BIOCHEMICAL CHARACTERIZATION AND PARTIAL PURIFICATION OF A BACTERIOCIN LIKE-INHIBITORY SUBSTANCE PRODUCED FROM Bacillus sp. T68 STRAIN

Abstract

Bacteriocins are peptides produced by various types of bacteria. Members of the Bacillus genus are known to produce compounds with inhibitory activity in protein structure against pathogenic and non-pathogenic microorganisms. In this study, a bacteriocin-like inhibitory substance (BLIS) synthesized by Bacillus sp. T68 strain, which was previously isolated from soil, was characterized. T68 strain was grown on different media to produce bacteriocin. Crude BLIS obtained was tested by well diffusion method against indicator bacterium. It was investigated within the temperature range of 10-121 C and pH range from 3.0 to 10.0. It was examined in terms of different organic solvents, enzymes and chemicals. Extracellularly produced BLIS was partially purified by ammonium sulphate precipitation method and analyzed on SDS-PAGE. Activity of partially purified BLIS was investigated. It was observed that BLIS produced in Luria Bertani Broth medium was higher as compared to the other media against indicator bacterium at 30 °C for 24 h. BLIS activity maintained at low temperatures (10-40 °C) and lost completely at high temperatures (> 60 °C). It was detected that BLIS exhibited activity in the pH range between 5.0 and 10.0. The effect of EDTA on BLIS activity was slightly positive. Proteinase K and trypsin inhibited BLIS activity. Among the detergents, sodium dodecyl sulphate and Triton X-100 reduced BLIS activity, while Tween 20 and Tween 80 retained it. Additionally, it was determined that application of Tween 20 at 30 °C for 5 hours increased the BLIS activity by 40%. It was found that the solvents used other than butan 1-ol preserved the BLIS activity over 80%. Chloroform and isopropanol increased the BLIS activity slightly. An inhibitory zone formed by the BLIS corresponding to a molecular weight of about 15 kDa was detected. This BLIS exhibited stability over wide pH and temperature ranges and in organic solvent treatments.