. (Ongena and Jacques, 2007). Structural groups are particularly involved in interaction with membranes and/or are supposed to be important for biological activity, an addition to the cyclic nature of the molecules (Ongena and Jacques, 2007).