Lipopeptide Antibiotic Iturin Research Articles

Evaluation of HPLC and MEKC methods for the analysis of lipopeptide antibiotic iturin A produced by Bacillus amyloliquefaciens

Two methods, high-performance liquid chromatography (HPLC) and micellar elec- trokinetic chromatography (MEKC), for analysis of multicomponent lipopeptide antibiotic iturin A are described. After conducting a series of optimizations, the HPLC separation of iturin A2, A3, A4, A5, A6, A7, and A8 isomers were achieved by a NPS RP-C18 column with an isocratic elution of acetonitrile/10 mM ammonium acetate (40:60, v/v), and flow rate of 1 mL/min. On the other hand, the MEKC separation achieved at 25 °C using an applied voltage of 20 kV, and back- ground electrolyte consisting of 20 mM boric acid buffer (pH 8.72) with 50 mM sodium dodecyl sulfate (SDS) and 10% (v/v) acetonitrile. The calibration curve was linear to 20-200 µg/mL and 50-500 µg/mL for HPLC and MEKC with R 2 = 0.9954 and R 2 = 0.9967, respectively. The detec- tion limits of iturin A from the strain B128 cultivation medium were in the 2.5-7.0 µg/mL and 5.0-13.0 µg/mL ranges, and the reproducibility of within-day assay was 98.1 and 98.7%, for HPLC and MEKC, respectively (a UV detector was applied in the both cases). Both methods were selective, robust, and specific, allowing reliable quantification of iturin A, and could be useful for clinical and biomedical investigations of related antibiotics.

Antifungal activity of lipopeptides from Bacillus amyloliquefaciens MG3 against Colletotrichum gloeosporioides in loquat fruits

This work aims to produce and purify antifungal lipopeptides from fermentation by B. amyloliquefaciens MG3, and verify antifungal mechanism on C. gloeosporioides. Using Quadrupole-Time of Flight Mass Spectrometry (Q-TOF-MS) analysis, the component with antifungal activity in the fermentation supernatant of the MG3 strain was determined to be the lipopeptide antibiotic iturin A (C14-C15). Antagonistic tests showed that iturin A could inhibit the mycelia growth and spore germination of C. gloeosporioides. The minimal inhibitory concentration (MIC), half maximal inhibitory concentration (IC50) and minimal fungicidal concentration (MFC) of iturin A against C. gloeosporioides were 36.47, 74.95 and 110 μg/mL, respectively. Iturin A induced an increase of cell membrane permeability and MDA content and a decrease of protein content in the fungal cell. It was observed that iturin A treatment resulted in smaller C. gloeosporioides lesion diameters on loquat fruit compared with the control group. Overall, the results of this study show that iturin A from B. amyloliquefaciens MG3 may be a potential biological control agent for postharvest preservation of loquat fruit.

Simultaneous hydrolysis with lipase and fermentation of rapeseed cake for iturin A production by Bacillus amyloliquefaciens CX-20

BackgroundRapeseed cake (RSC), as the intermediate by-product of oil extraction from the seeds of Brassica napus, can be converted into rapeseed meal (RSM) by solvent extraction to remove oil. However, compared with RSM, RSC has been rarely used as a raw material for microbial fermentation, although both RSC and RSM are mainly composed of proteins, carbohydrates and minerals. In this study, we investigated the feasibility of using untreated low-cost RSC as nitrogen source to produce the valuable cyclic lipopeptide antibiotic iturin A using Bacillus amyloliquefaciens CX-20 in submerged fermentation. Especially, the effect of oil in RSC on iturin A production and the possibility of using lipases to improve the iturin A production were analyzed in batch fermentation.ResultsThe maximum production of iturin A was 0.82 g/L at the optimal initial RSC and glucose concentrations of 90 and 60 g/L, respectively. When RSC was substituted with RSM as nitrogen source based on equal protein content, the final concentration of iturin A was improved to 0.95 g/L. The production of iturin A was further increased by the addition of different lipase concentrations from 0.1 to 5 U/mL into the RSC medium for simultaneous hydrolysis and fermentation. At the optimal lipase concentration of 0.5 U/mL, the maximal production of iturin A reached 1.14 g/L, which was 38.15% higher than that without any lipase supplement. Although rapeseed oil and lipase were firstly shown to have negative effects on iturin A production, and the effect would be greater if the concentration of either was increased, their respective negative effects were reduced when used together.ConclusionsAppropriate relative concentrations of lipase and rapeseed oil were demonstrated to support optimal iturin A production. And simultaneous hydrolysis with lipase and fermentation was an effective way to produce iturin A from RSC using B. amyloliquefaciens CX-20.

Evaluation of Yield and Surface Tension-lowering Activity of Iturin A Produced by Bacillus subtilis RB14.

Bacillus subtilis RB14 produces the lipopeptide antibiotic iturin A by submerged and biofilm fermentation. In this study, we optimized the conditions for iturin A production in a jar fermentor. The maximum yield of iturin A was 932 mg L-1 after 120 h. The surface tension of water decreased from 72.0 to 39.0 mN m-1 as the concentrations of C14 iturin A increased, indicating that C14 iturin A behaves as a surfactant in water. The critical micellar concentration obtained from the intersection of two fitted lines was 1.2 × 10-4 M. Moreover, the surface tension of water decreased as the length of the alkyl chain of iturin A increased.

Improvement of iturin A production in Bacillus subtilis ZK0 by overexpression of the comA and sigA genes.

This study provides new perspectives on improving iturin A production by Bacillus subtilis. Our newly engineered strains could be applied to commercial fermentation by enhancing yields of iturin A and reducing the rate of 'stuck fermentation'. Increased production would facilitate more widespread application of this powerful antibiotic.

Continuous enhancement of iturin A production by Bacillus subtilis with a stepwise two-stage glucose feeding strategy.

BackgroundThe lipopeptide antibiotic iturin A is an attractive biopesticide with the potential to replace chemical-based pesticides for controlling plant pathogens. However, its industrial fermentation has not been realized due to the high production costs and low product concentrations. This study aims to enhance iturin A production by performing a novel fermentation process with effective glucose feeding control using rapeseed meal as a low-cost nitrogen source.ResultsWe demonstrated that continuous and significant enhancement of iturin A production could be achieved by a novel two-stage glucose-feeding strategy with a stepwise decrease in feeding rate. Using this strategy, the ratio of spores to total cells could be maintained at a desirable/stable level of 0.80–0.86, and the reducing sugar concentration could be controlled at a low level of 2–3 g/L so that optimal substrate balance could be maintained throughout the feeding phase. As a result, the maximum iturin A concentration reached 1.12 g/L, which was two-fold higher than that of batch culture.ConclusionsThis is the first report which uses control of the glucose supply to improve iturin A production by fed-batch fermentation and identifies some important factors necessary to realize industrial iturin A production. This approach may also enhance the production of other useful secondary metabolites by Bacillus subtilis.

Improvement of production of lipopeptide antibiotic iturin A using fish protein

To enhance the production of lipopeptide antibiotic iturin A, nutrient contents of the culture mediums were investigated in both submerged and biofilm fermentations. As a carbon source maltose and as nitrogen source, fish protein was used. In submerged fermentation maltose uptake was found lower (12%) compared to biofilm fermentation (15%) that was associated with higher cellular growth in biofilm. However, requirement of nitrogen (fish protein) concentration was found similar in both submerged and biofilm fermentations. Production of iturin A in submerged fermentation with 12% maltose and 5% fish protein was 4450 mg/L, and in biofilm fermentation it was 5050 mg/L when 15% maltose and 5% fish protein was used.

Isolation of antifungal bacteria from Japanese fermented soybeans, natto

An inhibitory effect of a traditional Japanese fermented food, natto, was found against plant pathogens such as Rhizoctonia solani and Fusarium oxysporum, and the bacteria which showed inhibition were isolated from the natto. Among isolated bacteria, BC-1 and GAc exhibited a strong antagonistic effect in vitro against plant pathogens on an agar medium. The supernatant of bacterial culture also showed strong activity against R. solani, which meant the antimicrobial substances were produced and secreted into the medium. Both of the bacteria were estimated as Bacillus amyloliquefaciens from a partial sequence of the 16s rRNA gene. High performance liquid chromatography analysis clearly showed the production of the lipopeptide antibiotic iturin A by BC-1 and GAc.

Molecular and Biochemical Approaches for Characterization of Antifungal Trait of a Potent Biocontrol Agent Bacillus subtilis RP24

Bacillus subtilis strain RP24, isolated from rhizoplane of field grown pigeon pea, exhibited in vitro antagonism against a wide range of phytopathogenic fungi. An attempt was made to partially purify and characterize the diffusible antifungal metabolite/s produced by the strain RP24 and its negative mutant (NM) in potato dextrose medium. High performance liquid chromatography (HPLC) of partially purified extract of RP24 showed the presence of lipopeptide antibiotic iturin as a major peak that was comparable to that of standard iturin A (5.230 min) from Sigma-Aldrich whereas the corresponding peak was absent in extract of NM. The structure was further confirmed by liquid chromatographic mass spectrometric (LCMS) analysis as iturin A. LCMS analysis also showed the presence of surfactin and fengycin besides iturin A. Amplification of the lpa-14 (encodes the 4'-phosphopantetheinyl transferase required for the maturation of template enzyme of iturin A) and ituD (encodes a putative malonyl coenzyme A transacylase, whose disruption results in a specific deficiency in iturin A production) genes of iturin operon of strain RP24 was carried out and the sequences obtained were compared with the existing database of NCBI. The sequences of lpa-14 and ituD gene of RP24 showed 98% and 97% homology with lpa-14 and ituD genes of B. subtilis in the existing database. The results indicated that strain RP24 harbors iturin operon in its genome and a chemical mutation in this operon might have resulted in loss of antifungal activity in the negative mutant.

Application of malt residue in submerged fermentation of Bacillus subtilis

Malt residue is a common waste or byproduct from beer industries after brewing and milling of malted barley. In this work, Bacillus subtilis RB14 was used to study the microbial growth and production of secondary metabolites like lipopeptide antibiotic iturin A in the malt residue for its effective recycling. B. subtilis RB14 could grow in submerged fermentation of malt residue and significant growth (10 9 CFU/mL) was observed without any supplementation. In submerged fermentation iturin A production using malt residue was about 170 mg/L, which was found to be higher than its production in No.3 (Polypepton, glucose, KH 2PO 4, MgSO 4·7H 2O) medium where production was about 120 mg/L. More than 600 mg/L of iturin A production was observed when malt residue was combinedly used with No.3 medium. This production was significantly higher than their summation of their individual production. However, the growth of B. subtilis in combined medium was found to be similar to that of the submerged fermentation in simple malt residue. Therefore, the remarkable enhancement in production of iturin A in supplemented malt residue was attributed to the nutrients supplied from No.3 medium.

Biofilm formation and lipopeptide antibiotic iturin A production in different peptone media

Biofilm fermentation is a newly developed promising technique in fermentation technology. In this study no.3 and no.3S media have been used for the lipopeptide antibiotic iturin A production by Bacillus subtilis RB14. The main component of no.3 and no.3S media is Polypepton and Polypepton S, respectively. B. subtilis RB14 produces thick stable biofilm and high amount of iturin A in no.3S medium. Whereas, impaired biofilm formation and lower iturin A production was observed in no.3 medium. From the analytical information it was observed that the amounts of metal ions, such as K +, Ca 2+ and Mn 2+, cysteine and cellulose are lower in Polypepton compared to the Polypepton S. To investigate their effect on biofilm formation and iturin A production cysteine, cellulose, K +, Ca 2+ and Mn 2+ were added respectively into the no.3 medium at similar amount that Polypepton S contains. It was observed that individual addition of K +, Ca 2+, cysteine and cellulose had no effect on biofilm formation, cellular growth induction or iturin A production. However, when Mn 2+ was supplemented in no.3 medium, biofilm development was restored with an improved production of iturin A. Finally, combined addition of investigated substances into the no.3 medium resulted with highly folded, thick biofilm with high cellular growth and iturin A production compared to the original no.3 medium.

Solid state fermentation of lipopeptide antibiotic iturin A by using a novel solid state fermentation reactor system

A new solid state fermentation reactor (SSFR) for solid substrate was used for the production of lipopeptide antibiotic iturin A using Bacillus subtilis RB14-CS. Solid state fermentation (SSF) is the technique of cultivation of microorganisms on solid and moist substrates in the absence of free water. SSF has shown much promise in the development of several bioprocesses and products because of their several advantages like absence of free water that allows simplified downstream processing and low cost. SSFR allows agitation of the SSF culture with improved temperature control and air supply. Interestingly, when okara, the widely available waste product from the tofu industries, was used as the solid substrate for the SSFR, no iturin A production was observed. However, without agitation, production of iturin A was observed in the SSFR but the production level remained low. The low production of iturin A was found to be due to the heat generation and excess temperature rise inside the reactor system during the fermentation process. Maintaining the temperature within a range of 25–30°C, production of iturin A was significantly improved in the SSFR. This was comparable to the laboratory scale production, and signifies the potential application of the SSFR for SSF.

Biofilm fermentation of iturin A by a recombinant strain of Bacillus subtilis 168

Bacillus subtilis 168 produces thin and fragile biofilm in the static culture, however, it was found out that its transformant B. subtilis RM/iSd16 containing wild sfp, itu operon and degQ, which produced lipopeptide antibiotic iturin A, produced thick and much stable biofilm. Production of iturin A by RM/iSd16 in biofilm was almost two times higher compared to that in the submerged culture at 28 degrees C.

Second stage production of iturin A by induced germination of Bacillus subtilis RB14

Bacillus subtilis RB14, a dual producer of lipopeptide antibiotics iturin A and surfactin undergoes sporulation in the submerged fermentation and the production of these secondary metabolites becomes halted. In this study, production of lipopeptide antibiotics was investigated by induced germination of the spores by heat-activation and nutrient supplementation. The induced spores became metabolically active vegetative state and produced lipopeptide antibiotic iturin A that added up the total production at the end of the fermentation. However, additional production of surfactin was not observed. This second time iturin A production by the germinated cells from the spores was defined as second stage production.

Production of lipopeptide antibiotic iturin A using soybean curd residue cultivated with Bacillus subtilis in solid-state fermentation

Bacillus subtilis RB14-CS, which suppresses the growth of various plant pathogens in vitro by producing the lipopeptide antibiotic iturin A, was cultured using soybean curd residue, okara, a by-product of tofu manufacture in solid-state fermentation. After 4 days incubation, iturin A production reached 3,300 mg/kg wet solid material (14 g/kg dry solid material), which is approximately tenfold higher than that in submerged fermentation. When the okara product cultured with RB14-CS was introduced into soil infested with Rhizoctonia solani, which is a causal agent of damping-off of tomato, the disease occurrence was significantly suppressed. After 14 days, the number of RB14-CS cells remained in soil at the initial level, whereas almost no iturin A was detected in soil. As the okara cultured with RB14-CS exhibited functions of both plant disease suppression and nutritional effect on tomato seedlings, this product is expected to contribute to the recycling of the soybean curd residue.

A Biophysical Study of the Interaction of the Lipopeptide Antibiotic Iturin A with Aqueous Phospholipid Bilayers

Iturin A is a lipopeptide extracted from the culture media of Bacillus subtilis which shows a strong antifungal action. The interaction of iturin A with multilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) induced structures which did not sediment during centrifugation. Electron microscopy after negative staining showed that, at 30 mol%, iturin A/DMPC vesicles were visible but smaller than those formed by pure DMPC. Thermograms of DMPC/iturinA obtained after differential scanning calorimetry, at low concentrations of iturin A, were interpreted as indicating the presence of two laterally separated phases, one formed by pure phospholipid and the other by lipopeptide–phospholipid complexes, these two separated phases being already detected even at low concentrations such as 2 mol%. Fluorescence quenching experiments showed that the D-Tyr residue of the lipopeptide was fully accessible to the aqueous medium, indicating that the polar part of iturin A is located outside of the membrane hydrophobic palisade. It was concluded that the membrane barrier properties are likely to be damaged in the area where the lipid complexes are accumulated, due to structural fluctuations, and this may be one of the bases of its biological activity. Iturin-A was also able to greatly destabilize dielaidoylphosphatidylethanolamine (DEPE) membranes in the fluid form, producing a new structure which had a poor correlation in X-ray diffraction, and in 31P NMR spectroscopy gave rise to a spectrum containing a double isotropic signal. Iturin A was shown to induce DEPE to adopt phases other than HII inverted hexagonal, underlining that this lipopeptide is capable of modifying the curvature of the membrane, which may also be important in explaining the tendency of iturin A to create small vesicles and which may be another of the bases of its biological activity.

Interaction of iturin A, a lipopeptide antibiotic, with Saccharomyces cerevisiae cells: influence of the sterol membrane composition

The binding of the membrane-active lipopeptide antibiotic iturin A to yeast cells was studied using radioactive iturin A. Saccharomyces cerevisiae had a maximum binding capacity of 5.6 x 10(9) molecules per single cell. The Scatchard plot of binding showed a biphasic profile, with a lower dissociation constant for small concentrations of iturin A. The break of slope at 30 microM iturin A corresponds to the micellization of antibiotic in solution. The binding is also dependent on the nature of the sterol present in the membrane. A mutant yeast strain with a membrane containing cholesterol instead of ergosterol showed the highest affinity for iturin A and the highest sensitivity to this antibiotic, as measured by K+ ion release. In contrast the presence of stigmasterol increased the resistance of the cells to iturin A.