Purification and characterization of antibacterial surfactin isoforms produced by Bacillus velezensis SK

Abstract

Bacillus velezensis SK having broad-spectrum antimicrobial activity has been isolated from soil. The efficient extraction of antimicrobial compounds produced in various mediums has been done using Diaion HP-20 resin. Further, characterization of an antimicrobial compound by TLC, FTIR, in-situ bioautography analysis revealed the presence of cyclic lipopeptides, which is then purified by the combination of silica gel, size exclusion, dual gradient, and RP-HPLC chromatography techniques. Growth kinetic studies showed that Bacillus velezensis SK produces a mixture of lipopeptides (1.33 gL^-1). The lipopeptide exhibits good pH (2-10) and temperature stability up to 80 °C. LC-ESI-MS analysis of partially purified lipopeptide identified variant of surfactin, further analysis of purified chromatographic fractions revealed the occurrence of most abundant C₁₅-surfactin homologues (m/z 1036.72 Da). The isolated surfactin exhibits good antimicrobial activity (1600 AU/ml) against drug-resistant food-born B. cereus and human pathogen Staphylococcus aureus. Hence, identified strain B. velezensis SK and its potent antibacterial surfactin lipopeptide could be used in various food and biomedical applications.

Keywords: Antibiotic-resistance; Antimicrobial peptides; B. velezensis SK; LC–ESI–MS; Lipopeptide; RP-HPLC.

Fig. 1

Antagonistic activity of B. velezensis SK by cross streak method against a B. cereus NCIM 2703 b Staphylococcus aureus NCIM 2654, and c Colony morphology on nutrient agar d 1500 bp amplified 16 s rDNA. e Evolutionary relationship of B. velezensis SK based on 16 s rRNA and closely related species of Bacillus, phylogenetic tree was constructed by using Neighbour-Joining method by using MEGA7 software, bootstrap values on each branch point indicates 1000 pseudo replicates (scale represents 0.0001 nucleotide substitution). f Antimicrobial activity of partial purified (HP-20) lipopeptides by using agar well diffusion assay showing zone of inhibition (upper panel) in diameter in mm (lower panel) against Gram-positive and Gram-negative indicator bacteria including streptomycin resistant (R)

Fig. 2

a Time dependent growth curve analysis of B. velezensis SK, for growth (O.D at 660 nm), residual glucose concentration and pH. b Lipopeptides production and biomass production kinetics in MBM at 37 °C, 120 rpm agitation for 48 h. c Comparative antimicrobial activity of lipopeptides extracted by various methods from B.velezensis SK for efficient extraction against Gram-positive bacteria

Fig. 3

Antimicrobial activity of dual gradient (pH and acetone) fractions (1–11) by agar well diffusion assay against B. cereus ATCC 10,876 (6 mm well)

Fig. 4

Reverse-phase HPLC purification of lipopeptides produced by B. velezensis SK Chromatogram of a Standard surfactin, b Chloroform extracted lipopeptides using mobile phase 80% methanol in water over 30 min

Fig. 5

Thin layer chromatography (TLC) of partial purified lipopeptides from B. velezensis SK using a Solvent system-I and visualized with 0.2% ninhydrin, Lane 1, 2, 3, and 4 correspond to lipopeptide extracted with chloroform, lipopeptides produced in MBM, acid precipitated and methanol extracted lipopeptides and Diaion HP-20 lipopeptides respectively. b Solvent System-II and visualized by iodine vapour, TLC analysis of lipopeptide extracted by HP-20 lane 1 (MBM) and lane 2 (NB). c TLC plates of Diaion HP-20 extracted lipopeptide from NB visualized by UV 254 nm lane1, TSA lane 2, and Chloroform extracted lipopeptides lane 3 using solvent system-II. d In-situ antimicrobial activity of separated lipopeptides against B. cereus NCIM 2703 by using autobiography technique, and e Antimicrobial activity of TLC separated (upper spot) and eluted spots (disk 1) against B. cereus NCIM 2703 by paper disk assay. f Antimicrobial activity of TLC eluted (spot 1) bioactive spot by agar well diffusion assay. g TLC analysis of acid hydrolyzed purified lipopeptide fraction (Lane 2) and intact lipopeptide (Lane 1) as compared to amino acids Glutamate, Leucine, Aspartate and isoleucine lane 3, 4, 5, and 6 respectively after visualization by ninhydrin using solvent system-I

Fig. 6

Effect of a Temperature and b pH on antimicrobial activity of surfactin lipopeptide against B. cereus NCIM 2703 (pink and blue respectively) and S. aureus NCIM 2654 (violet) and E.coli NCIM 2832 (green). c Fourier transform infrared spectroscopy of B. velezensis SK lipopeptide extracted by HP-20 (blue) and purified by TLC (cyan), HPLC (forest green), and Silica gel chromatography (pink)

Fig. 7

Full ESI–MS scan of LC separated peak from dual gradient fraction (F5 80% acetone pH8) in positive ion mode showing molecular ion [M + H] of peaks at range of 62 to 64 min a C₁₂-Surfactin b C₁₄-Surfactin c C₁₅-Surfactin