국내 전통식품에서 분리한 Bacillus 속 세균이 생산하는 항세균 펩타이드의 분리, 정제 및 특성 연구

Abstract

Bacillus sp.CBS73 was selected from several numbers of Bacillus strains preserved in our laboratory isolated from Korean traditional foods. One of the reasons to study microorganism diversity is sequencing of the 16S rRNA gene, including which has several advantage size, and availability of databases for comparison. Fermented broth of CBS73 tested maximum antimicrobial activity against Mycobacterium smegmatis. Antimicrobial peptide production from the strain was optimized by using various nutritional parameters. 1% Fructose, 1% Beef extract, 0.1% NaCl were found to be the best carbon, nitrogen and metal ion sources. AMP CBS73 was purified by ammonium sulfate precipitation and gel chromatography respectively. In order to identify its structural characteristics, MALDI-TOF, and antimicrobial activity test against various Gram-positive and Gram-negative bacteria, including MRSA and E. coli were performed. The CBS73 was precipitated using 30-80% ammonium sulfate. AMP CBS73 was purified to homogeneity by using sequential gel chromatographic steps. i.e. Sepharose CL-6B, Sephadex G-50 and Sephadex G-25 respectively. The purified CBS73 exerted a molecular weight of 5210.2Da, as determined via mass spectrometry based on matrix-assisted laser desorptionionization–time-of-flight (MALDI–TOF) method and Tricine-SDS. AMP CBS73 was found to be stable over a broad range of pH 4.0 – 10.0 and thermos tolerant up to 100℃, where it lost its complete activity above 100℃. The effect of several chemicals on the AMP was evaluated. The AMP was resistant to various organic solvents and trypsin, α-chymotrypsin, lipase, proteinase K, protease, papain, pepsin and other various organic solvents. This AMP CBS73 exhibited broad-spectrum antimicrobial activity against tested Gram-positive and Gram-negative bacteria. Analysis of the minimal inhibitory concentration (MIC) revealed that CBS73 have antibacterial effects against Gram-positive bacteria including VRE, VRSA and Gram-negative bacteria including Escherichia coli, Salmonella. The antioxidant activity was evaluated by determining the reducing powers, total phenol content, and 2,2-diphenyl-1-picrylhydrazyl(DPPH) scavenging activity. DPPH scavenging activity and reducing power of the AMP CBS73 showed lower antioxidant activity when compared to the control group at the same concentration of ascorbic acid whereas total phenolic content determined that more than one-third of the phenol-containing Gallic acid at the same concentration. When the Raw 264.7 murine macrophage macrophages stimulated by LPS was a CBS73 antimicrobial peptide to produce an increase of the carbon monoxide did not show the effect of inhibiting the production thereafter by treatment at a concentration of 1-100㎍/㎖ and 1-50㎍/㎖ concentrations did not show cytotoxicity. Thus, from this study it can be concluded that, Bacillus sp. CBS73 strain isolated from domestic Korean traditional food able to produce antimicrobial peptide having high antibacterial effect, low molecular weight, stable over wide range of pH, temperature, enzyme & chemicals and moderate antioxidant activity.