Biochemical characterization and detection of antitumor activity of L-asparaginase from thermophilic Geobacillus kaustophilus DSM 7263T

Abstract

L-asparaginases, which are oncolytic enzymes, have been used in clinical applications for many years. These enzymes are also important in food processing industry due to their potential in acrylamide-mitigation. In this study, the gene for L-asparaginase (GkASN) from a thermophilic bacterium, Geobacillus kaustophilus, was cloned and expressed in E. coli Rosetta (TM) 2 (DE3) cells utilizing the pET-22b(+) vector. The 6xHis-tag attached enzyme was purified and analyzed both biochemically and structurally. The molecular mass of GkASN was determined as similar to 36 kDa by SDS-PAGE, Western Blotting, and MALDI-TOF MS analyses. Optimum temperature and pH for the enzyme was determined as 55 degrees C and 8.5, respectively. The enzyme retained 89% of its thermal stability at 37 degrees C and 75% at 55 degrees C after 6 h of incubation. The enzyme activity was inhibited in the presence of Cu2+, Fe3+, Zn2+, and EDTA, while the activity was enhanced in the presence of Mn2+, Mg2+, and thiol group protective agents such as 2-mercaptoethanol and DTT. The structural modeling analysis demonstrated that the catalytic residues of the enzyme were partially similar to other asparaginases. The therapeutic potential of GkASN was tested on hepatocellular carcinoma cells, a solid cancer type with high mortality rate and rapidly increasing incidence in recent years. We showed that the GkASN-induced asparagine deficiency effectively reduced the metastatic synergy in HCC SNU387 cells on a xCELLigence system with differentiated epithelial Hep3B and poorly differentiated metastatic mesenchymal HCC SNU387 cells.