Isolation and characterization of L-asparaginase of Bacillus sp. isolated from the riverside of Seyhan

The therapeutic enzyme L-asparaginase has received great attention as an antitumor agent for its treatment of tumor cells, particularly in acute lymphoblastic leukemia. In addition, as a food processing aid, L-asparaginases can significantly reduce the level of the carcinogen acrylamide in starchy foods. In this study, 40 Bacillus strains were isolated from the soil of the Seyhan riverside in Adana. Modified M9 medium containing L-asparagine as a substrate was used in order to evaluate the strain's L-asparaginase producing potential. 4 strains were determined as enzyme producing strains and among them, isolate No. Z12 showed the greatest activity in both qualitative and quantitative experiments. Isolate No. Z12 was identified as Bacillus sp. by microscopic (Gram +, endospore +, bacil) and biochemical tests (VITEK-2, 88%). The optimal enzyme production was carried out in a medium containing 0.75% L-asparagine as a substrate (pH 7.0) at 40°C for 5 days. Although L-asparaginase showed the activity over a wide range of pH values and temperature, optimum activity was observed at pH 8.0 at 40oC. When the enzyme was treated with different buffers (pH 7.0-12.0) for 60 min, it was observed that the activity was 100% preserved at pH 7.0. After pre-incubating the enzyme at different temperatures (40oC-100oC) for 60 min, the enzyme was 100% stable at 50oC, whereas the enzyme lost 50% of its activity in temperatures over 70oC. Urea and EDTA decreased the enzyme activity up to approximately 50%, however, MgCl2 increased the enzyme activity up to 20%. Using Zymogram and SDS-PAGE analyses, a single band was observed for the enzyme with the molecular weight of 128 kDa. According to the results, the native Bacillus Z12 strain can be used as a producer of L-asparaginase with industrial purposes.