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. 2021 May;24(5):666-674.
doi: 10.22038/IJBMS.2021.53419.12022.

In vitro identification of antimicrobial hemolytic lipopeptide from halotolerant Bacillus by Zymogram, FTIR, and GC mass analysis

Shekoofeh Sadat Etemadzadeh  1 , Giti Emtiazi  1
Affiliations

In vitro identification of antimicrobial hemolytic lipopeptide from halotolerant Bacillus by Zymogram, FTIR, and GC mass analysis

Shekoofeh Sadat Etemadzadeh et al. Iran J Basic Med Sci. 2021 May.

Abstract

Objectives: The multi-drug resistant bacteria and clinical infections are some of the biggest global concerns, so new drugs are needed. Antimicrobial peptides and lipopeptides are new bioactive agents with great potential that can become a new strategy for clinical applications.

Materials and methods: Some Bacillus strains were isolated based on hemolytic antimicrobial production from the soil. The extracellular proteins were extracted by acidic precipitation and chloroform/methanol method and analyzed by SDS-PAGE electrophoresis and stained with Sudan black. The black fragment was purified and characterized by FTIR, GC/MS, and HPLC analysis to demonstrate the presence of lipids and proteins. The anti-microbial ability and stability of the purified lipopeptide were assayed by the Kirby-Bauer method. Also, it was examined for metal removal.

Results: A new Bacillus halotolerans strain SCM 034 with hemolytic antimicrobial production was isolated. According to GC/MS (detecting C16, C17) and HPLC (detecting leucine, glutamic acid, valine, arginine, glycine, and aspartic acid) data, the black fragment was lipopeptide. Polyacrylamide hydrogel containing lipopeptide and gel purified lipopeptide showed anti-microbial activities against S. aureus and S. cerevisiae that were stable for a few months. Also, the lipopeptide was useful for cation removal and decreased cobalt, nickel, and calcium by 10.81 %, 24.39 %, and 34 %, respectively.

Conclusion: Production of antibacterial lipopeptide hemolysin from this strain is reported for the first time and according to the results, lipopeptides have unique properties with biomedical and pharmaceutical applications. Also, polyacrylamide hydrogel lipopeptide is a promising candidate for wound healing.

Keywords: Antimicrobial; Bacillus halotolerans; Hemolysin; Hydrogel; Lipopeptide; Polyacrylamide gel.

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Conflict of interest statement

The authors declare that they have no conflicts of interest.

Figures

Figure 1
Figure 1
SDS-PAGE gel electrophoresis of the extracted protein from the bacterial supernatants (A1 and L1); a) Gel without staining, where the light protein fragment was observed in yellow color especially in isolated A1, b) Gel staining by Coomassie brilliant blue on a light page, c) Gel staining by Coomassie brilliant blue on a dark page, where the light protein fragment was observed as the bright clear fragment and it was sharper in isolated A1
Figure 2
Figure 2
Growth curve of the isolated strain in N.B during 72 hr. The maximum growth was after 24 hr
Figure 3.
Figure 3.
Zymography of the SDS-PAGE gel of isolated A1 in different media to determine the effective fragment; a) Hemolysis activity of the bright clear fragment on the B.A plate, b) Anti-Staphylococcus aureus effect of the bright clear fragment, c) Anti- Saccharomyces cerevisiae effect of the bright clear fragment, d) Role of bright clear fragment in calcium absorption on a light page, e) Role of bright clear fragment in the calcium absorption on a dark page, f) Cobalt adsorption activity of the bright clear fragment
Figure 4
Figure 4
SDS-PAGE gel staining with Sudan Black B where the bright clear fragment was observed in black color because of the presence of lipid
Figure 5
Figure 5
Fourier transform infrared (FTIR) spectrum of the purified lipopeptide illustrating the functional groups of lipid and peptide
Figure 6
Figure 6
Major lipid compounds of the purified lipopeptide by GC/MS analysis: C16 and C17 which were found in bacterial lipopeptides
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