Isolation and Anti-Leukemic Characterization ofExtracellular L-asparaginase From EndophyticBacterium, Brevibacterium sp. M-R21 Isolated Glycyrrhizaglabra Root

Abstract: L-Asparaginase (L-ASPase) is known as a potent anti-cancer drug against L-Asparagineauxotroph tumor cells. In this study, an endophytic L-ASPase producing bacterium of the genus Bervibacillus from the root of Glycyrrhiza glabra was screened and characterized. After purification of the enzyme by ammonium sulfate precipitation, dialysis, and silica gel column chromatography, anti-cancer studies were performed against MRC-5 (normal lung cells) and U937 cell (leukemia cell line). Additionally, optimization fermentation was performed in terms of significant variables screened from a one-factor-at-the-time (OFAT) approach. The interactions of different experimental parameters were investigated using the response surface methodology (RSM) with the central composite design (CCD) algorithm. Cytotoxicity study showed that the dose-dependent effect of the L-ASPase at 100 IU/ml had a lethality of about 80% against leukemia cells. Therefore, the IC50 of the enzyme for leukemia cells was calculated to be approximately 33.54 IU/ml. Interestingly, the cytotoxicity of L-ASPase against normal lung cells was only about 20% at L-ASPase activity of 60-100 IU/ml. Based on the quadratic model, the optimal fermentation conditions were predicted to be 2% glucose, 2% NaCl, pH7, and incubation temperature 30 °C. Under these conditions, the highest enzyme activity was 90 IU/ml, which had an efficiency of about 30% compared to non-optimized conditions. The results showed that L-ASPase isolated from Brevibacterium sp. M-R21 with selective cytotoxicity against the leukemia cell line may be a potential candidate as an anti-cancer drug after further study.