SCREENING, PURIFICATION, AND CHARACTERIZATION OF L-ASPARAGINASE PRODUCED FROM LACTOBACILLUS CURVATUS

Background: L-asparaginase, an enzyme employed in the food and cancer treatment industries, breaks down L-asparagine into L-aspartate and ammonia. It preserves food quality and prevents the formation of acrylamide. Beneficial microorganisms in probiotics offer biological benefits, including the removal of mutagenic and carcinogenic substances. Probiotics are commonly defined as helpful bacteria such as Lactobacillus and Bifidobacterium species. Aim: This study aims to screen, purify, and characterize L-Asparaginase from Lactobacillus curvatus isolates. Methods: The study screened Lactobacillus curvatus isolates for their ability to produce the highest L-ASNase activity. The enzyme was extracted, and its molecular weight was estimated using SDS-PAGE. The effect of pH, temperature, metal ions, and detergents on L-ASNase stability was also examined. The study found that L-ASNase activity was influenced by various factors, including pH, temperature, metal ions, and detergents. Results: Among the 11 bacterial isolates screened, 7 demonstrated the ability to produce L-asparaginase, forming a distinct pink zone (17.5–20 mm) with Lactobacillus curvatus isolate no. 11 exhibiting the highest specific activity of 4.86 µg/mg. Enzyme purification was optimized using ammonium sulfate precipitation at 70%, yielding a 6.6-fold purification and an 86.6% total yield. Purification via ion exchange chromatography and gel filtration chromatography enhanced the enzyme’s specific activity to 6 µM/mg, achieving a 12-fold purification with a 72% overall yield. SDS-PAGE analysis determined the molecular weight of the enzyme to be 37 kDa. The enzyme exhibited maximum activity at pH 7 and 40 °C, with stability in the pH range of 6–7 and temperatures between 35 °C and 40 °C. Enzyme activity was significantly enhanced by Mg²? and P?? ions (111%). Mercaptoethanol, EDTA, and Tween-80 increased enzyme activity to 109%, 103%, and 100%, respectively. Discussion: The study found that only 7 out of 11 bacterial isolates developed a large pink zone diameter of (17.5-20) mm, with isolate No.11 being the highest producer of L-ASNase. The specific activity of active L-ASNase-producing isolates varied, with the highest specific activity measured at 4.86 U/mg. The purified enzyme had a molecular weight of 37 kDa, with optimum activity at pH 7, stability at pH 6, 6.5, and 7, and a temperature of 40 °C. Enzyme activity increased significantly with metal ions, reaching its highest level at 111% when Mg and P were present. Conclusions: The study identifies L-Asparaginase-producing Lactobacillus curvatus from previous sources, which was purified and found to have a specific activity of 6 µg/mg with a 12-fold purification.