Optimization of concaminant production of Bacillus pacificus proteolytic and lipolytic enzymes for industrial applications

In the preliminary study, mesophilic bacteria isolated from soil were screened for protease and lipase enzymes and as a result of the study, 2NP6 isolate was found to be positive for protease and lipase production. 16S rRNA gene sequence analysis was performed to determine the species of 2NP6 isolate and it was found to be over 99% similar to bacteria belonging to the genus Bacillus. Whole genome sequence analysis was performed to determine the exact species of 2NP6 isolate and to determine its protease and lipase gene sequences. As a result of the analysis, it was determined that the isolate showed 88,35% ANI and 84,8% dDDH similarity to Bacillus pacificus species, so the isolate was named Bacillus pacificus 2NP6. Production conditions were optimized to optimize the production of both enzymes of the isolate. Simultaneous production conditions were determined as 1 g/mL 1% skim milk for carbon source and 0,5% (v/v) sesame oil for lipase. The nitrogen source for both enzymes was 0,5% g/mL yeast extract. 4% overnight culture inoculation and incubation at 30 °C for 48 hours resulted in optimum production of both enzymes. The optimum temperature and pH values of protease produced under optimized conditions were determined as 55 °C and pH 10,0, respectively, while the optimum temperature and pH values of lipase were determined as 60 °C and pH 8,0, respectively. The Km and Vmax values of lipase enzyme for p-nitrophenyl butyrate substrate were 0,053185 mM and 4349,01 U/mg, respectively; the Km value of protease for azocasein was 5,841085 mg/mL and Vmax value was 13514,54 U/mg. In order to investigate the potential for industrial use, the potential of protease for hair removal from skin and milk coagulation and the stain removal potential of protease and lipase on various stains were evaluated.