Determination of some enzymes activities and molecular characterization of Bacillus species isolated from soil and water samples in Sinop region

Bacteria are capable of producing enzymes that play an important role in organic matter decomposition and nutrient cycling. Especially, Bacillus strains with ability to produce and secrete large quantities of extracellular enzymes constitute the most important industrial enzyme producers. In the study; a total of 126 Bacillus spp. isolated from soil and water samples collected from different regions in Sinop were identified according to morphological, physiological and biochemical tests. The activities of amylase, cellulase and protease were determined from all the characterized Bacillus spp. isolates. From a total of 126 strains, 54 isolates displayed enzyme activity and SDS-PAGE total cell protein and plasmid DNA profiles of these isolates were also determined. The numerical analysis of SDS-PAGE whole cell protein profiles revealed that Bacillus strains were separated to 2 major groups at similarity levels 63% or above. Plasmid DNA analyses results confirmed that total of 54 Bacillus spp. strains 41 in a harbored plasmids with molecular weights changing between 2.156 to 20.678 bps. For each of amylase, cellulase and protease enzymes, one strain with the highest activities was determined. Partial purification and characterization of the enzymes were carried out by determining the optimum temperature and pH conditions of these strains for the best enzyme production. Besides, the effects of different chemicals and NaCl concentrations on the enzyme activity were determined. The maximum enzymes activities were obtained at 40°C and pH:8.0 for amylase, at 60ºC and pH 10.5 for cellulose and at 40°C and pH:8.0 for protease. All the enzymes showed maximum activity in presence of %3 NaCl with 60 minutes pre-incubation. At the %3-20 salt concentrations, amylase, cellulose and protease were stabile with the remaining activities of 62.86%; 79.79%; 82.19% respectively. In the presence of MnCl2, amylase, cellulose and protease had the highest remaining activity about 101.06%; 148.83% and 109.09%, respectively. In addition, CaCl2 ions displayed the highest remaining activities of amylase, cellulose and protease with 138.29%; 151.16%; 113.63%, respectively. On the other hand, the enzymes showed resistance against various detergents, inhibitor, metal ions, chelators The molecular weight of the enzymes were determined to be 120 kDa for amylase, 64 kDa for cellulose and 51 kDa for protease as a single band, by zymogram analyses. Maltose and maltose-derived oligosaccharides were determined as hydrolysis products of amylase and cellulose by thin layer chromatography. As a result, the investigated enzymes with high stability have the potential of use in many industrial fields such as detergent production, brewing, bakery, and textile since them are resistant to various detergents, inhibitor, metal ions, and chelators. Key Words: Bacillus spp., Soil, Water, SDS-PAGE, Cellulase, Protease, Amylase