- Email: [email protected]
Tostadin, a novel antibacterial peptide from an antagonistic microorganism Brevibacillus brevis XDH
Bioresource Technology 111 (2012) 504–506
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Tostadin, a novel antibacterial peptide from an antagonistic microorganism Brevibacillus brevis XDH Zhen Song a, Qingxin Liu b, Hui Guo a,c, Ruicheng Ju a, Yuhua Zhao a, Jinyu Li b, Xunli Liu b,⇑ a
College of Life Science, Shandong Agricultural University, Taian, China College of Forestry, Shandong Agricultural University, Taian, China c Institute of Environment and Sustainable Development in Agriculture, Chinese Academy of Agricultural Sciences, Beijing, China b
a r t i c l e
i n f o
Article history: Received 27 December 2011 Received in revised form 5 February 2012 Accepted 11 February 2012 Available online 18 February 2012 Keywords: Brevibacillus brevis Antibacterial peptide Purification Characterization
a b s t r a c t A novel small antibacterial peptide was obtained from the liquid culture of Brevibacillus brevis XDH, which is a broad-spectrum antagonistic bacterium isolated from the soil of Mountain Tai, China. This peptide was purified from the fermentation medium of strain XDH via ammonium sulfate precipitation, cation exchange chromatography, and reversed-phase high-performance liquid chromatography (HPLC), successively. The structure of the active linear peptide was elucidated using mass spectra (MS) and nuclear magnetic resonance (NMR) analyses that consisted of nine amino acids. This peptide was easily soluble in water, thermally stable and strongly inhibited the growth of Escherichia coli and Staphylococcus aureus in vitro. The present data support the identification of a novel antibacterial peptide, which was named Tostadin. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Escherichia coli and Staphylococcus aureus, which are common and damaging pathogens of humans and animals, have a wide range of hosts and are a major threat to public health and food safety (Eaton et al., 2008). Chemical control currently remains as the primary means for preventing diseases caused by E. coli and S. aureus. Numerous synthetic chemicals comprise the major market share of antibiotics (Cambau et al., 2009). However, the demand for more effective and safer compounds with novel modes of action has been increasing because of the rapid emergence of drug-resistant pathogens. Consequently, the selection of natural antibiotics from antagonistic microorganisms is considered as an alternative method for the disease control. Antimicrobial peptides are the most important and effective antibiotics for combating the increase of drug-resistant bacteria (Wang et al., 2010). In the past few years, biologically based drugs have aroused a wide public concern because of its environmental protection and security features. Scientists have focused on the use of antagonistic bacteria and their active substances (Viscardi et al., 2008). Several classes of proteins and peptides that inhibit the growth of fungi and bacteria in vitro assays have been identified in the last decade. In 1996, the genus Brevibacillus, which is a well-known rod-shaped ⇑ Corresponding author. Postal address: Department of Sericulture, College of Forestry, Shandong Agricultural University, 61 Daizong Street, Taian, Shandong Province 271018, China. Tel.: +86 538 8249131; fax: +86 538 8249164. E-mail address: [email protected] (X. Liu). 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2012.02.051
Gram-positive bacterium, was established as an independent genus from the reclassification of Bacillus brevis (Shida et al., 1996). Brevibacillus is omnipresent in agricultural soils, and it can secrete structurally diverse secondary metabolites with broad antibiotic spectra. Some of these metabolites, such as chitinase and gramicidin S, have been extensively studied, and numerous Brevibacillus species, which have the potential as antimicrobial agents, have become research hotspots in the recent years (Mogi and Kita, 2009). Brevibacillus brevis XDH (Genbank accession number: DQ279 738), which can inhibit many pathogens both in vivo and in vitro, was isolated from the soil of Mountain Tai, China. In the present study, a novel antibacterial peptide, which was named as Tostadin, was isolated and purified from the liquid culture of strain XDH. Moreover, the structure of Tostadin was elucidated and some of its properties were analyzed.
2. Methods 2.1. Microorganism and culture conditions Brevibacillus brevis XDH was isolated from the soil of Mountain Tai and was stored in the College of Forestry, Shandong Agricultural University. It was maintained at 4 °C on potato dextrose agar (PDA: 200 g potato, 20 g glucose, 5 g beef extract, 5 g sodium chloride, and 20 g agar in 1 L distilled water) plate and was subcultured every month prior to use.
Z. Song et al. / Bioresource Technology 111 (2012) 504–506
The liquid medium used in the present study was composed of 20 g glucose, 30 g soybean meal, 6 g starch, 2 g calcium carbonate, 4 g magnesium sulfate, and 1 L distilled water. The seed liquid in a 250 mL Erlenmeyer flask with 50 mL medium was cultured in a rotary shaker under 30 °C at 200 rpm for 18 h. Fermentation was then performed in 250 mL flasks containing 50 mL medium with a 2% aseptic inocula under similar conditions described above. 2.2. Determination of the antibacterial activity The mixed-germs-plate method was used to determine the antibacterial activity of the antibacterial substances using the Oxford plate assay system. An E. coli strain, which was kindly provided by the College of Animal Sciences and Technology, Shandong Agricultural University, was used as the indicating microorganism with the concentration controlled at 2–6 104 CFU/mL in the medium. The fermentation broth was heated at 100 °C for 10 min and was centrifuged at 9500g for 20 min. Up to 250 lL supernatant was then injected onto the Oxford cup, and the diameters of the inhibition zones were measured after being incubated at 30 °C for 6 h. The fitting equation for antibacterial activity (titer) counting is given by Y = 10[(X 2.6475)/5.9311] N, where Y represents the titer (using concentration unit lg/mL), X represents the diameter of the inhibition spot (11.3 mm < X < 14.5 mm), and N represents the dilution times of the fermentation broth. 2.3. Isolation and purification of the antibacterial substance The fermentation broth of B. brevis XDH was sterilized at 100 °C for 10 min and was centrifuged at 4200 rpm for 30 min. The supernatant, which was mixed with ammonium sulfate to the saturation of 45%, was kept at 25 °C overnight and was centrifuged at 4200 rpm for 20 min. The salting-out sediment was dissolved using distilled water and was condensed using a rotary evaporator. The CM Sepharose Fast Flow gel was rinsed with NaCl (1 mol/L) and distilled water successively for 2–3 times before being loaded onto the column (1.6 30 cm). It was then equilibrated with deionized water for 12 h. Approximately 15 mL of the salting-out samples was added onto the surface of the gel, and was rinsed absolutely with plenty of distilled water to clean the substances that were not adsorbed. Linear gradient elution of NaCl (0–1.0 mol/L) was then performed at a flow rate of 60 mL/h. The eluate was monitored using a UV detector, and a bioassay was used to examine the antibacterial active peaks of the elution. The effective components were collected and desalinated using Sephadex G-25 chromatography, based on the same method as the CM Sepharose FF in loading, monitoring, and collecting, except that it was only rinsed with distilled water. Ultimately, the antibacterial substances were condensed and lyophilized. The crude material was then prepared using reversed-phase HPLC. The condition was as follows: Sinochrom ODS-BP column (300 10 mm, 10 lm), injection volume of 200 lL, temperature of 30 °C, flow rate of 2.0 mL/min, water and acetonitrile containing 0.1% trifluoroacetic acid (v/v: 68/32) as the mobile phase, and absorption wavelength of 210 nm. The active component was collected for property analysis. 2.4. Property analysis of the antibacterial substance The antibacterial component was sent to Shandong Analysis and Test Center, China, and the structure was elucidated via electrospray ionization mass spectrometry (ESI-MS) and experiments of TOCSY, NOESY, ROESY, 13C HSQC 2D NMR. After obtaining the detailed structure, China Peptides Co., Ltd. in Shanghai synthesized 100 mg of the pure sample. The modified 100 mg sample (acetylation at N-terminal and amidation at C-terminal) was also produced
505
by the company to investigate its antibacterial ability. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of the natural product and the two kinds of synthetic peptides were determined according to literature (Gauri et al., 2011). The active peptides, both natural and synthetic, were prepared to 100 lg/mL with distilled water and were incubated at 100 °C for 0, 10, and 20 min, respectively, to determine the thermal stability. After cooling to room temperature, the residual antibacterial activities were measured as described above. 3. Results and discussion 3.1. Isolation and purification of antibacterial component Brevibacillus species generally exist in many environments. As a group, this genus offers several advantages for protection against pathogens, because of the broad-spectrum activity of their antibiotics (Song et al., 2011). In general, the mechanisms of pathogen control are classified as competition, parasitism or predation, and antibiosis. B. brevis XDH is a potent antimicrobial agent isolated from the soil of Mount Tai, and it has a strong antibacterial activity against many pathogens of humans and animals (Song et al., 2012). B. brevis XDH showed great potentials in application, and thus it was chosen for further investigation. The crude antibacterial substances were precipitated using ammonium sulfate from the supernatant of fermentation broth. The extracts were then used for ion exchange chromatography on a CM FF column, which produced two unadsorbed fractions (P1 and P2) and an adsorbed fraction (P3), to enrich the active components from the culture of B. brevis XDH. The three fractions were assayed for growth inhibition on the test pathogens (E. coli and S. aureus). No antibacterial activity was detected in P1 and P2, whereas, it was observed in P3, resulting from the linear NaCl gradient elution at the concentration of around 0.3 mol/L. Further purification for the P3 was conducted using the reversed-phase HPLC, and four main peaks (A, B, C, and D) were obtained. After each peak was assayed for the antibacterial activities of E. coli and S. aureus, the third fraction (C) was found to show strong inhibitory activities. This compound was purified and collected using preparative HPLC on a Venusil XBP C18 preparative column under the same conditions mentioned above. About 35 mg purified component C was obtained from the 5 L liquid culture of B. brevis XDH. 3.2. Property analysis for the antibacterial compound The experiments of ESI-MS and NMR were performed, and the experts from the Shandong Analysis and Test Center analyzed the results. The molecular mass of the active peptide was determined to be about 1102.35 Da. Structure elucidation revealed that the active component was a linear peptide that consisted of nine amino acids. The detailed sequence was illustrated as Ser1-Leu2-Tyr3Lys4-Leu5-Thr6-Cys7-Lys8-Phe9 (SLYKLTCKF). Further unscrambling of the NMR spectra showed that residues 4 and 9 are of Dconfiguration, and others are of L-configuration. No same or similar sequences were found in the protein databases online. The present data support the identification of a novel antibacterial peptide, which was named Tostadin. Tostadin demonstrated significant antibacterial activities against E. coli and S. aureus. Pure Tostadin samples, both unmodified and modified, were synthesized in China Peptides Co., Ltd. to further analyze some of its properties. The MIC and MBC values of the unmodified peptide were 16 lg/mL and 32 lg/mL, respectively, with E. coli as the indicating microorganism. S. aureus was less sensitive to Tostadin with the MIC and MBC values of 32 lg/
506
Z. Song et al. / Bioresource Technology 111 (2012) 504–506
mL and 64 lg/mL, respectively. However, the modified peptide (AC-SLYKLTCKF-NH2) showed a stronger antibacterial activity, of which the MIC and MBC values against both of the two pathogens were 8 lg/mL and 16 lg/mL, respectively, basically the same with those of the natural product. This demonstrated that the synthetic peptides have the similar antibacterial abilities with the natural Tostadin. The results of the thermal stability tests suggest that both the natural and synthetic Tostadin are considerably thermally stable. The antibacterial activity was almost unchanged (remained more than 95% activity) even after being maintained at 100 °C for 20 min. Hundreds of antimicrobial peptides have been isolated and identified from a great number of microorganisms (Dale and Fredericks, 2005). Their modes of actions, including disrupting membranes, interfering with metabolism, and targeting cytoplasmic components have been well studied (Saikia et al., 2010). Brevibacillus is a well-known antibiotic-producing species that can also produce a wide variety of metabolites with antimicrobial activities (Zhang et al., 2008). A number of the active metabolites were bactericidal or bacteriostatic peptides, which were non-ribosomally synthesized using the multi-enzyme-catalyzed systems. Most of these peptides are very stable because of their low molecular weight and specific structure (Frueh et al., 2008). Gramicidin S is such a cyclic decapeptide with wide antibiotic spectra, which is a well-studied antimicrobial compound produced by B. brevis (Elmar et al., 1999). Moreover, these compounds have become research hotspots in the recent years because of their broad antibacterial spectrum, high potency, and no-crossing drug resistance with other antibacterial drugs (Zhao et al., 2010). In the present study, the antibacterial peptide Tostadin was isolated using a three-step isolation procedure, including ammonium sulfate precipitation, cation exchange chromatography on CMSepharose Fast Flow column, and the reversed-phase HPLC. This purification process was successfully used for this antibacterial peptide from strain XDH. The pure active component, namely Tostadin, which showed strong inhibition against E. coli and S. aureus, was purified and identified. Moreover, the modifications at both ends of Tostadin were found to display higher activities than those of the unmodified compounds. This phenomenon may be caused by the stable structure of the modified peptide since it is not easily resisted or decomposed. Besides, both the natural and synthetic Tostadin exhibited thermal stability, which can be found in other non-ribosomal peptides. In summary, this novel compound is a soluble peptide with great antibiotic effects and high stability, and it is easily synthesized because of its low molecular weight and linear structure. Thus, Tostadin may have great potential applications as a novel antibacterial agent. 4. Conclusions In this work, a linear peptide namely Tostadin was isolated and identified from a B. brevis strain XDH, which showed strong antibacterial activities against E. coli and S. aureus. Thirty five milligram
purified Tostadin was obtained by ammonium sulfate precipitation, ion exchange chromatography, and reversed-phase high-performance liquid chromatography. MS and NMR analysis demonstrated that this compound consisted of nine amino acids, of which two amino acids residues were of D-configuration. Further studies illustrated that both the natural and synthetic Tostadins were thermally stable. When modified at both ends, it exhibited higher activity and stability. These findings supported the characterization of a novel antibacterial peptide. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biortech.2012.02.051. References Cambau, E., Matrat, S., Pan, X.S., Bettoni, R.R., Corbel, C., Aubry, A., Lascols, C., Driot, J.Y., Fisher, L.M., 2009. Target specificity of the new fluoroquinolone besifloxacin in Streptococcus pneumoniae, Staphylococcus aureus and Escherichia coli. J. Antimicrob. Chemother. 63, 443–450. Dale, B.A., Fredericks, L.P., 2005. Antimicrobial peptides in the oral environment: expression and function in health and disease. Curr. Issues Mol. Biol. 7, 119– 133. Eaton, P., Fernandes, C.J., Pereira, E., Pintado, M.E., Malcata, F.X., 2008. Atomic force microscopy study of the antibacterial effects of chitosans on Escherichia coli and Staphylococcus aureus. Ultramicroscopy 108, 1128–1134. Elmar, J.P., Ruthven, N.L., Ronald, N.M., 1999. The interaction of the antimicrobial peptide gramicidin S with lipid bilayer model and biological membranes. Biochim. Biophys. Acta 1462, 201–221. Frueh, D.P., Arthanari, H., Koglin, A., Vosburg, D.A., Bennett, A.E., Walsh, C.T., Wagner, G., 2008. Dynamic thiolation–thioesterase structure of a nonribosomal peptide synthetase. Nature 454, 903–906. Gauri, S.S., Mandal, S.M., Pati, B.R., Dey, S., 2011. Purification and structural characterization of a novel antibacterial peptide from Bellamya bengalensis: activity against ampicillin and chloramphenicol resistant Staphylococcus epidermidis. Peptides 32, 691–696. Mogi, T., Kita, K., 2009. Gramicidin S and polymyxins: the revival of cationic cyclic peptide antibiotics. Cell Mol. Life Sci. 66, 3821–3826. Saikia, R., Gogoi, D.K., Mazumder, S., Yadav, A., Sarma, R.K., Bora, T.C., Gogoi, B.K., 2010. Brevibacillus laterosporus strain BPM3, a potential biocontrol agent isolated from a natural hot water spring of Assam. Indian Microbiol. Res.. doi:10.1016/j.micres. 2010.03.002. Shida, O., Takagi, H., Kadowaki, K., Komagata, K., 1996. Proposal for two newgenera Brevibacillus gen. nov. and Aneurini-bacillus gen. nov. Int. J. Syst. Bacteriol. 46, 939–946. Song, Z., Liu, K., Lu, C., Yu, J., Ju, R., Liu, X., 2011. Isolation and characterization of a potential biocontrol Brevibacillus laterosporus. Afr. J. Microbiol. Res. 18, 2675– 2681. Song, Z., Liu, X., Lu, C., Yu, J., Ju, R., Zhao, Y., Li, J., Liu, K., 2012. Medium optimization for the antibacterial substances production from Brevibacillus Brevis XDH using response surface methodology. Adv. Mater. Res. 345, 355–360. Viscardi, M., Perugini, A.G., Auriemma, C., Capuano, F., Morabito, S., Kim, K.P., Loessner, M.J., Iovane, G., 2008. Isolation and characterisation of two novel coliphages with high potential to control antibiotic-resistant pathogenic Escherichia coli (EHEC and EPEC). Int. J. Antimicrob. Agents 31, 152–157. Wang, J., Haddad, N.I., Yang, S.Z., Mu, B.Z., 2010. Structural characterization of lipopeptides from Brevibacillus brevis HOB1. Appl. Biochem. Biotechnol. 160, 812–821. Zhang, B., Xie, C., Yang, X., 2008. A novel small antifungal peptide from Bacillus strain B-TL2 isolated from tobacco stems. Peptides 29, 350–355. Zhao, Z., Wang, Q., Wang, K., Brian, K., Liu, C., Gu, Y., 2010. Study of the antifungal activity of Bacillus vallismortis ZZ185 in vitro and identification of its antifungal components. Bioresour. Technol. 101, 292–297.
Contents lists available at SciVerse ScienceDirect
Bioresource Technology journal homepage: www.elsevier.com/locate/biortech
Short Communication
Tostadin, a novel antibacterial peptide from an antagonistic microorganism Brevibacillus brevis XDH Zhen Song a, Qingxin Liu b, Hui Guo a,c, Ruicheng Ju a, Yuhua Zhao a, Jinyu Li b, Xunli Liu b,⇑ a
College of Life Science, Shandong Agricultural University, Taian, China College of Forestry, Shandong Agricultural University, Taian, China c Institute of Environment and Sustainable Development in Agriculture, Chinese Academy of Agricultural Sciences, Beijing, China b
a r t i c l e
i n f o
Article history: Received 27 December 2011 Received in revised form 5 February 2012 Accepted 11 February 2012 Available online 18 February 2012 Keywords: Brevibacillus brevis Antibacterial peptide Purification Characterization
a b s t r a c t A novel small antibacterial peptide was obtained from the liquid culture of Brevibacillus brevis XDH, which is a broad-spectrum antagonistic bacterium isolated from the soil of Mountain Tai, China. This peptide was purified from the fermentation medium of strain XDH via ammonium sulfate precipitation, cation exchange chromatography, and reversed-phase high-performance liquid chromatography (HPLC), successively. The structure of the active linear peptide was elucidated using mass spectra (MS) and nuclear magnetic resonance (NMR) analyses that consisted of nine amino acids. This peptide was easily soluble in water, thermally stable and strongly inhibited the growth of Escherichia coli and Staphylococcus aureus in vitro. The present data support the identification of a novel antibacterial peptide, which was named Tostadin. Ó 2012 Elsevier Ltd. All rights reserved.
1. Introduction Escherichia coli and Staphylococcus aureus, which are common and damaging pathogens of humans and animals, have a wide range of hosts and are a major threat to public health and food safety (Eaton et al., 2008). Chemical control currently remains as the primary means for preventing diseases caused by E. coli and S. aureus. Numerous synthetic chemicals comprise the major market share of antibiotics (Cambau et al., 2009). However, the demand for more effective and safer compounds with novel modes of action has been increasing because of the rapid emergence of drug-resistant pathogens. Consequently, the selection of natural antibiotics from antagonistic microorganisms is considered as an alternative method for the disease control. Antimicrobial peptides are the most important and effective antibiotics for combating the increase of drug-resistant bacteria (Wang et al., 2010). In the past few years, biologically based drugs have aroused a wide public concern because of its environmental protection and security features. Scientists have focused on the use of antagonistic bacteria and their active substances (Viscardi et al., 2008). Several classes of proteins and peptides that inhibit the growth of fungi and bacteria in vitro assays have been identified in the last decade. In 1996, the genus Brevibacillus, which is a well-known rod-shaped ⇑ Corresponding author. Postal address: Department of Sericulture, College of Forestry, Shandong Agricultural University, 61 Daizong Street, Taian, Shandong Province 271018, China. Tel.: +86 538 8249131; fax: +86 538 8249164. E-mail address: [email protected] (X. Liu). 0960-8524/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.biortech.2012.02.051
Gram-positive bacterium, was established as an independent genus from the reclassification of Bacillus brevis (Shida et al., 1996). Brevibacillus is omnipresent in agricultural soils, and it can secrete structurally diverse secondary metabolites with broad antibiotic spectra. Some of these metabolites, such as chitinase and gramicidin S, have been extensively studied, and numerous Brevibacillus species, which have the potential as antimicrobial agents, have become research hotspots in the recent years (Mogi and Kita, 2009). Brevibacillus brevis XDH (Genbank accession number: DQ279 738), which can inhibit many pathogens both in vivo and in vitro, was isolated from the soil of Mountain Tai, China. In the present study, a novel antibacterial peptide, which was named as Tostadin, was isolated and purified from the liquid culture of strain XDH. Moreover, the structure of Tostadin was elucidated and some of its properties were analyzed.
2. Methods 2.1. Microorganism and culture conditions Brevibacillus brevis XDH was isolated from the soil of Mountain Tai and was stored in the College of Forestry, Shandong Agricultural University. It was maintained at 4 °C on potato dextrose agar (PDA: 200 g potato, 20 g glucose, 5 g beef extract, 5 g sodium chloride, and 20 g agar in 1 L distilled water) plate and was subcultured every month prior to use.
Z. Song et al. / Bioresource Technology 111 (2012) 504–506
The liquid medium used in the present study was composed of 20 g glucose, 30 g soybean meal, 6 g starch, 2 g calcium carbonate, 4 g magnesium sulfate, and 1 L distilled water. The seed liquid in a 250 mL Erlenmeyer flask with 50 mL medium was cultured in a rotary shaker under 30 °C at 200 rpm for 18 h. Fermentation was then performed in 250 mL flasks containing 50 mL medium with a 2% aseptic inocula under similar conditions described above. 2.2. Determination of the antibacterial activity The mixed-germs-plate method was used to determine the antibacterial activity of the antibacterial substances using the Oxford plate assay system. An E. coli strain, which was kindly provided by the College of Animal Sciences and Technology, Shandong Agricultural University, was used as the indicating microorganism with the concentration controlled at 2–6 104 CFU/mL in the medium. The fermentation broth was heated at 100 °C for 10 min and was centrifuged at 9500g for 20 min. Up to 250 lL supernatant was then injected onto the Oxford cup, and the diameters of the inhibition zones were measured after being incubated at 30 °C for 6 h. The fitting equation for antibacterial activity (titer) counting is given by Y = 10[(X 2.6475)/5.9311] N, where Y represents the titer (using concentration unit lg/mL), X represents the diameter of the inhibition spot (11.3 mm < X < 14.5 mm), and N represents the dilution times of the fermentation broth. 2.3. Isolation and purification of the antibacterial substance The fermentation broth of B. brevis XDH was sterilized at 100 °C for 10 min and was centrifuged at 4200 rpm for 30 min. The supernatant, which was mixed with ammonium sulfate to the saturation of 45%, was kept at 25 °C overnight and was centrifuged at 4200 rpm for 20 min. The salting-out sediment was dissolved using distilled water and was condensed using a rotary evaporator. The CM Sepharose Fast Flow gel was rinsed with NaCl (1 mol/L) and distilled water successively for 2–3 times before being loaded onto the column (1.6 30 cm). It was then equilibrated with deionized water for 12 h. Approximately 15 mL of the salting-out samples was added onto the surface of the gel, and was rinsed absolutely with plenty of distilled water to clean the substances that were not adsorbed. Linear gradient elution of NaCl (0–1.0 mol/L) was then performed at a flow rate of 60 mL/h. The eluate was monitored using a UV detector, and a bioassay was used to examine the antibacterial active peaks of the elution. The effective components were collected and desalinated using Sephadex G-25 chromatography, based on the same method as the CM Sepharose FF in loading, monitoring, and collecting, except that it was only rinsed with distilled water. Ultimately, the antibacterial substances were condensed and lyophilized. The crude material was then prepared using reversed-phase HPLC. The condition was as follows: Sinochrom ODS-BP column (300 10 mm, 10 lm), injection volume of 200 lL, temperature of 30 °C, flow rate of 2.0 mL/min, water and acetonitrile containing 0.1% trifluoroacetic acid (v/v: 68/32) as the mobile phase, and absorption wavelength of 210 nm. The active component was collected for property analysis. 2.4. Property analysis of the antibacterial substance The antibacterial component was sent to Shandong Analysis and Test Center, China, and the structure was elucidated via electrospray ionization mass spectrometry (ESI-MS) and experiments of TOCSY, NOESY, ROESY, 13C HSQC 2D NMR. After obtaining the detailed structure, China Peptides Co., Ltd. in Shanghai synthesized 100 mg of the pure sample. The modified 100 mg sample (acetylation at N-terminal and amidation at C-terminal) was also produced
505
by the company to investigate its antibacterial ability. The minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values of the natural product and the two kinds of synthetic peptides were determined according to literature (Gauri et al., 2011). The active peptides, both natural and synthetic, were prepared to 100 lg/mL with distilled water and were incubated at 100 °C for 0, 10, and 20 min, respectively, to determine the thermal stability. After cooling to room temperature, the residual antibacterial activities were measured as described above. 3. Results and discussion 3.1. Isolation and purification of antibacterial component Brevibacillus species generally exist in many environments. As a group, this genus offers several advantages for protection against pathogens, because of the broad-spectrum activity of their antibiotics (Song et al., 2011). In general, the mechanisms of pathogen control are classified as competition, parasitism or predation, and antibiosis. B. brevis XDH is a potent antimicrobial agent isolated from the soil of Mount Tai, and it has a strong antibacterial activity against many pathogens of humans and animals (Song et al., 2012). B. brevis XDH showed great potentials in application, and thus it was chosen for further investigation. The crude antibacterial substances were precipitated using ammonium sulfate from the supernatant of fermentation broth. The extracts were then used for ion exchange chromatography on a CM FF column, which produced two unadsorbed fractions (P1 and P2) and an adsorbed fraction (P3), to enrich the active components from the culture of B. brevis XDH. The three fractions were assayed for growth inhibition on the test pathogens (E. coli and S. aureus). No antibacterial activity was detected in P1 and P2, whereas, it was observed in P3, resulting from the linear NaCl gradient elution at the concentration of around 0.3 mol/L. Further purification for the P3 was conducted using the reversed-phase HPLC, and four main peaks (A, B, C, and D) were obtained. After each peak was assayed for the antibacterial activities of E. coli and S. aureus, the third fraction (C) was found to show strong inhibitory activities. This compound was purified and collected using preparative HPLC on a Venusil XBP C18 preparative column under the same conditions mentioned above. About 35 mg purified component C was obtained from the 5 L liquid culture of B. brevis XDH. 3.2. Property analysis for the antibacterial compound The experiments of ESI-MS and NMR were performed, and the experts from the Shandong Analysis and Test Center analyzed the results. The molecular mass of the active peptide was determined to be about 1102.35 Da. Structure elucidation revealed that the active component was a linear peptide that consisted of nine amino acids. The detailed sequence was illustrated as Ser1-Leu2-Tyr3Lys4-Leu5-Thr6-Cys7-Lys8-Phe9 (SLYKLTCKF). Further unscrambling of the NMR spectra showed that residues 4 and 9 are of Dconfiguration, and others are of L-configuration. No same or similar sequences were found in the protein databases online. The present data support the identification of a novel antibacterial peptide, which was named Tostadin. Tostadin demonstrated significant antibacterial activities against E. coli and S. aureus. Pure Tostadin samples, both unmodified and modified, were synthesized in China Peptides Co., Ltd. to further analyze some of its properties. The MIC and MBC values of the unmodified peptide were 16 lg/mL and 32 lg/mL, respectively, with E. coli as the indicating microorganism. S. aureus was less sensitive to Tostadin with the MIC and MBC values of 32 lg/
506
Z. Song et al. / Bioresource Technology 111 (2012) 504–506
mL and 64 lg/mL, respectively. However, the modified peptide (AC-SLYKLTCKF-NH2) showed a stronger antibacterial activity, of which the MIC and MBC values against both of the two pathogens were 8 lg/mL and 16 lg/mL, respectively, basically the same with those of the natural product. This demonstrated that the synthetic peptides have the similar antibacterial abilities with the natural Tostadin. The results of the thermal stability tests suggest that both the natural and synthetic Tostadin are considerably thermally stable. The antibacterial activity was almost unchanged (remained more than 95% activity) even after being maintained at 100 °C for 20 min. Hundreds of antimicrobial peptides have been isolated and identified from a great number of microorganisms (Dale and Fredericks, 2005). Their modes of actions, including disrupting membranes, interfering with metabolism, and targeting cytoplasmic components have been well studied (Saikia et al., 2010). Brevibacillus is a well-known antibiotic-producing species that can also produce a wide variety of metabolites with antimicrobial activities (Zhang et al., 2008). A number of the active metabolites were bactericidal or bacteriostatic peptides, which were non-ribosomally synthesized using the multi-enzyme-catalyzed systems. Most of these peptides are very stable because of their low molecular weight and specific structure (Frueh et al., 2008). Gramicidin S is such a cyclic decapeptide with wide antibiotic spectra, which is a well-studied antimicrobial compound produced by B. brevis (Elmar et al., 1999). Moreover, these compounds have become research hotspots in the recent years because of their broad antibacterial spectrum, high potency, and no-crossing drug resistance with other antibacterial drugs (Zhao et al., 2010). In the present study, the antibacterial peptide Tostadin was isolated using a three-step isolation procedure, including ammonium sulfate precipitation, cation exchange chromatography on CMSepharose Fast Flow column, and the reversed-phase HPLC. This purification process was successfully used for this antibacterial peptide from strain XDH. The pure active component, namely Tostadin, which showed strong inhibition against E. coli and S. aureus, was purified and identified. Moreover, the modifications at both ends of Tostadin were found to display higher activities than those of the unmodified compounds. This phenomenon may be caused by the stable structure of the modified peptide since it is not easily resisted or decomposed. Besides, both the natural and synthetic Tostadin exhibited thermal stability, which can be found in other non-ribosomal peptides. In summary, this novel compound is a soluble peptide with great antibiotic effects and high stability, and it is easily synthesized because of its low molecular weight and linear structure. Thus, Tostadin may have great potential applications as a novel antibacterial agent. 4. Conclusions In this work, a linear peptide namely Tostadin was isolated and identified from a B. brevis strain XDH, which showed strong antibacterial activities against E. coli and S. aureus. Thirty five milligram
purified Tostadin was obtained by ammonium sulfate precipitation, ion exchange chromatography, and reversed-phase high-performance liquid chromatography. MS and NMR analysis demonstrated that this compound consisted of nine amino acids, of which two amino acids residues were of D-configuration. Further studies illustrated that both the natural and synthetic Tostadins were thermally stable. When modified at both ends, it exhibited higher activity and stability. These findings supported the characterization of a novel antibacterial peptide. Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at doi:10.1016/j.biortech.2012.02.051. References Cambau, E., Matrat, S., Pan, X.S., Bettoni, R.R., Corbel, C., Aubry, A., Lascols, C., Driot, J.Y., Fisher, L.M., 2009. Target specificity of the new fluoroquinolone besifloxacin in Streptococcus pneumoniae, Staphylococcus aureus and Escherichia coli. J. Antimicrob. Chemother. 63, 443–450. Dale, B.A., Fredericks, L.P., 2005. Antimicrobial peptides in the oral environment: expression and function in health and disease. Curr. Issues Mol. Biol. 7, 119– 133. Eaton, P., Fernandes, C.J., Pereira, E., Pintado, M.E., Malcata, F.X., 2008. Atomic force microscopy study of the antibacterial effects of chitosans on Escherichia coli and Staphylococcus aureus. Ultramicroscopy 108, 1128–1134. Elmar, J.P., Ruthven, N.L., Ronald, N.M., 1999. The interaction of the antimicrobial peptide gramicidin S with lipid bilayer model and biological membranes. Biochim. Biophys. Acta 1462, 201–221. Frueh, D.P., Arthanari, H., Koglin, A., Vosburg, D.A., Bennett, A.E., Walsh, C.T., Wagner, G., 2008. Dynamic thiolation–thioesterase structure of a nonribosomal peptide synthetase. Nature 454, 903–906. Gauri, S.S., Mandal, S.M., Pati, B.R., Dey, S., 2011. Purification and structural characterization of a novel antibacterial peptide from Bellamya bengalensis: activity against ampicillin and chloramphenicol resistant Staphylococcus epidermidis. Peptides 32, 691–696. Mogi, T., Kita, K., 2009. Gramicidin S and polymyxins: the revival of cationic cyclic peptide antibiotics. Cell Mol. Life Sci. 66, 3821–3826. Saikia, R., Gogoi, D.K., Mazumder, S., Yadav, A., Sarma, R.K., Bora, T.C., Gogoi, B.K., 2010. Brevibacillus laterosporus strain BPM3, a potential biocontrol agent isolated from a natural hot water spring of Assam. Indian Microbiol. Res.. doi:10.1016/j.micres. 2010.03.002. Shida, O., Takagi, H., Kadowaki, K., Komagata, K., 1996. Proposal for two newgenera Brevibacillus gen. nov. and Aneurini-bacillus gen. nov. Int. J. Syst. Bacteriol. 46, 939–946. Song, Z., Liu, K., Lu, C., Yu, J., Ju, R., Liu, X., 2011. Isolation and characterization of a potential biocontrol Brevibacillus laterosporus. Afr. J. Microbiol. Res. 18, 2675– 2681. Song, Z., Liu, X., Lu, C., Yu, J., Ju, R., Zhao, Y., Li, J., Liu, K., 2012. Medium optimization for the antibacterial substances production from Brevibacillus Brevis XDH using response surface methodology. Adv. Mater. Res. 345, 355–360. Viscardi, M., Perugini, A.G., Auriemma, C., Capuano, F., Morabito, S., Kim, K.P., Loessner, M.J., Iovane, G., 2008. Isolation and characterisation of two novel coliphages with high potential to control antibiotic-resistant pathogenic Escherichia coli (EHEC and EPEC). Int. J. Antimicrob. Agents 31, 152–157. Wang, J., Haddad, N.I., Yang, S.Z., Mu, B.Z., 2010. Structural characterization of lipopeptides from Brevibacillus brevis HOB1. Appl. Biochem. Biotechnol. 160, 812–821. Zhang, B., Xie, C., Yang, X., 2008. A novel small antifungal peptide from Bacillus strain B-TL2 isolated from tobacco stems. Peptides 29, 350–355. Zhao, Z., Wang, Q., Wang, K., Brian, K., Liu, C., Gu, Y., 2010. Study of the antifungal activity of Bacillus vallismortis ZZ185 in vitro and identification of its antifungal components. Bioresour. Technol. 101, 292–297.